Homologous recombination was described in Escherichia coli in the mid-1940s (351), and for many years it was thought to be the result of a sexual process, analogous to that found in eukaryotes. When the sensitivity to DNA damage of the first recombination-deficient mutants was noticed, it was realized that recombination in this bacterium may serve the needs of DNA repair as well (105, 107, 266, 267). Subsequently, genetic studies delineated two recombinational pathwaysthe primary, RecBC pathway, serving the needs of "sexual" recombination, and the secondary, RecF pathway, kicking in when the primary pathway is inactive and moonlighting at "postreplication repair" of daughter strand gaps (102, 106, 108). Still later, biochemical characterization of recombinational activities suggested that their primary role is in DNA repair (131, 132). Finally, the realization that disintegrated replication forks are reassembled by recombination justified the "repair" purpose for the RecBC pathway (130, 333) and prompted a revision of our ideas about the relationships of DNA replication and recombination.

The goal of this review is to consolidate genetic data on homologous recombination, physical data on DNA damage and repair, and biochemical data on recombinational enzymes under a different idea in an attempt to highlight new areas for the future in vitro and in vivo experiments. The different idea is that the primary role of the homologous recombination system in E. coli is to repair lesions associated with DNA replication of damaged template DNA (130, 336). Therefore, this review differs from other recent reviews on homologous recombination in E. coli (108, 320, 377) in that its two main emphases are on (i) the evidence for recombinational repair in bacteria and (ii) the interactions of various recombinational repair proteins with each other and with the replication machinery. The recombinational repair machinery is conserved among eubacteria, and so the same two basic pathways are present in such dissimilar species as E. coli and Bacillus subtilis. Therefore, although concentrating on the E. coli recombinational repair paradigm, occasionally I use evidence from other eubacteria.

Damage reversal and one-strand repair. Bacterial genomic DNA, like any macromolecule, is subject to constant chemical and physical assault. Repair of the resulting lesions is essential if DNA is to serve as the template for transcription and its own reduplication. In the course of evolution, a complex enzymatic machinery has evolved to maintain this centrally important molecule in usable form (195). Repair of some DNA modifications simply reverses the damage, returning DNA directly to its original state. For instance, photolyase, using near UV-visible light, splits UV-induced pyrimidine dimers (reviewed in reference 545). Another example is the suicidal Ada protein of E. coli, which transfers a methyl group from the modified base O⁶-methylguanine to itself (reviewed in reference 580).

Two-strand repair. Although the bulk of DNA damage affects one strand of a duplex DNA segment, occasionally both DNA strands are damaged opposite each other, resulting in two-strand damage, a term proposed by Howard-Flanders (266). To repair two-strand damage without the loss of sequence information, a cell needs a higher level of redundancy, an extra homologous sequence whose strands could be used to fix both DNA strands of the damaged sequence. The principle of such two-strand repair is depicted in Fig. 1. An affected duplex homologously pairs and exchanges strands with an intact homologous duplex (Fig. 1B). The resulting joint molecule is "resolved" by symmetric single-strand cuts in homologous strands, yielding two new DNA molecules, each containing a single one-strand lesion (Fig. 1C). Now the damaged strands can be mended by one-strand repair with the complementary strands as templates (Fig. 1D).

View larger version (17K): [in this window] [in a new window]   FIG. 1.   The idea of two-strand repair. (A) A DNA molecule with a two-strand lesion (small open rectangles in the solid duplex) is shown side-by-side with an intact homolog (open duplex). (B) The two sequences have exchanged strands in a homologous region, converting the two-strand lesion into a pair of one-strand lesions. (C) Junction resolution (the strands to cut are shown in panel B) separates the chromosomes from each other. (D) Excision repair removes the one-strand lesions, completing the overall repair reaction. Note that if black and white "parental" DNAs are not identical, the resulting chromosomes may become "recombinant."

Homologous recombination versus recombinational repair. Since the machinery for the two-strand repair is complex and not copious and since the repair incidents are rather infrequent, this type of repair is more accessible to genetic than to biochemical study. The principal genetic assay for two-strand repair is to monitor the formation of new chromosomes resulting from alternative resolution of joint molecules. A joint molecule (Fig. 2A) can be redrawn to show that the DNA junctions are able to isomerize (Fig. 2B). This isomerization of the junctions creates two possible ways of resolving each junction (shown by numbers beside the arrows [Fig. 2B]). If the resolution is random, in 50% of the cases the participating chromosomes will exchange shoulders, forming two "recombinant" chromosomes (Fig. 2C). If the parental chromosomes were genetically marked, progeny carrying recombinant chromosomes would be detected genetically as having traits that initially resided on separate parental chromosomes.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   The four ways to resolve a joint molecule with a double junction. Lowercase letters w, x, y, and z designate unique sites which serve as markers on the homologous chromosomes. A junction is resolved by two symmetrical single-strand cuts (small black arrows in panel B) across each other. Each such diagonal pair of cuts is numbered either 1 or 2 for each junction. If junctions freely isomerize and are resolved independently of each other, four outcomes of the resolution are expected. In two of the outcomes, the chromosome arms will be exchanged, resulting in recombinant chromosomes. (A) A joint molecule with two junctions as shown in Fig. 1B. (B) The same joint molecule isomerized to show both junctions in the open planar configuration (498). (C) The four resolution outcomes, numbered according to the resolution options realized at the left and the right junctions.

The two mechanisms of two-strand damage. Two-strand lesions appear in DNA in two distinct ways. DNA synthesis in a region increases recombination in this region (447), suggesting that one source of two-strand lesions is DNA replication. The fact that replication of DNA containing one-strand lesions stimulates recombination between this DNA and an intact homolog (360) suggests that DNA replication causes two-strand lesions when it runs into unrepaired one-strand lesions. There are at least two mechanisms of replication-dependent conversion of one-strand damage into two-strand damage. In vivo, a noncoding lesion (for example, an abasic site) is an absolute block to DNA replication in growing cells (347); similarly, in vitro, a noncoding lesion in template DNA blocks the progress of the major E. coli DNA polymerases (59). In the chromosome, replication is likely to reinitiate downstream of a noncoding lesion (see "Elongation phase of DNA replication in E. coli" below), leaving behind an unfillable single-strand gap (Fig. 3) (see "Origin of daughter strand gaps and mechanism of their repair: early studies" below). Such an unfillable gap is called a daughter strand gap, since it appears in one of the two daughter branches after the replication fork passage (538, 734). Another type of one-strand lesion, a single-stranded interruption in template DNA, is proposed to cause a disintegration (collapse) of a replication fork (see "Evidence for replication fork disintegration" below) (234, 597). As a result, a double-strand end is detached from the full-length duplex molecule (Fig. 3). Finally, inhibited replication forks are broken, similarly releasing one of the replicating branches as a free double-stranded end (263, 334).

View larger version (20K): [in this window] [in a new window]   FIG. 3.   The two major types of replication-induced two-strand lesions. A replication fork moves from left to right along the template DNA with unrepaired one-strand lesions. The left template contains a noncoding lesion (T=T, thymine dimer), the right template has a single-strand interruption. Additional explanations are given in the figure and in the text.

The two recombinational repair pathways of E. coli. The two types of replication-induced two-strand lesions are repaired in E. coli by two separate pathways, both dependent on the recA gene but named after the critical genes that distinguish between them (Fig. 4). Daughter strand gaps are repaired by the RecF pathway (see "Repair of daughter strand gaps" below), while disintegrated replication forks are repaired by the RecBC pathway (see Double-strand and repair" below). The three common phases (see "Two-strand repair" above) of the two repair reactions are (Fig. 4) (i) presynapsis, during which the damaged DNA is prepared for homology search, followed closely by synapsis, during which homologous pairing and strand exchange with the intact sister duplex occur; (ii) DNA replication restart; and (iii) postsynapsis, during which the recombination intermediates are resolved.

View larger version (20K): [in this window] [in a new window]   FIG. 4.   The two pathways of recombinational repair in E. coli. On the left, the RecF (daughter strand gap repair) pathway is shown; on the right, the RecBC (double-strand end repair) pathway is shown. Additional explanations are given in the text.

Frequency of two-strand lesions. Under conditions of laboratory growth, two-strand lesions are too infrequent to be detectable in wild-type (WT) cells directly by physical techniques, although they are detectable in recombinational repair mutants (434). After massive DNA damage, daughter strand gaps are detected as single-stranded regions of several hundreds of nucleotides in the chromosomal DNA (278, 710) or as interruptions in the newly synthesized DNA (538, 714), double-strand breaks are detected as an immediate chromosome fragmentation (61, 323, 683), and disintegrated replication forks are detected as replication-induced chromosome fragmentation (60, 715).

Recombinational repair capacity of E. coli cells. WT E. coli cells grown in a nutritionally poor medium are able to survive 53 to 71 cross-links per chromosome (595). It can be calculated on the basis of the data with excision repair-deficient strains (714) that E. coli cells are still viable after repairing 100 to 200 daughter strand gaps per chromosome. E. coli cells should also be able to tolerate multiple disintegration of replication forks, because recombinational repair should reattach the resulting double-stranded ends to the circular domains of the chromosome. The only two-strand DNA lesion that has proved to be deadly for E. coli is a double-strand break. E. coli survives only two or three double-strand breaks in its chromosome (325, 683), which suggests that whenever a double-strand break occurs in an unreplicated portion of the chromosome, it cannot be repaired. Whether E. coli is an exception among bacteria in its inability to repair multiple double-strand breaks remains to be determined. There is a eubacterium, Deinococcus radiodurans, which can repair >100 double-strand breaks per chromosome (437), but this extreme resistance to DNA damage stands out in the bacterial world.

The two main E. coli reservoirs in nature are (i) the animal gut, where the microbe is dividing and concentrated; and (ii) the natural water of lakes and ponds, where the microbe is starving and diluted (560). In the gut, that is, in the environment rich in nutrients and protected from the elements, E. coli is likely to replicate its DNA for many generations without much need to repair it. However, when E. coli finds itself in the water, where DNA replication stops and DNA repair is anemic while the possibilities for damage of DNA are significant, the E. coli genome must accumulate a tremendous amount of DNA damage. Unfortunately, the gut is a discontinuous niche, since the animal the gut belongs to will eventually die; therefore, to survive in the long run, E. coli has to exit the old gut and recolonize a young one. When the battered E. coli from the water eventually makes it to a new gut and starts replicating, it finds its DNA riddled with unrepaired lesions.

The sporadic occurrence of massive DNA damage separated by long periods of undisturbed growth calls for a modest standby repair system, capable of rapid induction in response to increased DNA repair needs. Such an arrangement is indeed found in E. coli; the rapid increase in its DNA repair capacity is called the SOS response (506).

Repair instead of DNA damage checkpoints: the prokaryotic strategy. The bulk of two-strand DNA lesions in enterobacteria are probably generated as a result of DNA replication on template DNA containing one-strand lesions. An easy way to prevent this aggravation would be to stop DNA synthesis altogether when one-strand lesions are sensed. This is exactly what eukaryotic cells dothey employ checkpoint mechanisms to delay chromosomal replication when their DNA is damaged (reviewed in references 295 and 401). Since prokaryotes would also benefit from such a strategy, it was argued that E. coli might have a system to delay DNA synthesis when its chromosome is damaged (73).

Organization of the SOS regulon. DNA lesions inhibit DNA replication. Inhibition of DNA replication in E. coli induces the SOS response: an increased expression of some 20 genes aimed at restoring the capacity of the chromosome to replicate (Table 1). The resulting enhancement of the ability of the cell to repair and tolerate DNA damage is achieved in several independent ways. The capacity of the cell for excision repair (see "Damage reversal and one-strand repair" above) is enhanced by overproduction of the UvrD helicase and the UvrA and UvrC subunits of the UvrABC excinuclease. Induced amounts of DNA polymerase II increase the capacity of the cell for DNA synthesis across abasic sites (58, 484, 660). Up to a 50-fold increase in the amount of RecA protein (292, 543) and a similar increase in the expression of RecN protein (494) enhance the recombinational repair. The SOS induction makes possible repeated disengagement of replisomes stalled at the lesions in template DNA to allow resumption of the synthesis downstream, a phenomenon known as replisome reactivation (see "Replisome reactivation and model for RecFOR catalysis of RecA polymerization at daughter strand gaps" below). When recombinational repair cannot fix certain DNA lesions, the UmuD'C complex catalyzes translesion DNA synthesis (see "Backup repair of daughter strand gaps: translesion DNA synthesis" below). Overproduction of SfiA protein inhibits cell division (41), providing extra time for completion of recombinational repair. If all these measures fail to restore DNA replication, the lingering SOS induction awakens colicinogenic plasmids and dormant prophages, whose expression lyses the cell. The lysis of doomed cells benefits the viable cells of the same clone when resources are limited, since inviable bacterial cells can multiply for several generations, wasting precious nutrients. The lysis by induction of a prophage or colicinogenic plasmid is therefore an example of "bacterial apoptosis," which could have evolved to increase the number of viable cells in a clone.

Levels of SOS induction. The SOS response is by no means a desperate attempt to stay alive, as its name inaccurately implies (506), but, rather, an orderly and measured reaction of the cell to DNA synthesis inhibition. General information on the E. coli genes induced during the SOS response is summarized in Table 1. The strength of SOS boxes in the operator regions of the SOS genes correlates well with the in vitro LexA repressor affinities for the corresponding promoters and is likely to determine the timing of expression of a given gene during the SOS induction. According to thus inferred order of expression during the SOS induction, the genes of the SOS regulon could be loosely grouped into three categories. The first genes to be induced are mostly those responsible for one-strand repair (uvrA, uvrB, and uvrD) or damage tolerance (polB) (Fig. 5). The LexA repressor itself is also induced immediately. The DinI gene product, which delays activation of translesion DNA synthesis (753), is likely to be synthesized at this stage, too. Increase in expression of the immediately induced genes is usually less than 10 times that of their constitutive expression. If the increased expression of the one-strand repair genes does not help to regain normal rates of DNA synthesis, the genes of recombinational repair, recA and recN, are induced (Fig. 5). The maximal induction of these genes is higher, 20- to 50-fold over their regular levels. When DNA damage is massive, so that even the enhanced recombinational repair cannot overcome the inhibition of DNA replication, the third group of genes, represented by sfiA and umuDC, is called into action. Since these genes are expressed at very low levels during regular DNA synthesis, their SOS induction could be more than 100-fold. Expression of the umuDC operon inhibits recombinational repair and makes possible translesion DNA synthesis (see "Backup repair of daughter strand gaps: translesion DNA synthesis" below), while SfiA protein delays cell division. As DNA replication rates return to normal, the three expression groups of the SOS genes are likely to become repressed in the reverse order (Fig. 5). Alternatively, if a cell cannot repair its DNA damage and is doomed to generate a dead lineage, prophages and colicin plasmids are induced to lyse it (Fig. 5).

