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Microbiology and Molecular Biology Reviews, September 2005, p. 501-526, Vol. 69, No. 3
1092-2172/05/$08.00+0     doi:10.1128/MMBR.69.3.501-526.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Replication Termination in Escherichia coli: Structure and Antihelicase Activity of the Tus-Ter Complex

School of Chemistry, University of Southampton, Southampton SO17 1BJ, United Kingdom,¹ Research School of Chemistry, Australian National University, A.C.T. 0200, Australia,² Gene Technologies Sector, HortResearch, Auckland, New Zealand,³ Department of Microbiology and Immunology, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, North Dakota 58202-9037⁴

SUMMARY

INTRODUCTION

Scope

Origins of the Concept of Replication Termination

COMPONENTS OF THE REPLICATION TERMINATION SYSTEM

The Terminator (Ter) Sequences

A trans-Acting Factor

The tus Gene and Tus Protein

PROPOSED MECHANISMS OF REPLICATION FORK ARREST

Evidence for Specific Protein-Protein Interactions

STRUCTURE OF THE Tus-Ter COMPLEX AND MOLECULAR BASIS OF REPLICATION ARREST

The Crystal Structure of the Tus-Ter Complex

Protein-DNA Binding Interactions

DNA Modification and Protection Studies

Nucleotide Substitution Studies

Mutants of Tus

A Stepwise Mechanism for Tus-Ter Binding and Unbinding

Comparison of Tus Sequences

STRUCTURAL INSIGHTS INTO THE INTERACTIONS OF Tus WITH HELICASES

Structures of DnaB and Related Hexameric Helicases

Interaction of Tus with Helicases

MECHANISMS OF POLAR REPLICATION ARREST

Prospects for the Future

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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View larger version (25K): [in this window] [in a new window]   FIG. 1. Positions of Ter sites and the tus gene on the E. coli chromosome. All Ter sites are oriented so that the replication forks can travel in the origin-to-terminus direction but not the opposite direction. The tus gene is just downstream of TerB.

Several aspects of replication termination (7, 13, 19, 26, 58, 67, 78, 108, 120, 145, 153) and Tus-Ter interaction (85, 170) have been reviewed previously. Although discussion here is limited to the system as it has evolved in E. coli and closely related eubacteria, understanding of termination in E. coli has developed in parallel with work on the mechanistically related system in Bacillus subtilis (26, 169). The B. subtilis termination system is the only other one where the molecular structure of the replication terminator protein (RTP) in complex with a cognate Ter site is known and the only one where structures of both the free (27, 134) and DNA-bound (172) forms of the protein have been determined. Although the Ter sites in B. subtilis were initially thought to be similar to those from E. coli (71), the two terminator proteins are completely unrelated in sequence and in structure and bind their respective Ter sites in quite different ways (85, 172). RTP binds as a dimer of dimers to two symmetric half-sites within a full B. subtilis Ter site (discussed recently in detail in reference 44), while as described below, Tus binds as a monomer to a full (asymmetric) E. coli Ter site.

DNA synthesis at replication forks is mediated by a multiprotein assembly called the replisome, which accomplishes concerted DNA synthesis on both the leading and lagging strands (Fig. 2). The roles of the individual protein components of the replisome and the macromolecular interactions that determine its structure and function have been the subject of intensive study over the past 25 years, and this has led to sophisticated models for how the complex works. These have been the subject of recent reviews (8, 15, 34, 35, 118, 153).

View larger version (27K): [in this window] [in a new window]   FIG. 2. Protein-protein interactions in the Escherichia coli replisome as it approaches the Tus-Ter termination block. (A) The DNA polymerase III (Pol III) holoenzyme is an asymmetric dimer containing 10 different subunits that include the twin polymerase () subunits that simultaneously replicate the two strands of the DNA template. (B) The replisome is a multiprotein complex made up of the DnaB helicase, the DnaG primase, and the Pol III holoenzyme. Each replicated strand commences with a short RNA primer synthesized by DnaG primase recruited from solution by interaction with DnaB. Single-stranded DNA is protected by SSB. Adapted from Fig. 2 of reference 153 with the permission of the authors.

The question of whether replication terminated at a specific site was examined in various experimental systems, and the first indication of the existence of a discrete terminus was found in studies with the conjugative R plasmid R6K and a deletion mutant of it, RSF1040 (33, 112). Electron microscopic examination of RSF1040 replication intermediates showed two origins ( and ß) and a single terminus (33). Replication was initiated from the origin, progressing first towards the "right," halting at the terminus, and then progressing towards the "left" from the same origin to the same terminus. Replication could also occur from the ß origin in the same asymmetric, bidirectional manner to the same terminus. The terminus is thus responsible for converting unidirectional replication into a sequential bidirectional mode (33). These conclusions are unaffected by recent studies that show the initiation of R6K replication to be more complicated, involving looping interactions of the replication initiator protein bound at a third origin () with the and ß origins (1, 2).