View larger version (21K): [in this window] [in a new window]   FIG. 5.   An idealized induction kinetics of the four groups of LexA-controlled genes (also, see reference 611). The graph illustrates the well-regulated nature of the SOS response. Both the x axis (time) and y axis (the level of the SOS induction) are in arbitrary units; therefore, the heights of the three curves are not to be compared.

The entire 4.7-Mbp circular chromosome of E. coli is traversed by a single replication bubble emanating from the unique replication origin. Both replication forks of the replication bubble are active; they meet in a chromosome region called the terminus, which is situated across from the origin. The terminus is delineated by termination sites arranged so as to form a replication fork trapreplication forks can enter the terminus, but they cannot exit it (253). To replicate the whole chromosome within a less-than-1-h bacterial cell cycle, replication forks have to proceed at about 650 bp/s (68). However, even the higher speed of almost 800 bp/s is insufficient when the cell cycle of E. coli is squeezed into 24 min in a rich medium. To prevent underreplication, E. coli starts a new round of DNA replication well before the completion of the ongoing round. Thus, in cells growing in a rich medium, there are one to three replication bubbles (two to six replication forks) (244).

Conceptually, replication of the E. coli chromosome is subdivided into three major phases: initiation, elongation, and termination (reviewed in references 25 and 406). Termination is the least understood phase (253) and is not immediately relevant to the needs of recombinational repair, although inhibition of DNA replication associated with termination sometimes causes disintegration of replication forks with their subsequent recombinational repair (263, 573). Elongation is the phase at which the two-strand lesion formation occurs and the recombinational repair machinery meets the replication machinery. Initiation of chromosomal DNA replication is helpful in defining interactions of the key replication proteins. An initiation strategy of multicopy plasmids is relevant because it utilizes the host reinitiation mechanism after the completion of recombinational repair.

Initiation of chromosomal DNA replication in E. coli. For a replication fork to start, the DNA duplex must be open. At the origin of chromosomal DNA replication, this opening is effected by binding of the initiator protein, DnaA. DnaA recognizes and binds to a degenerate nonanucleotide (T/C)(T/C)(A/T/C)T(A/C)C(A/G)(A/C/T)(A/C) (562). At the origin of chromosomal DNA replication, four DnaA recognition sites are found in a cluster. Binding of 10 to 20 DnaA monomers to this cluster of DnaA binding sites leads to an opening of DNA duplex nearby.

Nucleoid segregation and the problem of accessibility. Nucleoid segregation sets the "window of opportunity" for recombinational repair. In rapidly growing E. coli cells, nuclear bodies, or nucleoids, are seen in the electron microscope as dimers (688, 741), and even after being freed from their "cells," many purified nucleoids have a doublet appearance (240, 458, 490), indicating that the separation of nascent nucleoids is concomitant with DNA replication. It has been proposed that replicated daughter branches of the parental chromosome do not stay entangled but from the very beginning form separate bodies, growing out from the replication point in opposite directions (381, 740). Spatial separation of the origin-proximal markers after origin duplication was visualized in live cells (215).

recA and peculiarities of recA null mutants. recA happened to be the first E. coli recombinational repair gene to be discovered (107). recA is not a part of any operon, is surrounded by genes unrelated to DNA metabolism, and has its own promoter and terminator (260, 546). Normally, recA expression maintains 1,000 to 10,000 RecA monomers per cell (292, 445, 543, 558). RecA production is induced by DNA-damaging treatments such as UV irradiation or nalidixic acid, resulting in up to a 50-fold increase in the amount of the protein (see "Organization of the SOS regulon" above) (292, 543).

The variety of phenotypes of recA mutant cells stems from a single deficiency, the inability to form an active RecA filament. The in vitro properties and activities of RecA filament still bewilder and fascinate those who study them. For in-depth treatment of the enchanting RecA biochemistry, see the excellent reviews by Kowalczykowski (319) and Roca and Cox (524).

RecA without DNA. In high-concentration solutions, RecA aggregates to form oligomers, filaments, and bundles (70, 71, 246, 737). One of the major species in these aggregates consists of rings of six to eight monomers (70, 71, 246). These rings are characterized by electron microscopy for RecA from Thermus aquaticus, due to their greater stability (758). Surprisingly, they resemble in gross details both the hexameric rings of helicases like DnaB or RuvB and the F₁-ATPase (168, 760).

Filament formation by RecA around ssDNA. In vitro, in the presence of physiological concentrations of Mg²⁺ and ATP, RecA assembles around ssDNA into a helical filament (Fig. 6), an entity proficient in all known RecA activities (in the absence of the nucleotide cofactor or in the presence of ADP, RecA forms similar filaments but with different parameters; since such filaments are inactive in RecA-promoted reactions, they are not discussed in this review). At physiological pH, RecA filament does not readily assemble on duplex DNA; however, a filament assembled on ssDNA extends into a contiguous double-stranded region (362, 572). Every RecA monomer within a filament binds a single ATP molecule (307). RecA filament assembled on ssDNA slowly hydrolyses ATP at a rate of about 30 ATP molecules per min per monomer (69); duplex DNA-bound filament has an even lower ATPase activity (362, 502).

View larger version (85K): [in this window] [in a new window]   FIG. 6.   RecA filament in vitro: photographs and molecular model. (A) A relaxed circular duplex DNA completely covered by RecA filament. Naked duplex DNA of the same length, lying mostly inside the RecA filament, illustrates that DNA within RecA filament is stretched 1.5 times. Reprinted from reference 625 with permission of the publisher. (B) A RecA-covered circular ssDNA molecule; the arrow points to a segment complexed by SSB. Below, another ssDNA circle of the same length can be seen, but since it is entirely complexed with SSB, it appears very different, i.e., much smaller and kinky. Note that while RecA filament stretches ssDNA, SSB compacts it. Reprinted from reference 246 with permission of the publisher. (C) A crystal structure-based molecular model of an 18-monomer segment of RecA filament with three symmetry-related monomers in gray. Each of these monomers is enlarged on the right to show residues conserved among eubacterial RecA proteins (see reference 524 for details). In such a filament, the 5' end of DNA1 would be at the top. Reprinted from reference 524 with permission of the publisher.

Two DNA-binding sites in RecA filament. Soon after the beginning of biochemical characterization of RecA protein, it was realized that, to promote homologous pairing, RecA must have at least two DNA-binding sites: the primary site accommodating DNA1, around which the filament was assembled, and the secondary site for DNA2, to be compared with DNA1 (269). Since then, the idea of at least two DNA-binding sites within RecA filament has been substantiated with a variety of evidence.

Cleavage of LexA repressor by RecA filament. RecA filament holding a single DNA strand promotes autocleavage of the SOS response repressor, LexA (259, 366), as well as autocleavage of phage repressors (172, 522, 559) and of the UmuD protein (77). It is said that in these reactions RecA plays a role of coprotease, because there are conditions under which LexA, phage  repressor, and UmuD cleave themselves in the absence of RecA (77, 364). The LexA-binding site lies deep within the filament groove and overlaps with the secondary DNA-binding site (759), explaining why, when both DNA-binding sites are occupied by ssDNA, LexA cleavage is inhibited (152, 515, 642). RecA filament assembled on duplex DNA promotes LexA cleavage at 5 to 20% of the rate observed with a filament assembled on ssDNA (152, 642). This feature of the SOS repressor cleavage makes biological senseif all the single-stranded regions associated with DNA lesions are made double stranded (supposedly by pairing with intact homologous sequences), there is no reason to boost the repair capacity of the cell any further.

Detection by RecA filament of homology to ssDNA bound in the primary site. Although RecA forms a filament around ssDNA1 in a sequence-independent manner, the filament itself is "a sequence-specific DNA-binding entity, with the specificity determined by the bound DNA" (524). The amount of nonhomologous DNA in vivo is overwhelming, since even an identical sequence, shifted a single nucleotide out of register, becomes perfectly heterologous to DNA1. Heterologous DNA is not neutral in homology searches: preincubation of presynaptic filaments with heterologous DNA inhibits subsequent homologous pairing (214). The problem of the complexity of natural DNA is compounded by the enormous intracellular DNA packing densities, measured in E. coli at 20 to 100 mg/ml (57), and the restricted accessibility due to the nucleoid segregation (see "Nucleoid segregation and the problem of accessibility" above). Under these conditions, RecA has to find homology to the damaged DNA within minutes, as illustrated by the extreme DNA damage sensitivity of a partially active recA allele, proficient in homologous recombination in vivo and capable of "slow" recombinational reactions in vitro (273). If not repaired quickly, the damaged DNA could be degraded or segregated from its intact sister or could bring about even greater damage if the upcoming replication fork runs into it. On the other hand, if recombinational repair mends DNA damage mostly at replication forks, the affected and the intact homologous DNA segments should initially be in close proximity with each other.

Strand exchange between DNA1 and DNA2 catalyzed by RecA filament. When homology is found (i.e., when a homologous duplex DNA2 is aligned with ssDNA1), RecA filament catalyzes the exchange of strands between the two DNA molecules. In this process, ssDNA1 forms hydrogen bonds with the complementary strand of DNA2 while the identical strand of the duplex is displaced (135) (Fig. 7). The outgoing strand of DNA2 is accommodated in the secondary ssDNA binding site at the periphery of the RecA filament to be extracted from there later by SSB (346, 420).

View larger version (24K): [in this window] [in a new window]   FIG. 7.   The distinguishable phases of RecA-promoted reactions, as they are thought to happen in vivo. Black lines indicate a damaged duplex; white lines indicate an intact duplex; the open rectangle with rounded corners indicates a RecA filament; the irregularity in one of the black DNA strands indicates a noncoding lesion. The left side represents daughter strand gap repair; the right side represents double-strand end repair. Explanations are given in the figure.

Assistance for RecA by SSB at all stages. In vivo, the ssDNA is promptly complexed with SSB (see "Initiation of chromosomal DNA replication in E. coli" above). Consequently, if RecA is to polymerize on ssDNA, it either has to displace SSB (Fig. 6B) or has to coexist with SSB on the same ssDNA. Peculiar patterns of SSB and RecA binding to ssDNA under different in vitro conditions are discussed below (see "Regular DNA replication" and "SOS-induced conditions"); for now it will suffice to say that under certain conditions SSB simply does not allow RecA onto ssDNA; under other conditions, SSB allows RecA polymerization on ssDNA in the presence of auxiliary proteins; while under a third set of conditions, it yields ssDNA to RecA without hesitation. Under the second and third sets of conditions, SSB also helps at all three stages of RecA-promoted in vitro reactions.

View larger version (15K): [in this window] [in a new window]   FIG. 8.   SSB helps in the assembly of the functional RecA filament. The thick line indicates ssDNA; stem-loop structures indicate secondary (duplex) structures in ssDNA at physiological Mg2+ concentrations; open rectangles with rounded corners indicate RecA filaments; quartets of small circles indicate SSB tetramers. Without SSB (the top and the left side), formation of long contiguous RecA filaments on ssDNA is compromised because the growth of nascent filaments is impossible past the secondary structures. SSB helps RecA to polymerize into long contiguous filaments (the right side and the bottom) by ironing out secondary structures in the ssDNA.

The efficiency of the in vitro RecA-promoted pairing is unexpectedly high. RecA can pair two sequences which share as few as 8 nucleotides of homology (270); as mentioned above, RecA can also promote extensive strand exchange between homeologous (homologous but not identical) DNA sequences. This quite indiscriminate nature of the RecA-promoted pairing poses a potential problem even for the generally nonrepetitive genomes of bacteria. For example, in the E. coli genome, there are several rRNA operons which are mostly homologous to each other (53, 251), as well as many short (20- to 30-nucleotides) perfect repeats (50). If not properly supervised, RecA could repair damage in one such repeat by using another one. Such improper pairing, if accompanied by crossing over, would lead to gross chromosomal rearrangements. The components of the major mismatch repair system in E. coli supervise the quality of RecA-promoted pairing.

Inhibition by MutS and MutL of pairing between homeologous sequences. The supervision of the legitimacy of RecA-promoted pairing between long sequences is likely to be a secondary function of the MutS and MutL proteins. The two proteins are the components of the major mismatch repair pathway in E. coli. MutS binds to mismatches, while MutL is believed to transmit the signal of the MutS-mismatch interaction to other parts of the correction system (439). mutS and mutL mutants have increased rates of recombination between homeologous sequences (179, 488, 512, 579). Mechanistically, this phenomenon is accounted for by the in vitro ability of MutS to inhibit RecA-promoted strand exchange between homeologous sequences (744, 745). MutL enhances the efficiency of this inhibition. It is suggested that MutSL complex binds to a newly formed mismatch still within RecA filament and that this binding inhibits further RecA-promoted strand exchange (745). MutL could also recruit the UvrD helicase (231) to actively disperse RecA filaments, one known in vitro activity of UvrD (446). Thus, in the event that RecA catalyzes strand exchange between homeologous sequences, completion of such a product in vivo is likely to be aborted by MutSL.

Possible disruption of pairing of insufficient length by helicase II. The in vitro ability of RecA to pair ssDNA and a duplex DNA which have in common fewer than 10 contiguous nucleotides (270) makes one wonder how RecA discriminates in vivo against a 10-nucleotide homology in favor of a long, genuine homologous sequence. The answer may be that it does not but that other enzymes supervise RecA-promoted pairing to disrupt recombination intermediates which are "too short" or have a specific, "banned" structure.

RecA catalyzes the central reaction of recombinational repair in E. coli; recA mutants are deficient in many aspects of DNA metabolism. recA genes are ubiquitous in eubacteria (524); they are seldom found inactive (404), but rarely are they indispensable for viability (459). The propensity of RecA to form hexameric circles in vitro betrays its structural relationship to DNA helicases and F₁-ATPase. RecA forms a helical filament around ssDNA, finds a duplex DNA homologous to this ssDNA, and catalyzes strand exchange between these two DNAs. The RecA-ssDNA filament also promotes self-cleavage of the SOS repressor, LexA. SSB assists RecA in all these in vitro reactions.

One unsolved issue in RecA biochemistry is the mechanism of the homology search by the RecA filament. Studying the structure and dynamics of individual DNA strands inside the filament could shed light on the homology search mechanism as well as on some RecA-dependent in vivo phenomena. Another area of interest is the in vivo supervision of the RecA-promoted strand exchange; in vitro characterization of this important function has just begun.

When two DNAs trade strands within their internal segments, a pair of DNA junctions is formed. Independently of whether these are three-strand or four-strand junctions, such a pair of DNA junctions can be resolved in three possible ways. One way to disengage the recombining DNAs is to simply pull them apart, reversing the RecA-catalyzed strand exchange (464, 664) (Fig. 9A). In reality, instead of pulling DNAs apart, the two junctions are probably translocated towards each other, "squeezing out" the exchanged DNA segments. The alternative way to remove the junctions is to resolve them by cutting individual strands. Three-strand junctions can be resolved by cutting a single DNA strand (190), while to resolve four-strand junctions, two DNA strands of the same polarity must be cut symmetrically (256) (Fig. 9B). Finally, there is a hybrid way of removing a pair of DNA junctions: one of the junctions is resolved by cutting, but the cuts are not sealed right away, and the second junction is eliminated by being translocated to these cuts (Fig. 9C).

View larger version (20K): [in this window] [in a new window]   FIG. 9.   The three ways to remove a double DNA junction. A pair of four-strand junctions is shown, but the same applies to a pair of three-strand junctions or to a combination of one four-strand junction and one three-strand junction. The two DNA duplexes participating in the joint molecule are shown as either solid or open double lines. Small arrows near the junctions indicate the direction of junction translocation. Scissors mark the position of strand cuts. (A) Removal by translocation only (topoisomerase model). (B) Removal by symmetrical single-strand cuts only (resolution). (C) Removal by a combination of cuts and translocation. Additional explanations are given in the text.

When a DNA end trades strands with an internal segment of a homologous DNA, a single DNA junction is formed. A single DNA junction, whether it is a three-strand or four-strand junction, can be removed by pulling DNAs apart (Fig. 10A), although this is unproductive, or it can be resolved by cutting the DNA strand(s) at the junction (Fig. 10B) or by translocating the junction to the introduced interruption in the originally intact DNA strand (Fig. 10C).

View larger version (19K): [in this window] [in a new window]   FIG. 10.   Removal of a single DNA junction. A three-strand junction is shown, but the same applies to a four-strand junction. The two DNA duplexes participating in the joint molecule are shown as either solid or open double lines. Small arrows near the junctions indicate the direction of junction translocation. Scissors mark the position of strand cuts. (A) Removal by translocation only. (B) Removal by symmetric single-strand cuts only. Note that for the three-strand junction, the black strand already has an end across the cut in the white strand. (C) Removal by a combination of a cut and translocation. Additional explanations are given in the text. The last two options create a replication fork framework, whereas the first option seems to be nonproductive. However, if the original two-strand lesion was a double-strand break and the invading end has already been extended by DNA synthesis, the expelled extended end can now anneal with the other end of the break, permitting lesion repair (517).

In E. coli, there are at least two independent enzymatic systems for DNA junction removal: the RuvABC resolvasome and the RecG helicase. Their mechanisms of interaction with DNA junctions are quite different, and yet they partially complement each other, since mutants with single mutations in one or the other system show only a moderate defect in recombinational repair. This implies that more than one way of DNA junction removal is realized in vivo.

Certain mutations conferring sensitivity to mitomycin C and UV light were mapped to a locus called ruv (372, 473). ruv mutants repair daughter strand gaps normally and are able to reinitiate DNA replication after the repair, but they fail to resume cellular division, forming long nonseptate, multinucleate filaments (372, 473). The filamentation phenotype of ruv mutants can be mutationally suppressed without improving the resistance of the cell to UV irradiation (372, 474), arguing against the idea (473) that ruv mutants are deficient in some function needed for the reinitiation of cell division after DNA damage. Staining of ruv cells for DNA after UV irradiation shows that almost all DNA is concentrated in several long filamentous cells, while most normal-size cells are anucleoid (274), suggesting a defect in the chromosome partitioning. ruv mutants are deficient for conjugative recombination in recBC sbc genetic backgrounds (see "Double-strand end repair in the absence of RecBCD" below) and for plasmid recombination in otherwise WT cells (370, 372). recA null mutations reverse the lethal effect of ruv mutations in certain circumstances (37, 474) and also suppress the chromosome partitioning defect after UV irradiation (274), indicating recombinational poisoning of ruv mutants and suggesting that the product of ruv locus acts in the postsynaptic phase, after the RecA-catalyzed formation of joint molecules.

The ruv locus was shown to be induced during the SOS response (590). Molecular characterization of the locus revealed the presence of three genes: ruvA and ruvB are organized into an SOS-inducible operon, while ruvC belongs to an adjacent, noninducible operon (38, 575, 587). Mutations in any one of the three ruv genes confer the same phenotype (370, 575).

The biochemical activities of RuvABC proteins and the ways they interact with DNA junctions, both structurally and functionally, are now well characterized. The remarkable progress in our understanding of RuvABC has been the subject of several recent reviews, to which the reader is referred for details and references (336, 585, 586, 721, 723). Here, only the major moments relevant for recombinational repair will be outlined.

RuvA (22 kDa) forms tetramers (676) that bind Holliday junctions; it is the Holliday junction-recognizing activity of E. coli (276, 480). In vitro, at physiological Mg²⁺ concentrations, Holliday junctions assume a folded conformation (164, 703) (Fig. 11A), which impedes spontaneous branch migration (476). In solution, when Mg²⁺ concentrations are lowered below a certain level, junctions assume a spread-out conformation (164) allowing rapid spontaneous branch migration (476). Binding of RuvA to a "folded" junction even in the presence of Mg²⁺ forces it to spread out into the square planar conformation (478) (Fig. 11B). This unfolding of the junctions by RuvA is thought to facilitate their subsequent branch migration.

View larger version (19K): [in this window] [in a new window]   FIG. 11.   Interactions of RuvA, RuvB, and RuvC with Holliday junctions. The two homologous duplexes (solid and open double lines) are connected by a single Holliday junction. RuvA tetramer is shown as the four-petal flower, RuvB hexameric rings are shown as the trapezoid washers on DNA duplexes, RuvC dimer (E) is shown as the pair of open circles. The direction of DNA movement through RuvAB complex in panels D and E is indicated by arrows. (A) A Holliday junction in a folded conformation, observed in vitro under conditions mimicking physiological ones. (B) A RuvA tetramer binds the junction to open it into a square planar conformation, while two RuvB hexamers bind two opposite arms of the junction. (C) The RuvAB translocase: the same as in panel B, but the second RuvA tetramer binds to the unoccupied side of the junction, locking it in a turtle shell configuration. (D) One of the RuvA tetramers is removed to show junction isomerization, promoted by the pulling action of RuvB (compare with panel B). (E) RuvABC resolvasome: one of the RuvA tetramers has left to allow a RuvC dimer to assume a position for the junction resolution. The resolution sites (diamonds in the opposite DNA strands) are drawn into the junction by the action of RuvB. (F) RuvC cleavage at the resolution sites separates the interacting homologs. The RuvABC resolvasome is not shown.

The crystal structure of RuvA reveals a flower-like tetramer, with a negatively charged convex surface and a positively charged concave surface (508). The crystal structure of E. coli RuvA bound to a Holliday junction shows a single RuvA tetramer holding on its concave side a Holliday junction in the open-square conformation (235), although some in vitro studies indicate that at sufficient RuvA concentrations, the junction must be sandwiched between two RuvA tetramers (481, 761). The crystal structure of RuvA from Mycobacterium leprae shows the latter configuration: two RuvA tetramers form a "turtle shell" with four sideway holes, enclosing a Holliday junction (525).

RuvB (37 kDa) looks like a helicase by sequence gazing, exhibits a weak helicase activity (678, 679), and, like several other helicases (and RecA [see "RecA without DNA" above]), forms hexameric "doughnuts". RuvB hexamers bind duplex DNA like beads on a string (628). At high concentrations and under special conditions RuvB inefficiently branch migrates (translocates) Holliday junctions (438, 453). RuvB with a mutation in one of the helicase motifs forms hexameric doughnuts but is defective in DNA binding (432). No atomic structure is yet available for RuvB.

RuvC (19 kDa) binds a Holliday junction as a dimer, spreading the junction, almost like RuvA, in a planar conformation (it is not exactly square, perhaps because RuvC is a dimer, not a tetramer like RuvA) (36). RuvC is the long-sought Holliday junction resolvase; it nicks two strands of the same polarity, the same distance from a presumed crossover junction (122, 277). The nicking occurs most efficiently at a degenerate sequence 5'-(A/T)TT(G/C)-3' (568); the minimal requirement for the cleavage is a single thymine on the 3' side of the break (584). RuvC binds to but does not cleave junctions that lack the nicking sequence (568, 584). The resolution is most efficient when the cleavage site coincides with the position where DNA strands trade partners (34). Nicks introduced by RuvC can be directly sealed by the E. coli DNA ligase (33, 277).

The atomic structure of RuvC resolvase reveals a dimer formed by two wedge-shaped subunits with two positively charged valleys on one side of the dimer (16). A Holliday junction must unfold before it can fit into the two valleys of the RuvC dimer. Studies of RuvC mutants that are able to bind Holliday junctions but are resolution deficient suggest that catalytic domains in the RuvC dimer are situated at the bottoms of the valleys and comprise four closely spaced negatively charged residues (16, 541). The DNA strands that are proposed to lie across the active center (and so are likely to be cut during the resolution) are the "noncrossover strands". RuvC cleavage of the noncrossover strands was demonstrated with model Holliday junctions, whose branch migration was restricted by tying together two arms at a time (35).

The indistinguishable phenotypes of ruv mutants in recombination (402, 575) suggested that all three proteins work in a single complex. In vitro experiments with pairwise combinations of Ruv proteins elaborate this idea. RuvA and RuvB interact in solution (581) as well as at Holliday junctions (481). In the presence of RuvA, the concentration of RuvB required for Holliday junction translocation is lowered 20- to 40-fold (438, 453). RuvA function is not limited to RuvB loading at the junctions, since RuvA is required continuously throughout the translocation (438), perhaps to maintain the junctions in the spread-out conformation. Two RuvB doughnuts, sitting on the opposite sides of the RuvA tetramer, pull duplex DNA through their holes, causing the junction to branch migrate (255, 478) (Fig. 11B and D). The force of this pulling can translocate the junctions through extended regions of nonhomology (479). Since DNA is a helix, DNA "pumping" through RuvB is likely to be achieved by duplex rotation relative to the RuvB hexamer.

No cross-linking is detected between RuvA and RuvC in solution (171), suggesting that these two proteins do not interact with each other. Since they both bind Holliday junctions, the two proteins at least have to compete for them. RuvA binds to Holliday junctions more strongly than does RuvC, inhibits RuvC resolution, and, at high concentrations, completely displaces RuvC from the junctions, apparently forming an octameric shell around them (726). However, at subsaturating concentrations, RuvA and RuvC form a cocomplex on the junctions, with the RuvA tetramer apparently occupying a specific side of the junction and RuvC dimer binding to the unoccupied side (726). Holliday junctions in the unfolded conformation have two distinct sides, distinguished by the orientation of DNA strands around the center of the junction. On the one side, DNA strands go 5' to 3' clockwise, while on the other side, the 5'-to-3' orientation is counterclockwise (Fig. 12). RuvA-Holliday junction cocrystals (235) show that RuvA tetramer binds the "counterclockwise 5'-to-3' side" of the junction, which leaves the other side for RuvC.

View larger version (9K): [in this window] [in a new window]   FIG. 12.   The "flip" and "flop" sides of a Holliday junction in the open conformation. The same junction is shown from the opposite sides. The 3' and 5' ends of DNA strands are marked. In the center, the 5'-to-3' direction around the junctions is indicated by arrows. RuvC binds to the "clockwise" side of the junction, whereas RuvA binds to the "counterclockwise" side.

RuvC and RuvB proteins form complexes in solution (171) and enhance each other's reactions with small synthetic Holliday junctions: RuvB accelerates junction resolution by RuvC, while RuvC stimulates branch migration by RuvB (692). With longer, more natural DNA substrates, stimulation of RuvC resolution by RuvB requires the participation of RuvA (764). It is proposed that, due to the site specificity of RuvC resolution, RuvAB is needed to translocate Holliday junctions to resolution sites, where RuvC can resolve them (764). Coimmunoprecipitation allows the formation of RuvABC complexes around Holliday junctions to be detected (147), but it is unclear whether Holliday junctions stimulate interactions between RuvAB and RuvC or whether the junctions simply serve as a scaffold to hold the three proteins together.

In vitro, the presence of RuvC increases the proportion of the "productive" two-ring RuvAB complexes, formed on Holliday junctions at high RuvAB concentrations, under conditions where, in the absence of RuvC, three- or four-ring complexes predominate (691). At the same time, these two-ring RuvAB complexes impose a 20- to 40-fold specificity for the Holliday junction resolution by RuvC. Moreover, in the presence of RuvAB, RuvC resolves partially homologous Holliday junctions in the region of heterology, apparently because the resolution occurs during RuvAB-catalyzed branch migration (691). The name "resolvasome" was offered for the combined activity of RuvA, RuvB, and RuvC to reflect their functioning in a multisubunit complex capable of binding, isomerizing, translocating, and resolving Holliday junctions (337, 722).

The idea that RuvABC proteins work as a single complex explains why all ruv mutants have the same phenotype (370, 372), but it does not address the fact that only ruvA and ruvB expression is induced by DNA damage in E. coli (Table 1). Perhaps some SOS functions require significantly more RuvA and RuvB than RuvC. The relevant difference in cell physiology between normal and SOS-induced cells is discussed later (see "SOS expression as a compensation"); only the underlying enzymatic mechanisms are dealt with here. The in vitro observation that at saturating concentrations RuvA displaces RuvC from Holliday junctions (726) by assembling an octameric "turtle shell" around the junctions (525) (Fig. 11C) is a clue to these mechanisms, but it does not provide a rationale for them.

The rationale is suggested by the observation that recA mutants with enhanced ability to displace SSB from ssDNA (344, 396) exacerbate the UV sensitivity of ruv mutants (680), as if Ruv proteins normally counteract RecA filament assembly. In fact, one of the first discovered phenotypes of ruv mutants was their inviability in combination with the hyperactive RecA441 mutant protein (474). Therefore, it was proposed that RuvAB complex uses Holliday junctions to disperse the associated RecA filaments (337). Interactions of RuvAB translocase with RecA filaments in vitro support this notion: (i) RuvAB dissociates recombinational intermediates covered with RecA filaments (279, 677), and (ii) RuvAB disperses RecA filaments from duplex DNA (1).

Electron micrographs show that at high RuvB concentrations, four hexameric rings of RuvB surround a junction-bound RuvA tetramer (478, 691), suggesting that RuvA does not direct RuvB binding to particular arms. Although RuvC encourages the formation of the two-ring RuvAB complexes at Holliday junctions (691), the main factor in two-ring complex formation could be the availability of Holliday junction arms for the RuvB binding. In vivo, Holliday junctions are likely to be associated with RecA filaments, and RecA could preclude binding of RuvB to a particular pair of arms. By pumping through themselves the two available arms of a RecA-associated Holliday junction, RuvB hexamers will translocate the Holliday junction towards the RecA filament, which could cause filament dissociation. To complete this speculative picture, after RecA filament is dispersed, one of the RuvA tetramers could leave the junction, allowing access to RuvC resolvase (Fig. 11E).

ruvAB mutants are only moderately defective in homologous recombination or in repair of DNA damage (370, 372, 473), suggesting that other activities partially substitute for RuvAB in recombinational repair. Indeed, the moderate defect of ruv mutants in conjugational recombination or in DNA damage repair is aggravated by recG mutations (370). recG mutations by themselves cause only a moderate reduction in cell survival after UV or X-ray treatment (370, 373). recG is the last gene of the spoT operon, encoding a 76-kDa protein; there is no indication that the low-level expression of recG is enhanced during the SOS response (379).