Soon after, in 1977, evidence for a discrete site for termination in the E. coli chromosome was reported. Louarn et al. (110) changed the position of replication initiation by integrating R-plasmid origins at various sites in the chromosome of a temperature-sensitive mutant with a mutation in dnaA, the gene that encodes the replication initiator protein DnaA (119). These strains could not initiate replication from oriC at the nonpermissive temperature, but replication could still initiate at the integrated origins and proceed bidirectionally. It was found to terminate diametrically opposite oriC (between att80 at 28 min and attP2H at 45 min) even when the new origin was displaced by 26 min from it. Using a similar system, Kuempel et al. (103, 104) located the terminus between aroD and rac at 38 and 30 min, respectively, and Louarn et al. (111) later reduced this interval to the 6 min between man and rac.

It was still an open question whether the R6K and E. coli termini worked by the same mechanism. Both termini blocked replication at specific sites, and both seemed to work independently of the site of initiation and the type of origin. The E. coli terminus could block bidirectional replication initiated from oriC or (symmetric or asymmetric) replication from various integrated plasmid or phage origins at various locations (103, 104, 110), while the R6K terminus could block replication from the R6K origins in RSF1010 (33) and from a ColE1 origin in two different positions in a plasmid (98). Both termini apparently blocked replication forks arriving at the terminus from both directions. However, as described above, the modes of replication are quite different. In addition, while the R6K terminus region was located to a 216-bp segment of DNA (12), the continuing difficulty in pinpointing the precise location of the chromosomal terminus was beginning to suggest that it was a large region rather than a specific site.

This problem was solved in 1987 with the realization that the E. coli terminus was made up of discrete loci that separately blocked replication forks moving in opposite directions in a polar manner (38, 68, 138). The first two termination sites identified were situated at either end of the terminus region (Fig. 1); one was located close to trp at 28 min and the other near manA at 36 min (68, 138). This polar block to progress of the fork therefore appeared to be different from that at the R6K terminus, which was known to block fork movement from either direction (11, 33, 98).

Resolution of the similarity of the two systems had to wait one more year for nucleotide sequences from the E. coli terminus to become available (62, 71). The terminators that would eventually be named TerA and TerB (Fig. 3) had a strong similarity to the two halves of an imperfect inverted repeat in the R6K terminus (62, 71, 75). The two R6K sequences (named TerR1 and TerR2) were identical to TerA and TerB at 15 and 12 positions, respectively (Fig. 3). In both the R6K plasmids and the E. coli chromosome, the Ter sequences were placed so as to form a "replication fork trap" that would allow a replisome to enter the region between the two Ter sites but not to leave. Ter sequences were also found in a variety of other plasmids as well as in other bacteria (30), and the number of Ter sites identified in the E. coli chromosome also increased, first to 4 (48, 62), then to 5 (63), and finally to 10, after the publication of the entire genome sequence (23) and an in-depth study of nucleotide substitutions by Coskun-Ari and Hill (30).

View larger version (37K): [in this window] [in a new window]   FIG. 3. Nucleotide sequences of Ter sites from the E. coli chromosome and R6K plasmids. Base pairs that interact with the Tus protein are indicated by the shaded regions. In the orientation shown for these sequences, replication forks approaching from the left are blocked, while those entering from the right are unimpeded.

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Once small DNA fragments containing TerA and TerB as well as the two TerR sites were available, it was shown that they could block replication forks in ColE1 plasmids in vivo (139, 159), and proof that the minimal Ter sequences were indeed sufficient to block replication forks in a polar manner came after they had been inserted into plasmids as synthetic oligonucleotides (62, 71, 75).

The second line of evidence for involvement of a DNA-binding protein arose from deletion studies used to narrow down the locations of TerA and TerB. TerB was quickly located to a 4-kb region, while TerA was more difficult to locate precisely. However, deletion of the TerB region inactivated arrest activity at TerA, implicating a trans-acting factor encoded near the TerB arrest site (69). Kuempel and coworkers named the putative gene tus for "termination utilization substance."

The first description of the trans-acting factor was by Hill et al. (72), who isolated the gene encoding a DNA-binding protein by screening deletion and insertion mutants with mutations in the TerB region. They reported the gene sequences and the construction of tus strains that were deficient in termination activity. These mutants were complemented by plasmid-borne copies of tus. The gene was predicted to encode a 36-kDa polypeptide, and it directed overproduction of a protein estimated by gel electrophoresis to be this size (72).

Soon after, two other groups isolated a protein that bound to R6K Ter DNA. Sista et al. (159) purified an 40-kDa protein that bound the TerR sequence and defined its binding site by using copper-phenanthroline footprinting. A mutated Ter site with changes at six of the protected residues lost both the ability to bind the purified protein and the ability to arrest replication forks in vivo. Kobayashi et al. (96) reported isolation of a fragment of DNA encoding terminus-binding activity, together with insertion mutants that had lost the ability to bind a Ter site, whether on a plasmid or in the chromosome. The activity associated with the gene was sensitive to treatment with proteases and heat but not to treatment with RNase (96). They also determined the sequence of the gene, overproduced and purified the gene product, and demonstrated its binding to both TerR sites by DNase I footprinting (65).

All three activities were soon shown to be those of the same protein, encoded by the tus gene situated just following TerB (Fig. 1 and 4). The Tus protein bound to all known Ter sites and, once bound, could block the progress of a replication fork. A remaining question was how the moving fork was blocked. Did the Tus-Ter complex interact specifically with some component of the moving replisome, or did it merely act as a clamp on the DNA preventing its passage through the Ter site? With the gene, the protein, and hypotheses in hand, several groups tackled the mechanism of replication termination.