The synergism of ruv and recG mutations is partly explained by the fact that recG encodes another DNA helicase with in vitro activities mostly overlapping those of RuvAB but with some important differences. RecG helicase binds Holliday junctions and drives their branch migration, but, in contrast to RuvAB, the RecG-promoted reaction is blocked by a heterology in excess of 30 nucleotides (380, 729). RecG can also dissociate RecA-made joint molecules containing Holliday junctions, even those still covered by RecA filaments (730). However, the DNA-unwinding activity of the RecG helicase is anemic even in comparison with the weak helicase activity of RuvAB, and the two enzymes translocate along ssDNA in the opposite directions: RuvAB in the 5'-to-3' direction (678) and RecG in the 3'-to-5' direction (731).

The in vitro activities of RecG make it a possible substitution for RuvAB in the RuvC-promoted junction resolution in vivo. However, ruvC mutants are no more UV sensitive and recombination deficient than are ruvAB mutants, while ruvC recG double mutants are as deficient as ruvA recG or ruvB recG double mutants (370). This indicates that RecG does not substitute for RuvAB in junction translocation in vivo and suggests that RecG uses a different mechanism to resolve DNA junctions.

In ruv mutants, the RecG pathway of junction resolution can be stimulated by the expression of RusA resolvase, whose gene resides on a cryptic prophage (402, 576). In contrast to RuvC, which forms a single complex with a Holliday junction, RusA, depending on its relative concentration, forms four different complexes with four-way junctions (92), suggesting that RusA monomers bind four arms of the junction independently of each other, apparently recognizing DNA branching rather than Holliday junction as a single structure. RecG also tends to bind a Holliday junction from one side and shows a comparable affinity to three-way junctions (424, 729). RecG dissociates three-way junctions by binding to a particular arm and "extruding" the extra DNA strands complementary to the strands of the original arm ahead of the branching point. Consistent with this handling of three-way junctions, RecG dissociates R loops both in vivo and in vitro (197, 257, 699), although it is unable to unwind plain RNA-DNA hybrids of the same length (699). The "extruding" activity of RecG at the three-strand junctions suggests that the RecG-dependent resolution pathway works mostly with three-strand junctions.

This hypothetical mechanism for the RecG-dependent three-strand junction removal is compatible with the observation that while both RuvAB and RecG are able to translocate deproteinized three-strand junctions, only RecG can translocate them when they are still covered with RecA filament (728). In doing so, RecG disrupts RecA-promoted pairing, dissociating the joint molecules. The direction of RecG translocation towards the RecA filament is determined by RecA itselfwhen the filament is absent, RecG prefers to translocate the same junction in the opposite direction (728). These observations suggest that in vivo RecG pushes three-strand junctions towards the associated RecA filament (Fig. 13). For such a resolution to be productive, nicking of DNA strands at branching points has to occur (Fig. 10B and C and 13A).

View larger version (16K): [in this window] [in a new window]   FIG. 13.   Hypothetical removal of a single three-strand junction by RecG. RecA filament is shown as an open rectangle with rounded corners; RecA monomers (in panel C) are shown as open circles; RecG is shown as a dotted oval. The small arrow in panel A indicates the position of the required single-strand incision. Explanations are given in the text.

ruvABC genes are ubiquitous among eubacteria, but their arrangements tend to differ (721). RecG homologs are also found in other eubacteria (187, 194, 290). The hypothetical action of the two resolution systems during particular recombinational repair pathways is discussed in the appropriate subsections (see "Removal of DNA junctions and used Rec filaments" and "The two pathways for DNA junction removal in double-strand end repair") of the next two sections.

When excision repair-deficient E. coli cells are irradiated with low doses of UV, the rate of their DNA synthesis is barely affected (538, 605). Moreover, the molecular weight of nondenatured chromosomal DNA from these cells is not decreased. Even when total DNA from the irradiated cells is denatured with alkali, it still has the same molecular weight as the denatured DNA from unirradiated control cells, confirming the absence of excision repair. However, the newly synthesized DNA in UV-irradiated cells has a lower molecular weight even in excision repair-deficient mutants, indicating single-strand interruptions.

The single-strand interruptions in the newly synthesized DNA after UV irradiation are due to replisome encounters with pyrimidine dimers. When a replisome encounters a noncoding lesion (a pyrimidine dimer or an abasic site) in template DNA, its progress is blocked, as illustrated by the inability of the major E. coli DNA polymerases to bypass such lesions in vitro (59) and by the lower than 0.5% transformation efficiency of an ssDNA carrying a single lesion of this type compared with the lesion-free ssDNA (265, 348). Replication of both strands of the E. coli chromosome in vivo is believed to be discontinuous (see "Elongation phase of DNA replication in E. coli" above), and the other replisomes are likely to restart downstream from the lesion as scheduled, but the DNA segment between the site of the lesion and the position of replication restart will be left single stranded (Fig. 14B). Since such a single-strand gap forms in only one of the two daughter branches of the replicating chromosome, it is called a daughter strand gap. Daughter strand gaps were first detected physically by Rupp and Howard-Flanders (538) and genetically by Cole (117). Their average length was reported to be 800 nucleotides (278), which is approximately half the average length of Okazaki fragments in E. coli (317). A different method produced an estimate of 100 to 200 nucleotides for the size of daughter strand gaps in both the leading and the lagging strands in specific DNA sequences (710), indicating that the gaps might be quite small.

View larger version (20K): [in this window] [in a new window]   FIG. 14.   Experimentally established principles of the daughter strand gap repair in E. coli. Solid lines indicate parental DNA strands; open lines indicate newly synthesized daughter DNA strands; diamonds indicate thymine dimers; small horizontal arrows indicate single-strand scissions required to resolve the joint molecules. (A) A DNA molecule containing unrepaired noncoding lesions (for example, thymine dimers). (B) Replication of this molecule generates two molecules with single-strand gaps in the daughter strands opposite the lesions (538). Eventually, these gaps are repaired in a RecA-dependent way (606). (C to H) Experiments by Rupp et al. (539) had revealed that after gap closure, the newly synthesized strands are connected with the parental strands (strand exchange), suggesting a model for recombinational repair of daughter-strand gaps (C to E). Later, Ley (357) found that the gaps are transferred from the daughter strands to the parental strands, while Ganesan (199) found that the thymine dimers are transferred in the opposite direction, from the parental strands to the daughter strands, suggesting the formation and resolution of Holliday junctions (F to H).

One way to fill a daughter strand gap would be to modify a stalled replisome so as to allow it to carry out translesion DNA synthesis (see "Backup repair of daughter strand gaps: translesion DNA synthesis" below). However, experiments by Rupp, Howard-Flanders and colleagues revealed a peculiar feature of the daughter strand gap repair in E. coli: filling in the gaps was accompanied by formation of hybrid DNA strands in which segments of the newly synthesized strands were linked with segments of the template strands (539). The number of such exchanges of strands between sister duplexes roughly coincided with the number of UV lesions in the template DNA, indicating that daughter strand gap repair is accompanied by strand exchange (Fig. 14C to E). This phenomenon led the authors to propose that daughter strand gaps in E. coli are filled, with the help of the intact sister duplexes, by recombinational repair (266, 539). This conclusion was in line with the findings that conjugative transfer of UV-irradiated DNA stimulates genetic recombination (734) and that recA mutants, deficient in homologous recombination, are also deficient in daughter strand gap repair (199, 268, 507, 606).

The repair-associated strand exchange was further confirmed by the demonstration that in excision repair-deficient cells, irradiated parental DNA acquires patches of daughter DNA containing the gaps, whose number is slightly smaller than the number of UV lesions (357, 533). Together with the complementary demonstration that during the daughter-strand gap repair the initial lesions in the old DNA strands have a 50% probability of being transferred to the newly synthesized DNA strands (199) these results suggested the formation of Holliday junctions (see "Two-strand repair" above) and their alternative resolution (Fig. 14F to H).

It is noteworthy that the described repair reaction mends the daughter strand gaps but does not deal with the original one-strand lesions that have caused them (Fig. 14E and H). The original noncoding lesions have to be removed by excision repair after the gap has been filled in. In excision repair-deficient mutants, used in the physical studies of daughter strand gap repair, the original lesions persist in DNA for many generations after UV irradiation, being gradually diluted as cells multiply. Because of that, it is said that recombination in excision repair-deficient mutants is a mechanism of damage tolerance rather than repair.

In E. coli, daughter strand gaps are mended by the RecF pathway of recombinational repair, which is named after the first discovered gene specific for this pathway (200, 261, 535). A tentative sequence of postulated events during this process is as follows (108) (Fig. 15): in preparation for synapsis, the RecOR complex descends on the SSB-complexed daughter strand gap, perhaps guided by the RecFR complex. The presence of RecO allows RecA polymerization on the SSB-complexed ssDNA. During the synaptic phase of the reaction, RecA filament finds an intact duplex, homologous to the single-strand gap, and pairs them (Fig. 15B). The synapsis is facilitated by two different topoisomerases: DNA gyrase relieves positive supercoils, generated in the intact duplex due to the strand invasion, while topoisomerase I (Topo I) relieves negative supercoils in the new duplex between the invading and the resident strands. Pairing of the damaged and the intact DNA molecules allows filling in of the gap by a DNA polymerase. In the postsynaptic phase of the reaction, RuvABC resolvasome or RecG helicase removes Holliday junctions and the associated RecA filaments, completing faithful repair of the daughter strand gap. If an intact homologous duplex cannot be found, UmuD'C complex modifies the RecA filament and the replisome to allow translesion DNA synthesis, restoring the duplex at the price of possible mutagenesis. The flesh of experimental observations that animate this conceptual skeleton for daughter strand gap repair is presented below.

View larger version (26K): [in this window] [in a new window]   FIG. 15.   Overview of daughter strand gap repair. (A) A DNA molecule with a daughter-strand gap. T=T, the thymine dimer that caused the gap. (B) Synapsis of the gapped molecule with the intact homologous DNA. (C) Filling in of the gap and repair of the lesion that caused the gap. (D) Disengagement of the two DNA molecules. The RuvC cleavages are indicated by small vertical arrows in panel C. Additional explanations are given in the text and in the figure.

recF, recO, and recR: mutant phenotypes. Operationally, the mutants deficient in the presynaptic phase of daughter strand gap repair should be as deficient in daughter strand gap closure as are recA mutants, which are blocked at the subsequent synapsis. In vitro, SSB protein helps RecA at both presynapsis and synapsis; ssb mutants are deficient in recombinational repair of UV damage (359, 732) but have not been specifically tested for the deficiency in daughter strand gap closure. There are four genes which, when mutated, create mutants deficient in daughter strand gap closure. One of them is lexA (200); LexA is the repressor of the SOS regulon, and its role in daughter strand gap closure is likely to allow increased RecA production in response to DNA damage (see "Levels of SOS induction" above). The other three genes are recF (200, 534), recO (680), and recR (680). In addition to the deficiency in gap closure, recF mutants are deficient in the reciprocal process, the transfer of lesions from the parental to the daughter strands (716), suggesting that the reason why daughter strand gaps in recF mutants cannot be repaired is because homologous exchange is blocked.

recF, recO, and recR: possible replisome connection revealed by gene structure. recF is the third gene in a four-gene cluster which starts with dnaA (DnaA protein initiates chromosomal DNA replication at oriC) and also includes dnaN (a gene coding for the "sliding-clamp" subunit of DNA pol III) and gyrB (B subunit of DNA gyrase, the enzyme that introduces negative supercoiling into the E. coli chromosome [see "DNA gyrase" below]). Although recF has its own promoter inside the coding sequence of dnaN, it is thought that dnaA, dnaN, and recF constitute an operon under the control of dnaA promoters (485). The conserved structure of this chromosomal region in even distantly related eubacteria further argues for the biological significance of this combination of recF with replication genes (references 196 and 405 and references therein). The complex regulation of the recF gene expression (17, 485, 549) ensures that the RecF protein is maintained at a low level, less than 190 monomers per cell (394), which is, maybe coincidentally, close to the number of DnaN dimers (about 150 per cell) (79). When E. coli cells enter stationary phase, expression of dnaN and recF increases and becomes independent of dnaA expression (697). Overproduction of RecF protein adversely affects the viability and UV resistance of growing cells (221, 551).

Properties of RecF, RecO, and RecR and their influence on RecA-promoted reactions in vitro. The finding that UV sensitivity of recF mutants is partially suppressed by certain non-null recA mutations allowed Clark, as early as in 1980, to propose that RecF acts to stabilize RecA binding to ssDNA (104). The extension of this finding, on the one hand, to recO and recR mutants and, on the other hand, to other similar RecA mutants strengthened this idea and suggested that the RecFOR proteins function as a complex (708). Biochemical characterization of the mutant RecA proteins that do better in vivo in the absence of RecF, RecO, or RecR showed that they are more proficient than the WT RecA in displacing SSB from ssDNA in vitro (344). SSB is the main RecA competitor in vivo: SSB overexpression sensitizes cells to UV (65, 431), delays the SOS response, and inhibits recombination (445). Therefore, in vitro attempts at characterizing RecFOR activities were guided by the expectation that these proteins, working together, would help RecA to polymerize on ssDNA complexed by SSB.

View larger version (44K): [in this window] [in a new window]   FIG. 16.   Presynaptic phase of the daughter-strand gap repair: RecA filament assembly and replisome reactivation. The replisome is depicted as a big open oval, and its DnaN subunit (the clamp) is indicated as a smaller open rectangle with rounded corners. The 3' end of the nascent DNA is stalled at the noncoding lesion (the irregularity in the lower strand). SSB is shown as quartets of open circles around single-stranded region; RecFR (small black rectangles) are associated with the RNA primer (wavy segment in the upper strand at the very right). The RecA filament is shown as a hatched rectangle with rounded corners spreading to the left. Explanations are given in the figure and in the text.

Replisome reactivation and model for RecFOR catalysis of RecA polymerization at daughter strand gaps. In the WT E. coli cells irradiated with sublethal doses of UV, DNA synthesis is inhibited within 5 to 10 min (162, 298, 300, 738). It resumes shortly thereafter at the stalled replication forks but never again in ssb, recA, or lexA (Ind) mutants (162, 300, 712, 738) and only slowly in recF and recR mutants (128). recO mutants have not been tested for resumption of DNA synthesis after UV irradiation but are expected to be defective too. It is thought that when a replisome stops at a noncoding lesion in a template DNA, it needs to be disengaged from the damaged DNA ("reactivated") to restart downstream (300).

DNA gyrase. First, the positive supercoiling, generated when the two strands of the intact duplex are pulled apart to accommodate the invading strand, must be neutralized (Fig. 17). In vitro, negative supercoiling of the duplex facilitates the RecA-catalyzed invasion of a single strand (583). Since the E. coli chromosomal DNA is negatively supercoiled (489), the invasion of the third strand decreases the local negative supercoiling, which is then enzymatically restored to its original level.

View larger version (30K): [in this window] [in a new window]   FIG. 17.   Topoisomerase requirements during synapsis. Regular DNA superhelicity is shown in the top and bottom double helices. In the two middle panels, the helices are either overwound (too much coiling) or underwound (missing coils). DNA gyrase removes extra coils from the original (black-black) duplex, while DNA Topo I (TopA) introduces the missing coils in the hybrid (black-white) duplex.

Topoisomerase I. When two interacting strands of any nature run side by side without intertwining, they are said to be paranemic, in contrast to the situation when two interacting strands intertwine to form a double spiral (plectonemic interaction) (271). The first contacts of a daughter strand gap with the complementary strand of an intact duplex are necessarily paranemic (Fig. 17). In other words, the two DNA strands have so many negative supercoils that they can be freely separated from each other. The paranemic duplex is converted to a plectonemic duplex when the excess of negative supercoils is removed in a reaction catalyzed in E. coli by DNA topoisomerase I (Fig. 17).

One-strand repair: lesion removal and filling in of the gap. As RecA catalyzes the invasion of the single-stranded region of a gapped DNA molecule into an intact homologous duplex, a pair of branch points at which the invading strand trades places with the homologous resident strand is formed. The features of RecA polymerization in vitro (see "Filament formation by RecA around ssDNA" above) suggest that in vivo RecA filament assembles on daughter strand gaps in the direction of the lesion that caused the formation of the gap and probably spreads past the lesion into the neighboring duplex DNA. RecA will eventually drive the lesion-proximal DNA junction in the same direction, converting the corresponding three-strand junction into a four-strand (Holliday) junction (Fig. 18).

View larger version (57K): [in this window] [in a new window]   FIG. 18.   Synaptic and postsynaptic phases of daughter strand gap repair. This figure is a sequel to Fig. 16. The RecA filament is shown as a hatched rectangle with rounded corners. From top to bottom, the stages are pairing with an intact homolog, repair of the gap, and removal of the DNA junctions and the associated RecA filament. Explanations are given in the figure and in the text.

Removal of DNA junctions and associated RecA filaments. Genetic data suggest that both the RuvABC resolvasome and the RecG helicase participate in junction removal after the daughter strand gap repair, since mutating away either activity makes cells sensitive to UV, but neither mutation shows synergistic interactions with recF mutations in relation to UV sensitivity (372, 373). The postsynaptic phase of the daughter strand gap repair is understood in its gross details (see "Resolving recombination intermediates" above), but the real mechanisms have yet to be modeled in vitro, and so the description offered is inevitably speculative. When the gap is filled, the other three-strand junction could be converted into a Holliday junction, or it may stay three stranded for a while, if the repair DNA synthesis fails to traverse it. If this right three-strand junction is converted to a Holliday junction, both junctions and the RecA filament in between could be removed by the RuvABC resolvasome (see "Pairwise interactions of Ruv proteins: RuvABC resolvasome" above) (Fig. 9B). The alternative pathway for the junction and RecA filament removal does not depend on a particular configuration of the right junction. The right junction can be either converted into a Holliday junction or resolved by an unspecified cleavage activity, as is sometimes assumed (730), or even left as is. The translocation by the RecG helicase (see "RecG helicase" above) of the left (Holliday) junction towards the second junction will remove both junctions anyway (Fig. 9A or C).

Recombinational repair of daughter strand gaps is impossible when the intact sister duplex cannot be found; the likelihood of this situation increases with increasing DNA damage. For example, both daughter chromatids could be damaged in the same sequence, or the sister chromatid could be degraded due to a downstream lesion. Many bacteria have a backup mechanism to repair lingering daughter strand gaps without recombination, by translesion DNA synthesis. When this backup mechanism is turned on, RecA filament is dispersed from the gap and the replisome is modified so as to be able to bypass the lesion. Although nonrecombinational by its nature, this process is both regulated by RecA and requires the direct participation of RecA (reviewed in references 602 and 743).

RecA regulates translesion DNA synthesis at two levels. The first level of regulation is via LexA cleavage and SOS induction. Prolonged SOS induction increases the expression of the umuDC operon (23, 24) (see "Levels of SOS induction" above). Besides RecA itself, UmuC and UmuD are the only SOS proteins required for the translesion DNA synthesis (612); both proteins participate in this process directly. However, first UmuD has to bind to RecA filament and to cleave itself in a reaction similar to the autocleavage of LexA and prophage repressors (77). RecA-promoted autocleavage of UmuD is the second level of RecA involvement in translesion DNA synthesis. In vitro, this inefficient reaction, as well as autocleavage of prophage repressors, requires higher concentrations of Mg²⁺ than does RecA-promoted autocleavage of LexA; processing of UmuD in vivo is also rather inefficient (565). This is likely to ensure that UmuD and phage repressors are cleaved late during SOS induction (see also "Levels of SOS induction" and "Cleavage of LexA repressor by RecA filament" above). The product of autocleavage, UmuD', combines with UmuC to form a complex active in catalyzing translesion DNA synthesis (509, 647).

With the development of the in vitro system for translesion DNA synthesis, the molecular mechanisms by which the active UmuD'₂C complex catalyzes lesion bypass are beginning to be elucidated. Two observations, that translesion DNA synthesis is inhibited by overproduction of the DnaN "clamp" subunit of DNA pol III (640) and that overexpression of UmuC is poisonous for strains with defective DNA pol III (469), suggested that UmuD'₂C comes in direct contact with the DnaN clamp. UmuD'₂C turned out to be a nonprocessive error-prone polymerase (DNA pol V), capable of bypassing noncoding lesions (648). UmuD'₂C is hypothesized to replace the stalled DNA pol III at the lesion and to synthesize several nucleotides across the damaged template, to be promptly replaced by DNA pol III on the other side of the lesion (Fig. 19).

How does UmuD'₂C find the unfillable single-strand gap? UmuD'₂C complex binds ssDNA in the absence and the presence of RecA (75), but its way of getting to the unrepaired lesion appears to be via the RecA filament because, besides its two regulatory roles in translesion DNA synthesis, RecA is required directly, together with UmuD', UmuC proteins, and DNA pol III (509, 647). The clue to the mechanism of direct RecA participation in translesion DNA synthesis is the observation that expression of the Umu proteins inhibits recombinational repair by interfering with RecA action (62, 610). Therefore, RecA is proposed to direct UmuD'₂C towards the site of the unrepaired lesion, while UmuD'₂C destabilizes the RecA filament (Fig. 19) (62, 610).

View larger version (39K): [in this window] [in a new window]   FIG. 19.   Model for the translesion DNA synthesis. This figure is a sequel to Fig. 16 and shows an alternative to the mechanism illustrated in Fig. 18. The RecA filament is shown as a hatched rectangle; the DnaN clamp is shown as a small open rectangle; UmuC is shown as a small black rectangle associated with the DnaN clamp; UmuD is shown as small black dimer circles, some of them associated with UmuC; the replisome is shown as an open oval. Explanations are given in the figure and in the text.

The observation that RecA filaments stabilize UmuD'₂C complex against proteolysis in vivo supports the idea of a direct interaction of UmuD'C and RecA (192). Generally, both UmuD and UmuC are rapidly degraded in vivo by the Lon protease, while the majority of the newly formed UmuD' dimerizes with unprocessed UmuD and is degraded by the ClpXP protease (191). The strategy of UmuD proteolysis apparently prolongs the delay in formation of the active and stable UmuD' dimer, and for a good reason. Since DNA pol V has to insert random bases opposite some noncoding lesions, the translesion DNA synthesis often generates point mutations and is also known as the SOS mutagenesis. Translesion DNA synthesis is definitely not the best way to repair a lesion, but since umu mutations do confer moderate UV sensitivity (296), this error-prone DNA synthesis may be the life-saving option under conditions of massive DNA damage. Besides, translesion DNA synthesis is the only way to repair a lesion in a situation like conjugal transfer, when a single strand of a plasmid DNA is transferred from one bacterial cell to another. During the postconjugational synthesis of the complementary strand, there is no sister duplex to rely on if a noncoding lesion is encountered in the template strand. In fact, many conjugative broad-host-range plasmids carry their own genes for translesion DNA synthesis (633). This is helpful because even among the enterobacteria not all species are capable of induced mutagenesis, even though almost all of them carry genes homologous to the umuDC operon of E. coli (565).

Daughter strand gaps are formed when DNA replication encounters a noncoding lesion in a template DNA and reinitiates downstream, leaving behind a single-stranded region adjacent to the lesion. In E. coli, daughter strand gaps are closed by the RecF pathway of recombinational repair, via homologous pairing and strand exchange of the gapped chromatid with the intact sister chromatid. Other eubacteria seem to follow the same recombinational route: daughter strand gaps are repaired by recombination in Haemophilus influenzae (350, 600, 706) and in B. subtilis (159, 160). Moreover, judging by the universal presence of the recF homologs in other eubacteria (196, 405), the RecF pathway is ubiquitous. Genes for the translesion DNA synthesis (a backup repair of daughter strand gaps) are also widespread among eubacteria (633, 743).

The interactions of individual components of the daughter strand gap repair machinery are beginning to be elucidated in vitro. The prominent issue is the interaction of the RecF, RecO, and RecR proteins with the replisome, on the one hand, and RecA on the other. The replisome reactivation is another phenomenon that begs investigation, both in vivo and in vitro. The postsynaptic phase of the daughter strand gap repair is still a realm of pure speculations. The replacement of the recombinational repair machinery with the one for the translesion DNA synthesis recently became the area of exciting research.

DOUBLE-STRAND END REPAIR

Top Previous Next References

Origin and Repair of Double-Strand Ends

In contrast to cells which cannot excise pyrimidine dimers (247, 538, 605), excision repair-proficient cells stop DNA synthesis after UV irradiation (162, 605) and, if the UV dose was high, initiate a new round of DNA replication from the origin (46, 247, 397). Surprisingly, the old replication forks seem to be temporarily abandoned, since the DNA synthesized just before the irradiation becomes susceptible to degradation (128). This abandonment becomes permanent at higher UV doses (8, 162), and the total chromosomal DNA in excision repair-proficient cells becomes susceptible to a limited degradation (63, 105); in the absence of RecA, the chromosomal degradation after UV irradiation is uncontrollable (105, 128).

This bizarre behavior of the excision repair-proficient cells is rationalized by the data from neutral sucrose gradients, which allow the intact chromosomes to be separated from chromosomal fragments. Immediately after UV irradiation, the sedimentation pattern of the E. coli chromosome in neutral sucrose gradients is the same as that of an unirradiated control. However, if the irradiated cells are incubated in the growth medium, the neutral sucrose gradients indicate that the bacterial chromosome becomes fragmented. This fragmentation is suppressed in excision repair-deficient mutants (60, 667) or by inhibition of DNA replication (649), suggesting that DNA replication on template DNA that is undergoing excision repair causes chromosomal breakage.

The nature of this breakage is revealed in strains defective in the closure of ssDNA interruptions, like DNA ligase mutants (471, 483) or DNA pol I mutants (383, 635). If these mutants are allowed to replicate their DNA, their chromosome becomes similarly fragmented, as judged by its susceptibility to degradation by a nuclease specific for the double-strand ends (443, 450). Therefore, replication of a DNA template with single-stranded interruptions generates dsDNA interruptions.

Hanawalt may have been the first to propose how excision gaps interfere with DNA replication (234); later, similar schemes were advanced on several occasions (76, 142, 218, 333, 540, 567, 597). They all envision the collapse of a replication fork as it reaches the single-strand interruption in the template DNA, as a result of which the double-strand end is separated from the full-length duplex (Fig. 20A). If both forks of a replication bubble have collapsed due to nicks in the same DNA strand, in the neutral sucrose gradients this will look like chromosomal DNA fragmentation (Fig. 20B).

View larger version (18K): [in this window] [in a new window]   FIG. 20.   Replication-dependent origin of double-strand ends. (A) Replication fork collapse at a single-strand interruption in template DNA. The replisome is shown as the square group of circles, speeding from left to right along the DNA while replicating it. (B) Preexisting interruptions in the same DNA strand at both replication forks of a replication bubble cause chromosome fragmentation.

Exposure to ionizing radiation brings about chromosome fragmentation without DNA replication, indicating direct double-strand breaks (48, 219) (see "The two mechanisms of two-strand damage" above). In E. coli, double-strand breaks are sealed by recombinational repair in the replicated portion of the chromosome (323, 325, 683). In WT E. coli, the repair of double-strand breaks and reassembly of disintegrated replication forks is the function of the RecBC recombinational repair pathway (first reviewed in reference 102).

View larger version (12K): [in this window] [in a new window]   FIG. 21.   Replication fork reversal as the mechanism of replication fork breakage. A replication fork is shown moving from left to right; solid lines indicate parental strands; open lines indicate newly synthesized strands. (A) The progress of the replication fork is blocked (replisome mulfunctioning, a protein bound to DNA). (B) This results in replication fork reversal to generate a double-strand end, composed of the newly synthesized DNA strands, and a Holliday junction. (C) ExoV-catalyzed degradation of the double-strand end eliminates the Holliday junction. (D) Alternatively, resolution of the Holliday junction by RuvABC (small arrows in panel B) breaks the replication fork.

Evidence for replication fork repair by recombination. Skalka was probably the first to propose that disintegrated replication forks can be reassembled by recombinational repair (597). E. coli has two recombinational repair pathways, RecF and RecBC (see "The two recombinational repair pathways of E. coli" above), corresponding to two major classes of replication-induced two-strand DNA lesions (see "The two mechanisms of two-strand damage" above). As discussed in the section on daughter strand gap repair (above), the RecF pathway mends daughter strand gaps. The notion that disintegrated replication forks are reassembled in E. coli by the RecBC pathway explains two major phenomena. The first is that strains in which replication fork disintegration is expected to be frequent are dependent on recA and recBC but independent of recFOR. This is true for the "classical" replication fork disintegration mutants, such as polA (83, 225, 443), lig (119, 217), and dam (26, 408, 486, 487), as well as for the "classical" replication fork disintegration conditions, such as exposure to nalidixic acid (423). Throughout this section, inviability of a double polA geneX mutant will signify possible dependence of double-strand end repair on geneX (of course, the other phenotype of geneX mutants, which would also result in the inviability of polA geneX double mutants, could be increased DNA damage).

DNA replication primed by double-strand end-promoted recombination. The idea that disintegrated replication forks are repaired by recombination requires double-strand end invasion intermediates to be resolved by DNA replication. This prediction is at variance with the current models of homologous recombination at double-strand ends, which emphasize exchange without DNA replication (108, 320, 655). The current models are based on the old observations that inactivation of the replisomes in vivo still allows the completion of some double-strand end-promoted recombinational events by the RecBC pathway of E. coli, suggesting that the intermediates can be resolved without DNA replication (615, 617).