View larger version (13K): [in this window] [in a new window]   FIG. 4. Relationship between TerB and the tus gene. The tus gene and its –10 promoter region and ribosome-binding site (RBS) are shown. The Tus protein regulates tus gene expression by binding to the TerB sequence and blocking the initiation of transcription of tus. The TerB sequence is enclosed in the box, and base pairs that interact with Tus are shaded as in Fig. 3.

The gene coded for a protein of 308 amino acids (after removal of the N-terminal methionine residue) with a mass of 35,652 Da. The protein sequence showed no similarity to any known DNA-binding motif. The purified protein had a pI of 7.5, significantly lower than the value of 10.5 calculated from its amino acid composition. Since there was no indication that the protein was phosphorylated, this suggested that the tertiary structure had a large effect on the ionization state of several basic residues. Gel filtration and sucrose density gradient centrifugation showed that Tus was a monomer in solution with a Stokes radius of 23 Å and an axial ratio of two (31). This would allow it to cover 13 bp of DNA on binding, which was in good agreement with the results of the earlier footprinting studies (159).

Tus was shown by footprinting with copper-phenanthroline (159), DNase I (65, 130), and hydroxyl radicals (54, 158) to bind to several Ter sites. It bound extremely avidly to the TerB site; the Tus-TerB complex had a measured dissociation constant (K_D) of 3.4 x 10^–13 M and a dissociation half-life in vitro of 550 min at pH 7.5 in a buffer containing 150 mM potassium glutamate (54). Its binding to R6K TerR2 under identical conditions was weaker; the measured value of K_D was 30 times higher, primarily due to a higher dissociation rate (54). The protein was shown to bind to TerB as a monomer, which is unusual for a DNA-binding protein but consistent with the asymmetry of the Ter sites and replication fork arrest (31).

PROPOSED MECHANISMS OF REPLICATION FORK ARREST

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The basis of the mechanism of fork arrest was soon established. The Tus-TerB complex was shown to block the action of the major replicative DNA helicase, DnaB in vitro in an orientation-dependent manner (91, 106). The orientation of the block was the same as for the arrest of replication fork movement both in vivo and in vitro (61, 70, 106, 113).

In the normal process of replication, DnaB is at the front of the replisome (Fig. 2B). It is a ring-shaped homohexameric enzyme that translocates in the 5'-to-3' direction on the lagging-strand template to unwind double-stranded DNA in front of the DNA polymerase III holoenzyme, the multisubunit replicase (118, 153) that simultaneously synthesizes both strands (Fig. 2A). One strand (the leading strand) is replicated continuously, while the other (lagging) strand is synthesized discontinuously in a series of (Okazaki) fragments. The replicative RNA-priming enzyme, DnaG primase (49), is recruited by DnaB for the priming of each new fragment on the discontinuous strand (133). The single-stranded sections that result from helicase action are coated with single-stranded DNA-binding protein (SSB). DnaB is physically associated with the replicase through the subunit of the holoenzyme (93).

When progress of the replisome was halted by the Tus-Ter complex, both in vitro (70) and in vivo (126), DNA synthesis continued right up to 4 base pairs before the conserved GC6 base pair in the TerB site (Fig. 3). This is a surprising result given the size of the polymerase holoenzyme, let alone the enormity of the entire replisome. Since the leading-strand template is known to be excluded from the central channel of DnaB (80, 87), it is conceivable that the active site of the leading-strand polymerase is very close to the point of strand separation by the helicase (Fig. 2B). However, it appears more likely that dissociation of DnaB from the replisome occurs as part of the arrest process. In the presence of DnaG primase, the distribution of leading-strand stop sites changed, showing a degree of sensitivity of leading-strand synthesis to the protein complement of the lagging strand (70).

Lagging-strand synthesis stopped 50, 66, or 82 bp before the TerB site (70). The 50-bp (17-nm) gap could be envisaged as a loop bound by one or two tetramers of SSB on the lagging strand (Fig. 5). This implies that the loop is either topologically or physically constrained from closing any farther to allow priming by DnaG before dissociation of DnaB. The 16-bp spacing between the lagging-strand priming sites may reflect some aspect of protein organization on the lagging strand that affects the site of priming or subsequent primer extension or may simply be due to the sequence specificity of the DnaG primase (49, 70).

View larger version (21K): [in this window] [in a new window]   FIG. 5. Replisome of E. coli and mechanism of replication fork arrest by a Tus-Ter complex. (A) The replisome moving along the DNA template approaches Tus, and the DnaB helicase assists primase to lay down the last lagging-strand primer. (B) DnaB helicase action isblocked by Tus, and DnaB dissociates from the template. (C) DNA polymerase III (Pol III) holoenzyme completes leading-strand synthesis up to the Tus-Ter complex and (D) synthesizes the last Okazaki fragment on the lagging strand, which will eventually be ligated by DNA ligase to the penultimate fragment following removal of its RNA primer by DNA polymerase I (not shown). (E) The holoenzyme then dissociates, leaving a Y-forked structure that is single stranded on the lagging strand near the Tus-Ter complex.