Overview of double-strand end repair. Double-strand end repair can be used to mend double-strand breaks as well. The current model of recombinational repair of double-strand breaks postulates repair DNA synthesis (637); in fact, the scheme calls for the installation of two converging replication forks (309, 603). Therefore, double-strand break repair can be viewed as a combination of two initially independent double-strand end repair events and is not discussed separately.

In WT E. coli, rejoining of the chromosomal DNA fragmented by gamma irradiation is completely blocked by only three mutations: recA, recB, or recC (555). ssb mutants have not been tested yet, although they are also likely to be deficient in recA-dependent rejoining of fragmented chromosomes, since they are defective in recombination requiring double-strand end repair (174, 208, 504, 700). RecB and RecC combine with RecD to work in a single enzyme, whose complex behavior makes it a fascinating subject to study, surpassed only by RecA protein (reviewed in references 320 and 650). The RecBCD enzyme is a combination of a potent helicase with duplex DNA- and ssDNA-specific exonucleases. In vitro, the RecBCD enzyme degrades linear duplex DNA to oligonucleotides at an incredible speed. In vivo, it degrades bacteriophage DNA cut by the host restriction systems, and in recA mutants, it degrades the entire chromosome after it was fragmented as a result of DNA damage. "Professional" DNA degradation by the RecBCD nuclease is difficult to reconcile with the central role of this enzyme in double-strand end repair. E. coli employs a two-component system to turn this "Mr. Hyde" into "Dr. Jekyll" when double-strand breaks and disintegrated replication forks need to be repaired.

RecBCD: Genes and mutants. The three contributing genes of the RecBCD enzyme are closely grouped on the E. coli chromosome: recB and recD form an operon, while recC, although situated nearby, has its own promoter (11, 183-185, 557). In related microbes, the structure of the region is similar (519). A combination of extremely weak promoters and suboptimal codons maintains the level of 10 RecBCD nuclease molecules per chromosome (650). None of its three genes is known to be SOS inducible.

RecBCD: Biochemical activities. RecBCD enzyme is a heterotrimer (652) of RecB (134 kDa), RecC (129 kDa), and RecD (67 kDa). The prominent activities of the RecBCD enzyme include DNA helicase, dsDNA exonuclease, and ssDNA exonuclease. DNA hydrolysis does not need an input of energy, and most nucleases do not hydrolyze ATP. The trademark of the RecBCD nuclease is that it requires ATP hydrolysis for the degradation of duplex DNA. In fact, the RecBCD nuclease of E. coli, also known as ExoV (746), together with analogous enzymes of other eubacteria (and the SbcCD nuclease [121]), are the only known ATP-dependent exonucleases (102, 650, 658).

RecBCD: Mechanism of DNA hydrolysis before Chi. The amino acid sequences of RecB and RecD suggest that both proteins possess ATP-binding domains (183, 184); indeed, both proteins bind ATP (286). RecB is a DNA-dependent ATPase (412) and a weak helicase, which translocates 3' to 5' along ssDNA (56, 491). RecD is also a DNA-dependent ATPase (96); mutational inactivation of its ATP-binding domain results in an inactive reconstituted RecBCD enzyme (315, 316, 412). Experiments with hybrid RecBCD enzymes in which the ATP-binding sites were inactivated on either RecB or RecD or on both subunits established that both ATP-binding sites control the hydrolysis of ssDNA, with the RecB site controlling hydrolysis of the 3'-ending strand and the RecD site controlling hydrolysis of the 5'-ending strand (95).

View larger version (13K): [in this window] [in a new window]   FIG. 22.   Mode of RecBCD enzyme binding to a DNA end and patterns of RecBCD-promoted DNA hydrolysis. The direction of travel of the enzyme through the DNA duplex is from right to left. (A) Scheme of RecBCD binding to a double-strand end. The C-terminal domain of RecB, containing the nuclease active site, is shown as a separate subdomain. The final 5 to 6 bp of the duplex end are unwound. (B) Mode of the RecBCD action before it has seen a Chi. The top strand ends 3' at the right. (C) Mode of RecBCD action after the enzyme has seen a Chi. The enzyme itself is not shown.

RecBCD: Mechanism of DNA hydrolysis after Chi. The previous section described the way RecBCD treats DNA before it has seen a Chi (also written as ). Chi sites were discovered in bacteriophage  as mutations creating hot spots for E. coli recombination (reviewed in reference 461). red gam mutant  (see "SSA enzymes of phage " below) cannot inactivate ExoV (gam) and lacks its own recombination system (red), and so its DNA is savaged by RecBCD after being linearized for packaging by a phage-encoded terminase (175). red gam  grows poorly in WT E. coli cells but sports big-plaque variants, which all turn out to acquire point mutations creating Chi sites at one of the several locations along the  chromosome. Linearization of Chi-containing  DNA, instead of triggering its degradation, results in its homologous recombination with other  DNAs catalyzed, surprisingly, by the same RecBCD enzyme. The paradox of a single enzyme (RecBCD) carrying out two mutually exclusive activities on linear DNA (complete destruction versus preservation through recombination) led to the proposal that the encounter of the RecBCD enzyme with a Chi-site disables the dsDNA exonuclease activity, converting the enzyme into a "recombinase" by virtue of its helicase activity (622). This idea was confirmed experimentally, both in vivo (139, 338, 763) and in vitro (157, 653).

RecBCD: RecA filament assembly. Inhibition of the nuclease activities of RecBCD by Chi in vitro does not require any other protein (155, 157, 653). In contrast, disabling of the nuclease activity of RecBCD in vivo requires SSB and RecA proteins (139, 338), suggesting that RecBCD and RecA interact. Another indication of functional interactions between RecA and RecBCD is the fact that the optimal recombinational repair in vivo is achieved only when both enzymes are from the same or closely related species (150).

DNA-keeping enzymes. Genetic data suggest that completion of double-strand end repair (that is, recBC-dependent recombination) requires at least three DNA-keeping activities: DNA gyrase, DNA pol I, and DNA ligase (174, 766). DNA gyrase is important for maintenance of negative supercoiling in DNA (see "DNA gyrase" above); supercoiled DNA is a better substrate for RecA-promoted strand invasion (583). DNA pol I and DNA ligase, working together, fill in and seal single-strand interruptions which are generated in any DNA repair reaction. In addition, DNA pol I may play a more specific role, as suggested by its involvement in initiation of plasmid DNA replication (see "Initiation of plasmid DNA replication" above).

View larger version (13K): [in this window] [in a new window]   FIG. 23.   Overview of double-strand end repair. RecA filament is shown as an open rectangle with rounded corners. Small one-sided arrow indicates the direction of RecA filament assembly. The star indicates limited DNA synthesis by DNA pol I; little wavy segments indicate RNA primers. (A) RecA polymerizes on a 3' single-stranded overhang, generated by the RecBCD action after Chi. (B) RecA-catalyzed invasion of the 3' end allows limited DNA synthesis to be primed by DNA pol I. (C) As a result of this synthesis, a primosome assembly structure is created in the displaced strand. (D) PriA assembles a primosome at the displaced strand, which then lays down primers and unwinds DNA at the fork. (E) A replisome is loaded onto the primed replication fork; the three-strand junction is being converted into a Holliday junction. (F) Double-strand end repair is completed by resolution of the Holliday junction.

Replication fork restart. Following the limited displacement synthesis by DNA pol I, replication fork restart is believed to begin by binding of PriA to the displaced strand (Fig. 23C) (20, 309). polA priA double mutants are inviable (352); priA mutants are sensitive to DNA-damaging agents and deficient in homologous recombination requiring double-strand end repair (310, 552). In vitro, PriA is required to attract the replisome to replication fork frameworks (283). Certain point mutations in dnaC, which encodes the inhibitor of the DnaB replicative helicase, suppress recombinational repair defects of priA mutants, suggesting that the defects are caused by the inability of priA mutants to load the primosome (310, 552). The two principal components of the primosome are DnaB helicase, which drives replication fork unwinding, and DnaG primase, which lays the primers for the DNA synthesis (see "Initiation of chromosomal DNA replication in E. coli" above) (Fig. 23D and E). The availability of primers signals that a replisome can be loaded onto the replication fork framework, finishing the regeneration of a replication fork. Now, to complete the double-strand end repair, the DNA junction and the associated RecA filament must be removed.

The two pathways for DNA junction removal in double-strand end repair. polA recA or dam recB double mutants are inviable because the constantly required double-strand end repair is blocked at the presynaptic or synaptic phases. The postsynaptic phase turns out to be equally important, because polA ruv and dam ruv double mutants (274, 487) or polA recG double mutants (257, 275) are inviable, too. From a different perspective, although rejoining of the direct double-strand breaks resulting from treatment of WT E. coli with ionizing radiation requires the functions of only recA, recB and recC (555), the ultimate survival of E. coli cells after treatment with ionizing radiation is also compromised by mutations in the ruvA, ruvB, ruvC, and recG genes (372, 373, 473).

There is an old observation suggesting that DNA degradation by ExoV plays an important role in the viability of E. coli cells. Both recA and recBC mutants of E. coli are deficient in double-strand end repair; however, the viability of recA mutants is around 60%, while the viability of recBC mutants, which inactivate both the double-strand end repair and DNA degradation, is only 30% (85). Moreover, the viability of recA recB double mutants is reduced to 20%, confirming the rule that recA mutations decrease the viability by one-third while recBC mutations decrease the viability by two-thirds. What could be the role of ExoV in the chromosomal DNA metabolism in E. coli?

ExoV and stability of replication forks. In some replicative DNA helicase mutants, such as rep or dnaB mutants, replication forks are believed to be inhibited (434). Inhibited replication forks were suspected to be unstable (44, 263, 334) (see "Evidence for replication fork disintegration" above), which was later confirmed and shown to result in chromosomal DNA fragmentation (434). Remarkably, inhibited replication forks turned out to be less stable in recBC mutants than in the recBC⁺ cells (566). Moreover, the breakage of the inhibited replication forks depends on the RuvABC resolvasome, suggesting that the mechanism of the breakage is through the replication fork reversal with subsequent "resolution" of the resulting Holliday junction (Fig. 21B and D). ExoV is assumed to prevent the breakage of such a reversed replication fork by degrading the generated double-strand end and thus eliminating the Holliday junction (566) (Fig. 21C). Both the replication fork reversal with subsequent RecBCD entry (384) and the breakage of the reversed replication forks by RuvABC (334) have been considered previously.

Excessive DNA degradation affects survival after ionizing radiation more than after UV. Generally, recBC mutants are equally sensitive to both ionizing radiation and UV irradiation, which is thought to reflect their inability to repair both double-strand breaks and disintegrated replication forks. However, one allele of recB called rorA is more sensitive to ionizing radiation than to UV irradiation (209).

View larger version (14K): [in this window] [in a new window]   FIG. 24.   Lethality caused by double-strand breaks in the replicated portion of the genome in rorA mutants. The chromosome is depicted as a theta-replicating structure, with the single line representing duplex DNA. The origin of DNA replication is denoted by small open circles at the top of the chromosome; the terminus is shown as a solid circle at the bottom. If both daughter branches of a replicating chromosome are broken at positions close to each other, the double-strand breaks have more chances of being repaired in the WT cells than in rorA mutant cells, due to the increased DNA degradation in the latter.

DNA degradation as a possible backup strategy. Of the Chi sites in the E. coli chromosome, 75% are oriented so as to stop the RecBCD degradation coming towards the replication origin (54). To a certain degree, this preferential orientation reflects the facts that (i) the Chi sequence contains the primase-binding site, and primase binding sites are more frequent in the template for the lagging-strand DNA synthesis (54); and (ii) the trinucleotide composition is different for the transcribed and untranscribed strands, while the majority of the genes in the E. coli chromosome are cooriented with the DNA replication (116). However, these two factors cannot account for all the skew, especially around the replication origin, where it is 10:1. This preferential orientation of Chi sites suggests that the RecBCD repair pathway primarily deals with disintegrated replication forks (333), probably because this type of lesions is more frequent in the E. coli chromosome than are direct double-strand breaks.

View larger version (15K): [in this window] [in a new window]   FIG. 25.   Outline of the maintenance of circular chromosome. The chromosome is shown as a rectangle with rounded corners (the single line represents duplex DNA). The origin of DNA replication is denoted by small open circles at the top of the chromosome. (A and B) A theta-replicating chromosome (A) may suffer a collapse of one of the replication forks, becoming a sigma-replicating chromosome (B). (C) In a wild-type strain, the double-strand end is then degraded by RecBCD and reattached to the circular domain with the help of RecA. (D) In the absence of RecA, the linear replicating branch is completely degraded by RecBCD, while a new round of theta replication is initiated from the origin. (E and F) In the absence of RecBCD, the chromosome is trapped in the rolling-circle replication; initiation of a new bubble from the origin can only lengthen the linear tail.

RecE pathway. sbcA mutations activate the expression of a part of the cryptic Rac prophage (reviewed in reference 81), as a result of which at least two phage proteins relevant for recombinational repair are produced. The RecE is a duplex DNA-specific exonuclease which selectively degrades the 5'-ending strand, producing a DNA duplex with a 3' overhang (285). RecT promotes annealing of complementary single DNA strands and can catalyze three-strand branch migration (233). Possible roles of these two activities in bacteriophage recombination are discussed below (see "SSA enzymes of the Rac prophage"). A likely role of the RecE exonuclease in the E. coli repair is to resect double-strand ends so that they terminate with long 3' overhangs. These 3' single-stranded tails would be complexed by SSB and then degraded by ExoI, an ssDNA-specific nuclease with the 3'-to-5' polarity of degradation, whose activity is stimulated by SSB binding to ssDNA (441). In the RecBC pathway, the 3'-ending strand is held as a loop by RecBCD (Fig. 22), which apparently protects the 3' end from other nucleases. In the RecE pathway, the role of RecT could be to compete with SSB for binding to the RecE-generated 3'- single-strand overhangs, thus protecting them from degradation by ExoI.

View larger version (21K): [in this window] [in a new window]   FIG. 26.   Scheme for the presynaptic phase of double-strand end repair along the RecE pathway. Short wavy segments with the associated little caps indicate RNA primers with RecFR proteins; an open rectangle with rounded corners indicates the RecA filament. Explanations are given in the figure and in the text.

RecF pathway. The RecF pathway of recombinational repair of double-strand ends is turned on in recBC mutants by inactivation of two additional nucleases. One nuclease to be inactivated is ExoI (see the previous section), which degrades ssDNA starting from 3' ends (353) and is thought to participate in methyl-directed mismatch repair (439). The gene coding for ExoI is called sbcB/xonA (332). Another nuclease to be inactivated is SbcCD (204, 374) which is an ATP-dependent dsDNA exonuclease (121) distantly related to RecBCD (463). It is not known how the ExoI and SbcCD inactivation enables the RecF pathway to promote double-strand end repair. One idea is that in the absence of ExoV the nuclease activity for generating 3' overhangs is so weak that other nucleases that might degrade 3' overhangs will interfere (103). A complementary idea is that the RecF pathway has no protein like RecT in the RecE pathway or RecBCD in the RecBC pathway to protect the 3' single-strand overhangs from degradation.

Unified mechanism of double-strand end repair. The above mechanisms for the double-strand end repair along the RecBC pathway and the alternative RecE or RecF pathways have a lot in common. In fact, their distinction is only in the presynaptic reactions, which, although mechanistically similar, are catalyzed by different enzymes. Therefore, one can develop a unified mechanism for double-strand end repair (Fig. 27) (108).

View larger version (30K): [in this window] [in a new window]   FIG. 27.   Unified scheme for double-strand end repair. RecA filament is shown as an open rectangle with rounded corners; the positions of single-strand scissions are indicated by small arrows. Explanations are given in the figure and in the text.

Disintegration of replication forks occurs in response to replication fork inhibition or as a result of the presence of single-strand interruptions in the template DNA. In E. coli, disintegrated replication forks are reassembled by the RecBC pathway of recombinational repair, via the homology-directed invasion of the double-strand end into the intact sister duplex. Double-strand break repair in E. coli is possible in the replicated portion of the chromosome and is probably the sum of two independent double-strand end repair events. Repair of disintegrated replication forks and double-strand breaks in other bacteria is likely to follow the E. coli scheme. ATP-dependent nucleases (analogous to the RecBCD enzyme) are detected in many eubacteria (425, 658); Chi sites are characterized in three other microbes (49, 94, 613).

Among the prominent issues in the RecBC pathway is the formation of the two-strand DNA lesion itself: the possibility of replication fork disintegration has yet to be demonstrated in vivo. The presynaptic and synaptic phases of the RecBC-dependent double-strand end repair have been reconstituted in vitro. The replication fork restart is the area of active current research; its progress should allow us to bridge the RecABCD in vitro reactions with those of RuvABC and RecG. In contrast, the RecF and RecE pathways of the double-strand end repair have yet to be reconstituted in vitro. The role of ExoV in the eubacterial chromosome metabolism begs in vivo experimentation.

The inviability of the double polA dif mutants (328) could have suggested yet another gene of double-strand end repair, but dif turned out to be a short sequence related to the demultimerization sites of multicopy plasmids (51, 112, 328)! dif mutants experience difficulties with chromosome partitioning, but the difficulties disappear if recombinational repair is also inactivated (328). Recombinational repair is expected to result in crossing over in 50% of the cases (see "Homologous recombination versus recombinational repair" above); a single crossover between circular chromosomes will combine them into a dimeric chromosome (Fig. 28). The unique susceptibility of circular chromosomes to odd numbers of exchanges was first recognized by McClintock in her studies with maize ring chromosomes (422). Long considered to be an oddity, this problem turned out to be the real one for prokaryotes, with their almost invariably circular chromosomes.

View larger version (14K): [in this window] [in a new window]   FIG. 28.   Removal of DNA junctions after recombinational repair may result in a crossover which translates into chromosome dimerization. (A) A Holliday junction behind a replication fork. Positions of single-strand scissions due to the preferred mode of RuvC resolution are indicated by small arrows. (B) A crossover is formed behind a restored replication fork due to the RuvC resolution. (C and D) A single crossover in a replicated portion of a circular chromosome (C) translates into a dimer chromosome when replication is completed (D).

The two replication forks in the E. coli chromosome start at the unique origin and converge on the replication terminus situated across from the origin (see "Cellular processes that surround and complicate recombinational repair" above). In the middle of the terminus, there is a 30-bp dif site responsible for the chromosome monomerization (51, 112, 328). A pair of resolvase-like enzymes encoded by unlinked genes xerC and xerD work on this site to demultimerize the chromosomes (51, 52) (Fig. 29).

View larger version (15K): [in this window] [in a new window]   FIG. 29.   XerCD-dif site-specific recombination ensures chromosome monomerization in E. coli. The chromosome is shown as a rectangle with rounded corners, with the thick single line representing duplex DNA; replication origins are shown as small open circles at the top of the chromosome, and the replication terminus is represented by a small solid circle at the bottom of the chromosome. (A) A chromosome replicating in theta mode. (B) One of the replication forks has disintegrated. (C) The replication fork is reassembled by recombinational repair, resulting in a Holliday junction. (D) The Holliday junction is resolved, generating a crossover. (E and F) Completion of the chromosomal replication results in a dimer chromosome. (G and H) Segregation of the daughter nucleoids translocates the crossover to the terminus region, where it is resolved, permitting separation of the daughter chromosomes from each other.

Mutating away xerC or deleting the dif site causes cell filamentation due to problems with nucleoid partitioning. Mutating away recA or recB alleviates these problems of xerC or dif mutants (51, 328), suggesting that it is mostly the RecBC pathway of recombinational repair that generates sister chromatid exchanges. However, direct physical detection of the in vivo site-specific recombination at dif shows that it is decreased equally by either recB or recF mutations and is eliminated only in a recB recF double mutant (630).

It is calculated that some 20% of E. coli cells in an exponentially growing culture experience an inducing event (crossing over between sister chromosomes) which subsequently leads to partitioning problems in dif mutants (328, 629), putting the number of repair events at twice that value, around 40%. In good agreement with this assessment, available data (599) allow us to calculate that about 45% of recA cells (which probably degrade linear tails, since they cannot repair collapsed replication forks [see "DNA degradation as a possible backup strategy" above]) have at least one nucleoid missing.

The XerCD-mediated reaction can be studied in vivo with plasmid substrates bearing the 30-bp dif sequence (51, 52, 112, 328). In this setting, the reaction shows no resolution selectivity: two dif sites are recombined independently of whether they are on a single DNA molecule or on two separate DNA molecules. If this lack of selectivity in the plasmid system reflects a similar situation in the chromosome, it is unclear how the XerCD-dif system is able to faithfully resolve chromosomal dimers.

It was postulated that the XerCD-dif system is able to monomerize chromosomes as a result of frequent exchanges between the two dif sites on the sister chromosomes and the active separation of the sister chromosomes by a partitioning apparatus of the cell (51, 328). However, it was found that the site-specific recombination at dif does not occur at all if the cell division is blocked, suggesting that the chromosome monomerization is a highly regulated process (629). Indeed, no recombination between dif sites is catalyzed by XerCD in vitro (118), demonstrating that the chromosomal monomerization system is more complex than initially appeared.

Another idea of how the XerCD-dif system might function envisions a supramolecular structure at the chromosome terminus that renders the site-specific reaction unidirectional. Studies with reintroduction of dif at various locations in the chromosome of dif-deleted strains show that dif is active only in an interval 10 kb to each side of the natural dif location (125, 327). Contrasting with this positional specificity, both the psi demultimerization site of pSC101 plasmid and the loxP/Cre site-specific recombinational system of bacteriophage P1 can complement a dif deletion if inserted at the natural position of the dif site (126, 355). Although the location of dif coincides with the zone of meeting of the two replication forks, completion of DNA replication per se does not trigger the monomerization, since dif, in a strain with an inversion of almost half the chromosome, is situated at least 1 Mb away from the zone of replication fork meeting and still functions in the chromosome monomerization (125)!

The behavior of dif invites parallels with the terminal recombination zone, the several-hundred-kilobase chromosomal segment covering the terminus, characterized by the high orientation-dependent recBC-catalyzed homologous recombination, whose center coincides with the position of dif (127). If a supramolecular structure in the terminus region indeed exists, its dimensions must be on the order of several hundred kilobases, since deletions of up to 233 kb around dif are without consequences as long as dif is reinserted at the site of the deletion (125, 327).

Recombinational repair is frequently accompanied by crossing over, which, in dimeric eubacterial chromosomes, leads to the formation of chromosome dimershence the need for a chromosome monomerization system. XerCD is a site-specific recombinase that, acting at the dif site in the middle of the terminus region of the E. coli chromosome, monomerizes the latter in preparation for cell division. Homologs of the XerCD recombinases have been characterized from several eubacteria (reference 698 and references therein).

The in vitro biochemistry of the XerCD-dif system is challenging, probably because of the requirements for yet-to-be-characterized factors. The regional regulation of site-specific as well as homologous recombination within the terminal recombination zone is the subject of exciting in vivo research.

The major aspects of the two pathways of recombinational repair in E. coli are summarized for comparison in Table 2. The table underscores the fact that apart from the enzymes of DNA synthesis required for the replication restart, all "recombination" enzymes act in one way or the other to control RecA polymerization or depolymerization. However, regulating RecA activity during repair is only one aspect of the in vivo control over recombinational repair. The other aspect, to be discussed below, is suppression of RecA activity under conditions when no repair is needed (regular DNA replication) or stimulation of RecA activity when massive repair effort is required (SOS induction).

Some other proteins could discourage RecA polymerization during regular DNA replication. For example, LexA, the SOS regulon repressor (see "Organization of the SOS regulon" above), suppresses RecA polymerization in vitro under suboptimal conditions (237, 515). A recently characterized DinI protein is another candidate for such a negative regulation (753). Of course, the main RecA inhibitor is SSB (see "Assistance for RecA by SSB at all stages" above), the protein that controls access of other proteins to ssDNA (reviewed in reference 430). In vitro, in the presence of 1 mM Mg²⁺ and 1 mM ATP, RecA cannot displace SSB from ssDNA; however, if the concentration of Mg²⁺ is increased to 10 mM, RecA quickly displaces SSB from ssDNA (reviewed in reference 335). There are intermediate Mg²⁺ concentrations at which RecA will displace SSB slowly, due to a kinetic barrier for a nucleation event; the in vivo conditions are assumed to create such barrier. The reason why normal DNA replication does not elicit RecA polymerization could therefore be a kinetic one: although ssDNA is present at replication forks continuously, any particular DNA sequence stays single stranded only transiently, giving RecA insufficient time to overcome the nucleation barrier (558).

The information on the optimal Mg²⁺ and ATP concentrations for the various in vitro DNA replication and recombination reactions could reflect the active concentrations of these ions in vivo; such data, admittedly more illustrative than representative, are collected in Table 3. They give the impression that the optimal conditions for DNA replication, DNA degradation by ExoV, and some recombination-related reactions are Mg²⁺ in 10:1 excess over ATP and no dATP. In contrast, optimal conditions for multienzyme recombination reactions tend to require a higher ATP/Mg²⁺ ratio. RecG is active only when the ATP/Mg²⁺ ratio is reversed, while RecBCD under the "reversed" conditions retains only its helicase activity (see "RecBCD: biochemical activities" and "RecBCD: mechanism of DNA hydrolysis after Chi" above). In addition, at least two recombinational enzymes, RecA and RuvAB, prefer dATP over ATP.

Since the concentration of Mg²⁺ in almost all of these in vitro reactions can be lowered to 5 mM without affecting the outcome, the significant variable in these data is the concentration of ATP, which changes from as little as 0.03 mM for the maximal ExoV activity to around 10 mM for certain RecA- and RecG-catalyzed reactions. dATP adds further variation to the nucleotide theme of the equilibrium. The concentration of ATP in vivo is around 3 mM (55, 387, 413); that of dATP is only 0.2 to 0.3 mM (55, 413), but since DNA precursors are thought to be concentrated at the replication sites, the dATP concentration around the replication forks could be higher (415). In equimolar solutions with ATP, Mg²⁺ is tightly complexed by the anion (202, 228); therefore, since the in vivo free Mg²⁺ concentration in E. coli is 1 to 4 mM (3, 227, 392), it is present in a small excess over triphosphates. This gives the combined MgATP² + Mg²⁺ pool around 5 mM and the in vivo ATP/Mg²⁺ ratio of 0.5 to 0.8, the one that is preferred by the multienzyme recombination reactions in vitro. Therefore, "regular" intracellular conditions could be permissive for RecA polymerization if long-lived ssDNA is present. Indeed, introduction into E. coli cells of ssDNA which is unable to replicate induces the SOS response (205, 248, 249).

The ATP/Mg²⁺ ratio during favorable growth conditions is optimal for the balance between DNA replication and occasional recombinational repair; however, this ratio may change when rapidly growing cells experience massive DNA damage. Massive DNA damage triggers a rapid severalfold increase in the intracellular dATP concentration (144, 465, 634) which is likely to stimulate RecA-promoted reactions. Maybe even more importantly, the intracellular ATP concentration follows suit and rises two- to threefold, preceding SOS induction (28, 29, 140, 226, 465). The resulting increase in ATP concentration to 6 to 9 mM could temporarily increase the ATP/free-Mg²⁺ ratio, although it is unlikely that all the free Mg²⁺ will be exhausted, since the total intracellular pool of this cation stands at about 100 mM (442). The cause of this rise in the concentration of DNA and RNA precursors is proposed to be direct inhibition of DNA synthesis by the DNA damage and replisome idling at the lesions (167, 245, 335, 492). The demonstration that when DNA replication is blocked, the rate of a nucleotide pool expansion equals the rate of incorporation of this nucleotide immediately before the block, while when DNA replication is allowed, the rate of the nucleotide pool contraction is commensurate with the new rate of incorporation (414), substantiates this idea. The important change could be the combined MgATP² + Mg²⁺ pool, which raises over 10 mM.

It was suggested on several occasions that, through inhibiting replication, DNA damage induces the SOS conditions that favor recombinational repair over DNA replication and that the mechanism of SOS induction works by affecting the competition between SSB and RecA for ssDNA (167, 335, 492, 506) (Table 4). Thus, while recombinational repair is initiated by persistent ssDNA in situations when DNA replication is not generally inhibited, the SOS response could be induced when the first condition is followed by the nucleotide imbalance caused by replisome inhibition at frequent DNA lesions. From this perspective, recombinational repair and the following SOS response are the two stages in the same process of dealing with DNA synthesis irregularities. These ideas underscore the importance of measuring the intracellular Mg²⁺ and nucleoside triphosphate concentrations under conditions of normal and inhibited DNA replication.

The hypothesized increase in the MgATP² concentration in response to DNA damage should adversely affect cellular processes, which are dependent on the "regular" MgATP² concentrations. From this perspective, the increase in RuvAB production during the SOS response could be such a compensation for the decreased activity of the enzyme due to the suboptimal conditions. Furthermore, the very different in vitro ATP/Mg²⁺ optimum ratios for RuvAB and RecG Holliday junction translocases (Table 3) suggest that in vivo RuvAB is more active under "regular" conditions whereas RecG is more active under the SOS-induced conditions.

The possibility that the SOS-induced conditions are inhibitory for some required functions suggests that some of the SOS-induced proteins specifically counteract this SOS inhibition of other important proteins. For example, because of the precursor imbalance, DNA replisomes could be inhibited directly during SOS induction. The recently characterized gene dinB is responsible for mutagenesis of undamaged DNA in cells with an induced SOS response (304). The product of dinB is an error-prone DNA polymerase (pol IV), which could function to increase the replication speed or processivity under SOS conditions at the price of elevated mutagenesis.