What occurs when a replication fork approaches from the other (permissive) direction is much less clear. Khatri et al. (91) suggested that the Tus protein remains associated with one strand (the strand shown in Fig. 3) of the unwound DNA after DnaB has passed through the Ter site from the permissive side. However, Gottlieb et al. (54) found that Tus had no affinity for either strand of DNA in the single-stranded form, and Neylon et al. (131) also reported that the affinity of Tus for each separate strand of the TerB site was the same as that for a nonspecific single-stranded DNA under low-salt conditions where binding could be observed. Very little work has been reported on the process by which the helicase passes through the Tus-Ter complex when it approaches from the permissive direction.

Another remaining issue is the nature of the interaction between Tus and DnaB. Does Tus merely act as a clamp on the DNA, or are there specific protein-protein or protein-DNA-protein interactions between Tus and the oncoming helicase (or other component of the replisome)? These two possibilities can be broadly described as the "clamp model" and the "interaction model." These two simple mechanisms were initially proposed with the expectation that the question would be resolved rapidly. However, it still remains controversial in spite of publication during the ensuing years of a high-resolution crystal structure of the Tus-TerA complex (85). A third potential mechanism that has been recently suggested (131) is one in which Tus interacts with the helicase (or other elements of the replisome) through the DNA. That is, that Tus engineers a structure in the DNA on the nonpermissive side that prevents the further passage of the helicase. A fourth and related alternative, apparently yet to be tested experimentally, is that the helicase generates a structure in the DNA at the permissive face that actively promotes dissociation of Tus and/or a structure at the nonpermissive face that increases the affinity of Tus for the Ter site. In the remainder of this review, we will examine the available evidence for these possible molecular mechanisms of Tus-mediated polar replication fork arrest at Ter sites.

In the experiments of Bastia and coworkers, the Tus-TerR complex was observed to block the replicative helicases DnaB and simian virus 40 (SV40) T antigen, but it failed to block helicases involved in DNA repair or plasmid rolling-circle replication, including Rep, Dda, TraI, and UvrD (14, 91, 147). Even though the block to the action of T antigen (a 3'-5' helicase) seemed to be at the face permissive for DnaB, they nonetheless favored a mechanism that involves specific protein-protein interactions between Tus and a domain of the replicative helicases. In support of this, they cited the (unpublished) observation of a direct interaction between Tus and DnaB (114). More recently, the same group has described experiments using a yeast two-hybrid system that provide evidence of in vivo interaction between the two proteins (127). They also describe the binding of DnaB to an immobilized glutathione S-transferase-Tus fusion protein and isolation of mutants of Tus that have reduced binding to DnaB and similarly reduced fork-blocking activity but near-normal TerR binding. This is the strongest evidence to date for a specific interaction between Tus-TerR and the oncoming helicase.

In contrast to the results of Bastia and coworkers, in the experiments of Lee et al. and other groups studying the Tus-TerB interaction, the complex impeded the progress of both replicative and repair helicases (60, 105, 106). In addition, it did so in a polar manner. That is, the same face of the Tus-Ter complex blocked DnaB translocating in the 5'-3' direction but also blocked SV40 T antigen (5, 64), PriA (60, 105), UvrD (106), and TraI (64) translocating on the opposite strand in the 3'-5' direction. This would suggest that the action of the complex is either as a clamp or directed against some aspect of helicase structure and/or function that is sufficiently general to be exhibited by all those tested. The idea that a clamp might be sufficient is supported by a report that a mutant EcoRI restriction endonuclease that binds to its recognition sequence with a dissociation constant of 2.5 x 10^–13 M, but does not cleave DNA (95, 173), was capable of blocking the helicase action of DnaB, UvrD, and SV40 T antigen (14). The block was orientation independent, since EcoRI binds to DNA as a symmetric dimer. Later, it was shown that the lac repressor-operator complex can substantially inhibit the action of a range of helicases in vitro, including DnaB (175). The effectiveness of these unrelated protein-DNA complexes in blocking replication forks would appear to indicate that a simple clamp is sufficient to halt helicases in vitro.

Experiments with surrogate systems do not support this view. In an ingenious series of experiments, Andersen et al. (6) compared the effectiveness and polarity of the Tus-Ter complex in vivo in E. coli and B. subtilis. Alongside this, the functionally similar but unrelated replication termination system of B. subtilis was compared in both organisms. While B. subtilis RTP-TerI worked well to terminate replication in both organisms, the E. coli Tus-TerB complex was very much more effective in its natural host. In earlier similar experiments, Kaul et al. (90) had also shown the B. subtilis termination system to be effective in E. coli. These data might indicate a fundamental difference in mechanism between the two systems and support the existence of a specific interaction between Tus-Ter and a replisomal protein(s) in E. coli, at least.

On the other hand, in evolutionary terms, it is not surprising that the systems work somewhat better in their natural context. Natural systems under selection pressure would be expected to take advantage of opportunities to improve their efficiency. Indeed, it would be surprising in the specific case of the Tus-Ter acting against E. coli DnaB if there was not a functional interaction that had developed to improve the efficiency of replication arrest. However, it is not clear how highly specific interactions could develop to play a general role in antihelicase activity. Perhaps the more pertinent question is whether Tus-DnaB interactions are limited to small improvements in a single protein-protein interface or whether they play an important role in the more general case of Tus activity against the full range of helicases.