Another possible example of such an SOS-compensatory activity is RecN protein, whose function is still unknown. Viability of E. coli after exposure to ionizing radiation or mitomycin C (which cross-links DNA) is severely compromised in radB/recN mutants (493, 553, 554, 556). recN is not required for chromosomal rejoining after low doses of irradiation but becomes increasingly important at higher doses (555). recN expression, which is normally undetectable, is greatly induced during the SOS response (182). The recN promoter has two binding sites for the LexA repressor, making recN one of the most tightly regulated genes of the SOS regulon (532). Sequence analysis of recN reveals an ATP-binding domain but no other peculiarities that would hint to a possible function of its product (532). The function of RecN is unlikely to be RecBC pathway specific, since recN mutants are also defective in double-strand end repair along the RecF pathway (see "RecF pathway" above). The degradation of damaged DNA is unaffected in recN mutants (553). Since the initial rejoining of double-strand ends in recN mutants seems to be normal, while the massive rejoining efforts are blocked, RecN might be needed for slowing the nucleoid segregation (see "Nucleoid segregation and the problem of accessibility" above) in response to DNA damage, since double-strand break repair depends critically on the availability of sister duplex.

SINGLE-STRAND ANNEALING: THE PHAGE WAY TO LINK HOMOLOGOUS DOUBLE-STRAND ENDS

Top Previous Next References

Single-Strand Annealing in DNA Metabolism of Lambdoid Phages

View larger version (16K): [in this window] [in a new window]   FIG. 30.   SSA repair of a double-strand break between direct repeats. Direct repeats are shown as black arrows. Explanations are given in the figure and in the text.

SSA enzymes of phage . Soon after the discovery of the recA gene in E. coli (107), it was found that  recombination is not affected by recA mutations (74, 645, 689). The  genes red and red were found to be required for this RecA-independent  recombination (166, 193, 589, 592); the corresponding proteins are an exonuclease (88, 365, 589) and an ssDNA-annealing protein (306, 455). Later, yet another gene, gam, was found to be required for the maximal levels of the Red-promoted recombination (175, 767). If unchecked, the host RecBCD nuclease (see "Preparation of double-strand ends by RecBCD nuclease for RecA polymerization" above) degrades the linear concatemeric products of  rolling-circle DNA replication (175, 220). Gam binds to RecBCD and inhibits all the known activities of this enzyme (294, 457, 686). Gam analogs are produced by many bacteriophages of E. coli replicating their genomes as linear DNA (542).

View larger version (16K): [in this window] [in a new window]   FIG. 31.   Some reactions promoted by the SSA repair proteins. (A) Strand assimilation catalyzed by  exonuclease. The exonuclease is the washer marked exo; the small arrow below shows the direction of the exonucleolytic degradation. The exonuclease will degrade the 5'-ending strand of the duplex until the branching strand is completely assimilated into the duplex (91). The exonuclease stops its progress when strand assimilation generates a single-strand interruption, accounting for the inability of the enzyme to start DNA degradation from single-strand interruptions. (B) Three-stranded branch migration catalyzed by RecT or Beta. If the RecT- or Beta-promoted annealing of complementary strands encounters a duplex region overlapping the protein filament on the other DNA, the protein catalyzes strand exchange, displacing the resident strand by the incoming strand. The reaction resembles the three-strand branch migration promoted by RecA protein. However, in contrast to the RecA-promoted strand exchange, RecT and  Beta are able to start this reaction only if both participating molecules have complementary single-stranded regions (233, 358).

SSA enzymes of the Rac prophage. Rac is a cryptic lambdoid prophage residing in the E. coli chromosome (see "RecE pathway" above). It has at least two SSA repair genes, which are usually silent but can be activated by sbcA mutations. These two recombinational genes complement  red mutants; in fact, the two genes are picked up from the chromosome by  with its own recombinational genes deleted, resulting in a recombination-proficient revertant (216, 289, 767). The product of recE is ExoVIII, which degrades the 5'-ending strand of linear duplex DNA and, hence, is analogous to  exo (285). However, the two genes do not have significant sequence similarity, and the proteins are very different: while  Exo is a 26-kDa dwarf, ExoVIII is a 140-kDa monster (284).

Mechanisms of double-strand end repair in  infection: invasion versus annealing. Biochemical characterization of the recombination functions of  showed that in vivo this phage was likely to recombine by SSA mechanism (90, 358). In the proposed scheme,  exonuclease would degrade the 5'-ending strands of the two ends with overlapping homology while  Beta would anneal the unraveled complementary 3'-ending strands. The final processing of recombination intermediates requires strand assimilation and sealing of the single-strand interruptions by DNA ligase (90), which is provided by the host.

View larger version (19K): [in this window] [in a new window]   FIG. 32.   Comparison of the double-strand end invasion with single-strand annealing reactions. Duplex DNA is shown as double lines (open or solid); the cos (packaging) site of  is shown as a chevron. (A) Circular  chromosomes. (B) The chromosomes in panel A are cut at different and unique sites in vivo (marked with scissors in panel A. (C) The ends are processed by  exo to generate 3' overhangs. (D and E) The RecA-catalyzed invasion of the 3' overhang into an intact  chromosome with the following resolution of the joint molecule (190). (F) Alternatively, the 3' overhang is annealed by Beta with a complementary 3' overhang of another linear  chromosome, cut at a different location. (G) Completion of the SSA recombination. Note that the hybrid region in panel E is expected to be shorter than in panel G.

Possible role of SSA repair in the life cycle of . If the Lambda Red system could indeed catalyze strand invasion in the absence of RecA, it should be able to promote recombination and repair in the E. coli chromosome. Generalized phage transduction requires "double-strand end repair" of the linear transducing DNA with a circular chromosome. Consistent with the notion that any strand invasion into a circular duplex requires RecA, the frequency of generalized transduction is reduced 2 to 3 orders of magnitude in recA mutants (245, 765). Remarkably, the Red recombination system of phage  is able to catalyze a low level of transduction in the absence of RecA (705, 720). This Red-promoted recombination could occur at replication forks, where single-stranded pieces of DNA could be gradually assimilated via annealing with the template for the lagging-strand DNA synthesis (Fig. 33). The scheme predicts that the RecA-independent Red-mediated transduction will be influenced by the position of replication forks and will be inhibited if DNA replication is temporarily blocked.

View larger version (10K): [in this window] [in a new window]   FIG. 33.   Mechanism for the Red-mediated transduction in the absence of RecA. Open lines indicate the transducing duplex DNA; solid lines indicate the host chromosome; arrows indicate DNA synthesis at the 3' ends; the circle indicates  exo. (A) The transducing DNA is degraded by  exo, while a replication fork is passing through the homologous region in the chromosome. (B)  Beta catalyzes annealing of the transducing strand with the template for the lagging-strand DNA synthesis. (C) Assimilation of the incoming strand into the host DNA catalyzed by the combined action of  exo and Beta. (D) The strand is completely assimilated. There is a heterology (shown as a bump) between the transducing DNA strand and the host DNA strand. This heterology is probably resolved by the next round of DNA replication, since the incoming DNA is likely to be fully methylated (a poor substrate for the methyl-directed mismatch repair [see "Damage reversal and one-strand repair"]).

View larger version (24K): [in this window] [in a new window]   FIG. 34.   Role of invasion-type and annealing-type recombination in phage  DNA metabolism. Duplex  chromosome is shown as a thick single line or a circle; a mature phage particle is shown at the bottom. The two basic processes, DNA replication (steps 1, 2, and 9) and DNA degradation due to the acting of packaging enzyme terminase (step 12) and spurious strand breaks (steps 3, 5, and 7), are shown in stages on the left and on the right, eventually leading to packaging of the phage DNA into the capsid (step 10). Homologous recombination takes the degradation products and returns them into the DNA replication metabolism (steps 4, 6, 8, 11, and 13). Steps are as follows: 1, theta replication of the circular  chromosome; 2, completion of the theta replication; 3, collapse of one of the replication forks, converting the theta-replicating chromosome into a sigma-replicating one; 4, RecA- and Red-dependent recombinational repair of the collapsed replication fork, returning the chromosome to theta replication; 5, collapse of the replication fork due to the nick in the linear DNA strand of the sigma structure, yielding a circular chromosome and a short linear fragment; 6, RecA- and Red-dependent invasion-type recombination, generating a sigma-replicating chromosome from a circular chromosome and a short linear chromosomal fragment; 7, collapse of the replication fork due to the nick in the circular DNA strand of the sigma structure, generating a long linear chromosomal fragment; 8, RecA- and Red-dependent invasion-type intramolecular recombination, generating a sigma-replicating chromosome from a long fragment; 9, DNA replication lengthening the linear tail of the sigma structure; 10, packaging of a genome segment from linear concatemers; 11, Red-dependent annealing-type recombination, combining two short fragments into a longer fragment; 12, unproductive terminase cutting to reduce the size of the linear chromosomal pieces; 13, Red-dependent annealing-type recombination, combining two short fragments or a single long fragment into a circular chromosome, able to initiate DNA replication from the origin.

View larger version (11K): [in this window] [in a new window]   FIG. 35.   Possible role of SSA repair in a better utilization of  DNA during packaging. This scheme is an illustration of step 11 in Fig. 34. Products of the late DNA replication (linear concatemeric pieces) are represented by double lines; small open circles indicate  exo degrading the 5'-ending strands. Genome equivalents in the  DNA concatemers are indicated by the short vertical lines. A packageable genome would be bracketed by two intact vertical lines. Thus, from the two initial DNA pieces (top), two phage genomes could be packaged, while the product of SSA repair (bottom) contains three packageable genome equivalents.

Double-strand break repair in plasmids with direct repeats. In the recBC sbcA genetic background, where the recombinational system of the Rac prophage is expressed (see "RecE pathway" and "SSA enzymes of the Rac prophage" above), formation of deletions between direct repeats in plasmids does not require RecA (186, 341). It is thought that in this background, plasmids form linear multimers similar to concatemeric DNA of phage  (115) and then recircularize in a RecA-independent manner, deleting the DNA between the repeats (593) (Fig. 36). If transformed with a linear dimer plasmid, a recBC sbcA strain recircularizes the plasmid with an efficiency approaching unity (636). This efficient double-strand break repair depends only on the RecE exonuclease and is independent of any other tested Rec proteins (RecT has not been tested), including RecA (390, 636). Long heterologies at one end of such linear plasmids do not preclude efficient recircularization (389, 594). The last finding was interpreted to signify a mechanism based on double-strand end invasion, with the role of synaptase being played by the RecT protein (389, 594). However, since RecT alone does not promote RecA-like synapsis (233), SSA repair remains the more economical way to explain these data. SSA repair should be able to tolerate substantial end heterologies, with the single-strand "whiskers" being removed by ssDNA-specific nucleases.

View larger version (10K): [in this window] [in a new window]   FIG. 36.   SSA recircularizes linear DNA with terminal redundancies. This scheme is an illustration of step 13 in Fig. 34. Direct repeats are shown as arrows. (A) A linear piece of  concatemeric DNA with terminal redundancies (direct repeats), unable to initiate DNA replication. (B) Strand-specific resection of double-strand ends. (C) Annealing of the unraveled complements of the direct repeat. (D) Removal of the DNA excluded from the synapsis, sealing the interruptions. The resulting recircularized  chromosome is competent for initiation of DNA replication.

Double-strand break repair in plasmids with inverted repeats. In a plasmid with an inverted repeat, each inverted segment containing a drug resistance gene with nonallelic mutations, recombination between the segments can restore the functional drug resistance gene in one of them (751). When WT or recBC sbcA cells are transformed with such a plasmid and plated for determination of the drug resistance, recombinants are infrequent (308, 644). However, if recBC sbcA cells are transformed with the plasmid that was cut in the middle of one of the repeats, the recombination frequency increases 100-fold (WT cells show no increase) (308, 331, 644). Two recombinational products are formed: 30% of the recombinants have the original configuration of the outside markers, while the other 70% of the recombinants have the markers flipped (crossover) (Fig. 37) (308). This double-strand break-stimulated recombination absolutely requires the Rac prophage-encoded RecE and RecT proteins but is independent of any host recombinational gene, including recA (331, 644). The ratio of the two recombinational products is more or less constant across the "recombinational alphabet," with the exception of recJ mutants, in which the ratio is reversed (331). Cloned recombinational genes of phage  also promote this double-strand break repair reaction (643). RecA independence notwithstanding, the authors explain these results in terms of the invasion-type double-strand break repair, assigning the role of RecA to RecT of Rac or to  Beta (308, 331, 643, 644). However, the formation of the two types of recombinants in this peculiar system is explained by SSA repair as well (Fig. 38); this explanation is more attractive because it does not require a biochemically undemonstrated RecA-like activity. SSA repair also explains the influence of the RecJ exonuclease, which could normally degrade the 5' end of the intermediate in Fig. 38F, preventing the formation of the noncrossover product. In the absence of the RecJ nuclease, there is no discrimination against the noncrossover products, resulting in the flipped ratio of crossovers to noncrossovers. Finally, it is quite illustrative that the RecF pathway, which is so genetically similar to the RecE pathway in the RecA-dependent double-strand end repair (see "Double-strand end repair in the absence of RecBCD" above), is quite incapable of promoting this kind of recombination in the linearized substrates (750).

View larger version (16K): [in this window] [in a new window]   FIG. 37.   The two products of double-strand gap repair in a plasmid with inverted repeats. Inverted repeats are shown as two black-and-white arrows on the opposite sides of the plasmid circle. One side of the circle is marked by knots on DNA strands to facilitate the detection of the inversion (crossover). The letters A, B, C, and D serve the same purpose.

View larger version (14K): [in this window] [in a new window]   FIG. 38.   SSA repair of a double-strand break in a plasmid with inverted repeats. Inverted repeats are shown as black-and-white arrows on the opposite sides of the plasmid circle. The letters A, B, C, and D help to reveal the inversion. (A) A plasmid carrying two inverted repeats with a double-strand gap in the middle of one of the repeats. (B) Degradation of the 5'-ending strand from one of the ends up to the middle of the intact inverted repeat. (C) The unraveled strand anneals on itself at the inverted repeat. The degradation stops (Fig. 31A), and the nick is sealed, resulting in a linear molecule with a loop at one end. (D) Degradation of the same polarity from the other end to the middle of the intact repeat. (E) The unraveled strand anneals on itself again, using the inverted repeat. The resulting circular ssDNA is then converted to a double-stranded form (not shown). It has markers on the opposite sides of the inverted repeats flipped, as if there was a conservative repair with associated crossing over (Fig. 37, left). (F) Alternatively, the degradation in panel B goes all the way to the other end of the molecule, rendering it completely single stranded. (G) The broken repeat anneals with the intact repeat and is repaired off it. Resynthesis of the degraded strand (not shown) generates a product identical to the one of the conservative double-strand break repair without crossing over (Fig. 37, right).