STRUCTURE OF THE Tus-Ter COMPLEX AND MOLECULAR BASIS OF REPLICATION ARREST

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A large amount of data is available on the Tus-Ter interaction, including results of DNA footprinting, kinetic studies, effects of mutations to both Tus and the Ter sequence, and the gene sequences of Tus proteins from related bacteria. In this section, we will analyze the published data on the Tus-Ter interaction, starting with the crystal structure of the complex (85), followed by footprinting and kinetic studies. This will be followed by the data on Ter DNA mutations and mutational studies of the Tus protein itself and then by an analysis of the protein sequences from three related bacterial species, as well as two further proteins with sequence similarity to Tus. There has been no previous analysis of all the available data within the framework of the crystal structure. Finally, we will summarize the results and examine a series of models of protein-DNA and protein-protein interactions at the site of replication arrest.

The E. coli Tus-TerA complex was crystallized by Kamada et al., and the X-ray crystal structure was reported in 1996 (85, 86). The structure (Protein Data Bank code 1ECR), shown in Fig. 6, is a unique protein fold consisting of two discontinuous domains that straddle the TerA double helix. The two domains are joined by two antiparallel pairs of ß strands that make up the core DNA-binding domain (ßIF and ßGH) and also by the L4 loop. These two pairs of strands lie in the major groove of TerA. The structure of Tus in the complex is 37% helix, 28% sheet, and 35% loops and turns. The I, II, and III amphipathic helices form an antiparallel bundle that runs parallel to the DNA but makes no contact with it. The IV and V helices along with the L1 and L2 loops lie at the top of the larger (N-terminal) domain. With the VI-VII region in the smaller C-terminal domain, they complete the face of Tus that blocks the progressing helicase (the nonpermissive or fork-blocking face). Three of the four main loops (L1 to L3) are at the nonpermissive end of the complex. The remaining loop (L4) lies at the permissive end in the minor groove, making a number of DNA contacts.

View larger version (42K): [in this window] [in a new window]   FIG. 6. The crystal structure of the Tus-TerA complex, PDB code 1ECR (85). Three views of the Tus-Ter complex are shown. The top view is looking down the DNA from the nonpermissive face of the complex. The middle view is rotated 90° from the first to show the front of the complex. The bottom view, rotated a further 90°, is along the DNA from the permissive end of the complex. The permissive and nonpermissive faces are indicated in the middle view. The balls indicate the (5') strands that would pass through the central channel of the DnaB helicase. Images of protein structures in this and succeeding figures were generated in SWISS-PDB VIEWER version 3.7 (http://ca.expasy.org/spdbv/) (56) and rendered using POV-RAY version 3.1g.watcom.win32 (www.povray.org).

The double-stranded TerA captured within the complex is significantly deformed from the canonical structure of B-form DNA. The average helical twist is 29.5°, compared to the canonical value of 34.6° (85). The DNA backbone is also deformed between G17 and A14 (Fig. 7) due to it being sandwiched between the ßF and ßG strands and the L4 loop. The propeller angle of the AT16 base pair is –24.2°. The DNA is consequently underwound, making the major groove deeper and expanding the minor groove, and it is bent overall through about 20° (85). The TerA fragment in the crystal does not extend beyond the protein and therefore provides little information about the DNA structure at the permissive end of the complex; it is thus possible that the DNA would be further deformed by contacts with the protein beyond the extremity of the cocrystallized fragment (Fig. 7).

View larger version (39K): [in this window] [in a new window]   FIG. 7. Summary of contacts between Tus and TerA. Adapted from reference 30 with permission of the publisher. Arrows show interactions between amino acid side chains and groups in the base pairs. Residues in the TerA oligonucleotide used for determination of the crystal structure were A4 to T18 on one strand and T19 to T5 on the other and are shown with boldface outlines. Dashed lines indicate possible interactions at the permissive end that were not seen in the crystal structure (see the text for details).

View larger version (20K): [in this window] [in a new window]   FIG. 8. Sequence and secondary structure of the Tus protein (data are from reference 85). The 31 residues that make nonspecific contacts to the DNA backbone are in blue. The 17 residues that make direct or water-mediated specific contacts with the DNA bases are in red.

There are 17 residues that make sequence-specific contacts with TerA DNA (Fig. 8). Nearly half of these are hydrophobic, and the remainder are mainly hydrogen-bonded interactions between charged or polar amino acid side chains and polar donor/acceptor atoms of the bases in the major groove. Several of the latter interactions are mediated by water molecules. Only the hydrophobic contact between Thr136 and T8 involves a residue in an helix.

In contrast, no fewer than 31 residues make nonspecific contacts with the deoxyribose phosphate backbone of the DNA (Fig. 8). While these residues are still concentrated in the central DNA-binding motif, they are more widely distributed than those that make sequence-specific contacts. The majority of the phosphate interactions involve charged or polar side chains, particularly guanidine, amine, and amide groups, and nearly half are water mediated. Most of these residues lie in ß sheets or in loop regions. On the other hand, nearly all the protein-deoxyribose interactions are hydrophobic, usually involving the C4' and C5' atoms of the sugar, which protrude into the minor groove of the DNA. The only residue that interacts with the C1' and C2' of the deoxyribose in the major groove, Ile178, also makes a sequence-specific hydrophobic contact in the major groove. Arg198 makes the only hydrogen bond contacts with a sugar, from the side chain N()H2 to the O4' of A5 and G6. Other residues that may make contacts that are not explicit in the crystal structure are Lys249, His253, and His304, which could make water-mediated contacts, and Gln294, which can be rotated to make a contact with the 5'-phosphate of A14.

Notably, residues that make nonspecific contacts are often positioned such that they flank those that make sequence-specific contacts. It may be that the nonspecific interactions are required to position the backbone interactions correctly for optimal binding or, conversely, that the nonspecific interactions provide a means to allow Tus to slide along DNA searching for its specific binding contacts.

View larger version (33K): [in this window] [in a new window]   FIG. 9. Summary of the results of footprinting studies by Sista et al. (158) and Gottlieb et al. (54). Arrows indicate protection from hydroxyl radical cleavage. Filled circles indicate protection from methylation by dimethyl sulfate. Open circles show enhanced methylation. The base pairs that interact with Tus are shaded as in Fig. 3.

Although most of the protected nucleotides were within the region bound by Tus in the crystal structure, from G6 to A18 on the top strand and from T19 to C6 on the bottom strand (Fig. 3), both groups reported protected sites outside this region. Sista et al. (158) found four such sites, between 1 and 3 base pairs preceding and 1 following the Tus-binding site in the TerR2 sequence (Fig. 9). While Gottlieb et al. (54) described two protected sites preceding TerB, these were only 1 base pair from the binding site and could be explained by occlusion by the overhanging protein. The TerR2 protection sites are more difficult to explain on the basis of the static crystal structure.

The K_D of the Tus-TerR2 complex has been estimated to be 30-fold higher than that for Tus-TerB (54). If this is largely the result of the loss of sequence-specific interactions, then the protected sites on TerR2 may reflect greater mobility of the protein on this DNA. Conversely, it may simply be the case that the crystal structure does not accurately represent the mobility in solution of the amino acid side chains in the vicinity.

Another explanation is that Tus engineers structures in the DNA at each end of the complex that are resistant to hydroxyl radical cleavage. At the permissive face of the complex (Fig. 6), this may be the result of strand separation. This is suggested by the run of four AT base pairs, the twisted conformations of the AT16 and TA18 base pairs, and the nucleotide substitution data that will be discussed below. At the nonpermissive face, strand separation may be indicated by the severe twist induced in the AT5 base pair. The high AT content in DNA at the nonpermissive end of most Ter sites (Fig. 3), the nucleotide substitution data (below), and the very close approach of DNA polymerase inferred from the position of the end of leading-strand synthesis (70) may also suggest that strand separation occurs at this point.

Where a thymine methyl group is involved in a hydrophobic interaction, there was found to be a positive G (i.e., more rapid dissociation) for the substitution of halogenated uracil. The two main thymine methyl interactions are at nucleotides T12 and T16, and these are the two thymines with the highest G for conversion to uracil and the halogenated analogs. A negative G (slower dissociation) for iodo- and bromouracil substitution was observed for modifications of T8, T14, and T19 and indicates the presence of a polarizable group in the minor groove (41). This is confirmed by the crystal structure for T8 (interacts with Lys89) and T14 (interacts with Lys175), and a contact with T19 can be formed by rotating the side chain of Arg288. In most cases the Tus complex with TerB replaced with a 2-aminopurine:uracil base pair was observed to be slightly more stable than a 2-aminopurine · cytosine base pair, indicating that unfavorable base pairing contributes part (GC10) or most (CG17) of the increase in G. However at GC13, where the N4 of cytosine interacts with His176, the substitution of uracil for cytosine opposite 2-aminopurine greatly destabilized the Tus-Ter interaction (42).

Coskun-Ari and Hill (30) chose an alternative approach of replacement of base pairs in TerB with all three natural alternates and produced a near-complete set of all possible substitutions in the region GC6 to AT21. This allowed them to identify three new Ter sites in the E. coli genome sequence, to define in general terms which Ter sites are strong or weak Tus-binding sites, and to specify precisely which residues in the consensus sequence are important for binding as well as for replication fork arrest activity in vivo.

The nucleotide substitution data need to be interpreted carefully. A single substitution could affect DNA stability, the entropic cost of removing water from its hydration shell, and even the internal structure of the Tus protein, as well as directly affecting binding. As expected, the combined substitution data agree broadly with the crystal structure and conservation of residues within the Ter sites. The most important base pairs for Tus binding were found to lie in the most conserved regions (Fig. 3). For example, the TA7 base pair, which is not conserved and does not contact Tus in the crystal structure, was found to be dispensable, and the partially conserved AT8 base pair showed tolerance for the GC substitution found in a number of natural Ter sites (30).

In general, there was a correlation between binding energy and replication arrest activity in vivo. However, at the nonpermissive end, the three substitutions at GC6 all had a much larger effect on replication arrest than expected on the basis of the change in binding energy, indicating that this base pair is important for replication arrest for reasons that are not related primarily to the stability of the Tus-TerB complex (30).

It is also difficult to correlate the crystal structure (85) with the effects of some substitutions at the permissive face (30). Although changes to the conserved AT19 base pair caused a large change in G for binding and abolished replication arrest activity, the crystal structure shows no explicit sequence-specific interaction at this site. The Arg288 side chain can be brought into contact with either O2 and N3 or N3 and O4 of this thymidine, depending on whether its N is simultaneously positioned to interact with adenine or thymine at TA18 (Fig. 7). The N3-O4 interaction could also be strengthened if the strands were separated, but the quantitative data offer little guidance about which interactions are most likely. Substitution at AT20, which lies beyond the DNA used for crystallization, reduced arrest activity while having only a modest effect on binding (30). This may be due to interactions (not seen in the crystal structure) with Trp243 and Gln248 (Fig. 7) or to structural changes in the DNA required for fork arrest activity. Finally, at the adjacent AT21 site, now well away from the protein, there was also an effect on both binding and arrest activity, again suggesting a role for DNA structure in the binding reaction, the arrest reaction, or both (30). We note that Gln248 can be positioned for potential interactions with T21 (Fig. 7).

The role of the four base pairs GC6 and AT19 to AT21 may well be concerned with engineering of a structure in the DNA that affects helicase passage through the Tus-Ter complex. This structure might include the separation of the DNA strands at one or the other end of the complex, and this is supported by other elements of the crystal structure (85, 170). The results are also consistent with a dynamic complex in which partial unbinding processes play a role in the antihelicase activity. In this case these anomalous base pairs would be involved in binding of intermediates on the binding-unbinding pathway but not in the final steady-state Tus-Ter complex.

Genetic methods have been developed to select directly for Tus mutants defective in replication arrest (160, 161). In one of these, developed by Skokotas et al. (160) and also used by Kamada et al. (85), a Ter site was placed so as to disrupt replication of the chromosome of a tus recA strain, and tus mutants were introduced into cells on a plasmid. Active Tus binds to the Ter site and prevents chromosome replication, while Tus mutants defective in fork arrest allow replication and cell survival. Mutants of this type could reflect not only the effects of substitutions on specific and/or nonspecific DNA binding but also aspects of fork arrest not related to DNA binding or even the folding of Tus into a stable structure. These effects could of course be separated by further investigation of the properties of the isolated variant proteins, but this has not always been done.

Of the 18 residues (Table 1; Fig. 10) identified in this way as being important for activity, 11 (His50, Arg93, Tyr156, Ala173, Lys175, Arg232, Gln237, Arg241, Gln252, Ala254, and Pro256) are directly involved in DNA binding and 3 others (Glu49, Leu159, and Pro238) are adjacent to residues that make contact with the DNA. The other identified residues which are probably important in maintaining tertiary structure include four prolines (residues 42, 95, 238, and 256); Leu150, which contributes to the hydrophobic core of the helices in the N domain; and Gly171, which provides the flexibility necessary for ßF to twist as it bends through 90° to pass under ßHI to follow the major groove of the bound DNA molecule (Fig. 6). Thus, it is reasonably easy to explain why each residue affects replication arrest activity in terms of effects on Tus stability or Tus-DNA binding. While these studies clearly identify important residues, the absence of mutations at a particular site does not indicate that the residue is unimportant. Only one of the selected mutants (P238L) was obtained in more than one experiment (85, 161), indicating that the sampling processes were not exhaustive.

View larger version (35K): [in this window] [in a new window]   FIG. 10. The nonpermissive face of the Tus-Ter complex. Four equivalent views of this face are shown, highlighting the features that might come into contact with the DnaB helicase. (A) Secondary structure elements that could contact DnaB; (B) residues at the nonpermissive face of the complex, including Glu49; (C) space-filling representation colored by residue type (red, acidic; blue, basic; yellow, polar; gray, aliphatic); (D) charge distribution on the Tus surface at the nonpermissive face. Charge was calculated without the TerA DNA in place, using atomic charges and Poisson-Boltzman calculation as implemented in SWISS-PDB VIEWER version 3.7 (56).

The characteristics of this mutant prompted Henderson et al. (59) to use oligonucleotide-directed mutagenesis to examine a larger range of mutants with mutations in the L1 loop (residues 41 to 53). This loop is expected to be a reasonably autonomous folding unit, as it is separated from adjoining secondary structure elements by proline residues at positions 42 and 52. Only His50 interacts directly with Ter DNA, but other mutations in L1 may also destabilize interactions with L2; Lys46 has interactions with Asn85 and Ser88 (which makes a contact to the phosphate of A7), and in solution, Glu47 could interact with Asn85. The E43Q, V44T, K45A, K46A, E47Q, D48N, E49A, E49K, H50N, and N51D mutants were examined in a quantitative assay of arrested plasmid replication intermediates, a growth assay similar to the selection system described above, and for binding to TerB and inhibition of DnaB helicase activity in vitro (Table 1). Of the mutants that had defects in replication arrest (K46A, E47Q, E49K, and E49A), all except for K46A showed more stable rather than weaker TerB binding (59).

However, this in vivo replication arrest defect was not mirrored precisely in in vitro antihelicase assays. Both E47Q and E49A mutants were as effective as wild-type Tus in preventing helicase action, while the E49K and K46A mutants were less effective. These results were confirmed by Mulugu et al. (127), who found that the E47Q mutant protein was an effective block to DnaB helicase action and replication forks in in vitro assays but that the E49K mutant was defective in both. These data indicate a role for some residues (most especially Glu49) in replication arrest beyond simple DNA binding, and this strengthens the case for a role of Tus-DnaB interactions. The differences between results obtained with the in vitro assays and the more complex in vivo systems presumably reflect not only the ability of Tus to block progress of the replication fork but also the efficiency with which replication restart mechanisms operate to reestablish a functional fork following its stalling and dissociation of some of the replisomal components. Replication restart mechanisms are of current interest (32, 120, 148), and these studies have been extended to the specific case of forks stalled by a Tus-Ter block (18-20, 73, 74, 77, 78, 120-122, 140, 145, 155). However, these investigations are beyond the scope of this review and are not discussed further.

Mulugu et al. (127) reported additional mutational evidence for Tus-helicase interactions. An in vivo interaction between Tus and DnaB was detected using a yeast two-hybrid system. A library of randomly mutated tus genes was then screened using a reverse two-hybrid screen for reduced binding to DnaB. Three selected colonies all yielded the same mutation, a conversion of Pro42 to Leu. This mutation resulted in a slightly increased K_D for the complex of Tus with a TerR2 oligonucleotide, and the complex dissociated more rapidly. It also had a reduced in vitro affinity for DnaB and was incapable of blocking helicase activity. Pro42 is on the surface of the protein, well away from the helicase-blocking face of the complex. It is not clear from the structure how it could directly affect Tus-helicase interactions. Three other mutations in the L1 region were also examined for effect on Tus-DnaB binding. Like P42L, the E49K mutant had reduced Tus-DnaB binding and was almost completely defective in in vitro antihelicase activity. The P52L mutant had reduced Tus-DnaB binding and somewhat reduced antihelicase activity, while the E47Q mutant had increased binding and normal activity. The reduction in antihelicase activity correlated broadly with the measured strength of binding to DnaB.

In spite of the extensive work with these selected mutant proteins, no single mutation or combination of mutations has been observed to completely eliminate the fork arrest activity of Tus while retaining its strong binding to Ter DNA. In fact, the most defective, the E49K mutant, still showed significant replication arrest activity (59, 160). Taken together, these results suggest that part of the activity of Tus resides in the strength of DNA binding and part resides in interactions with a replisomal component that is probably DnaB.

Another recent study of Tus mutants focused on residues that make specific DNA-binding contacts. Neylon et al. (131) used SPR to measure the effect of converting three of the outlying DNA-binding residues, Lys89, Arg198, and Gln250, to Ala, as well as examining the previously characterized A173T mutant. These measurements were done with buffer conditions different from those used previously, most importantly having significantly higher salt concentrations. The measured K_D of the Tus-TerB complex under these conditions (in 250 mM KCl) was about 0.5 nM, while the values for the K89A, R198A, and Q250A mutants were in the range of 90 to 220 nM, and that for the A173T mutant was 2 µM. The large increase in K_D for the A173T mutant under these conditions compared well with that reported for the same protein in a very different buffer (161).

The change in the dissociation constant for the complexes of the K89A, Q250A, and A173T mutants with TerB was due mainly to very large increases in the dissociation rate constant (131), suggesting that these residues have an important role in maintaining the complex once formed. The effect of the R198A mutation, however, was due largely to a 50-fold decrease in the association rate. This mutation had only a modest (<4-fold) effect on the dissociation rate. In addition, the R198A mutant had markedly decreased binding to (nonspecific) DNA that did not contain a Ter site. The magnitude of the change in K_D for R198A-Tus binding to nonspecific DNA was comparable to the change seen in specific Ter binding, suggesting that a large part of the effect on specific binding was due to a defect in nonspecific binding (e.g., due to the decrease in positive charge). The other mutations had no significant effect on binding to DNA that did not contain a Ter site. The effect of mutations at these residues on antihelicase activity was not reported.

View larger version (16K): [in this window] [in a new window]   FIG. 11. Tus-DNA and Tus-Ter binding. The solution form of Tus binds nonspecifically to DNA and scans along the double helix searching for a Ter site. On finding a Ter site, a series of conformational changes leads to formation of the closed Tus-Ter complex.

In summary, the mutations isolated by screening procedures (Table 1) identify several residues that are important for in vivo fork arrest activity. Most confirm the importance of particular residues in DNA binding. The effects of the remainder can be explained in terms of disturbing the structure of the protein that provides the scaffolding for DNA-binding residues. The properties of the E49K and some other L1 mutants suggest strongly that there is more to the process of replication arrest than simple DNA binding, and there is evidence from the correlation of Tus-DnaB binding and replication arrest for a role for protein-protein interactions, at least in the specific case of the Tus-Ter complex blocking the DnaB helicase. On the other hand, the differential effect of some residues on specific Tus-Ter binding as opposed to nonspecific Tus-DNA binding suggests a dynamic model of the<