In spite of the large volume of information available on Tus and its involvement as an antihelicase in replication fork blockage, there are many mechanistic aspects of the process that are uncertain. Under these circumstances it makes sense to briefly summarize the established data.
(i) The details of antihelicase activity and replication arrest appear to be strongly dependent on the identity of the Ter site, the mode of action of the helicase, and the complement of other proteins in the translocating replisome. The in vitro experiments, while they shed light on the action of the Tus-Ter complex, probably do not fully reflect the details of replication arrest and subsequent replication restart processes in vivo.
(iii) For a monomeric DNA-binding protein like Tus, a simple thermodynamic clamp cannot account for the polarity of replication fork arrest. A plausible clamp model must include kinetic or structural details to explain polarity.
(iv) There is evidence from both protein mutant and nucleotide substitution studies that the effect of some substitutions on replication arrest cannot be explained in terms of their effect on DNA binding. In particular, substitutions at Pro42, Glu49, GC6, and AT19 have a much greater negative effect on replication or helicase arrest than would be expected from their effect on DNA binding. There is a general but not absolute correlation between the strength of Tus binding to DnaB and in vitro antihelicase activity (Table ).
Prospects for the Future
The mechanism of polar replication fork arrest by the Tus-Ter complex is a problem worthy of resolution, because it represents a well-developed model system for an unusual kind of protein-DNA interaction. In what follows, we describe some of the experiments required to further define the process. The most significant question reduces to how to explain the polarity of the process. Fundamentally, and it may seem to be stating the obvious, all that is required to explain polarity is that the pathway for dissociation of Tus from its complex with a Ter site should be different depending on whether the replisome is approaching from the permissive, as opposed to the nonpermissive, face.
We note that protein oligomerization (e.g., monomer to dimer or dimer to tetramer) during DNA binding occurs in many comparable systems, including many repressor-operator interactions, and that it often occurs in a stepwise fashion. A multistep (cooperative) process is capable of solving the problem of achieving high overall binding affinity and specificity while still allowing the dissociation rate to be high enough to allow quick physiological responses (141
). In the case of a replication fork approaching Tus-TerB
from the permissive direction, a high rate of dissociation of Tus could also result from breaking the process down into a series of steps.
Polarity can also be achieved in this way in the case of multimeric proteins. For example, the B. subtilis
replication termination system consists of a series of imperfect inverted repeat Ter
sequences and a protein, RTP, that binds sequentially as a homodimer to each of two adjacent half-sites during formation of the fork-arresting complex (44
). It is clear that in the case of RTP, polarity of replication termination could be adequately explained by cooperativity in binding of the second dimer, coupled with differential affinity of binding of the protein at each of the half-sites (43
). However, even in this case, this is not the whole story (reviewed in reference 44
). It may well be that the Tus-Ter
complexes share aspects of mechanism, even though their structures do not.
Can a clamp mechanism be used to explain polarity of action of a monomeric protein-DNA complex, such as that between Tus and Ter sites? Within the class of clamp mechanisms there are a variety of possibilities. The simplest (Fig. ) is one for which the passage of a helicase through the Ter site requires the complete dissociation of Tus in a single step. With this mechanism, however, it is not possible to explain polarity. A helicase approaching either face of the complex would have to overcome the same energetic barrier to pass through. Kinetic or other aspects need to be added to explain polarity.
FIG. 14. A “complete dissociation” model of Tus action. As shown, the permissive face of the Tus-Ter complex is on the left and the nonpermissive face is on the right. DnaB approaching the permissive face for replication comes into contact with (more ...)
More complicated clamp mechanisms involve a concerted stepwise process by which the helicase moves into the Ter
site, removing Tus. The simplest form of a stepwise dissociation model, a two-step mechanism, can explain the polarity of the Tus-Ter
complex (Fig. ). Here, the binding residues are divided into two classes, residues at the permissive end of the complex and residues at the nonpermissive (fork-blocking) face. A helicase arriving from the permissive side can successfully compete with the Tus residues binding to this end of the Ter
DNA, causing a conformational change in the remaining DNA-binding residues that either removes Tus from the DNA directly or allows the helicase to further compete successfully for the remaining binding sites. When the helicase approaches from the other side, the competition between helicase and Tus is such that Tus cannot so easily be removed. The most complex model of this type would describe the conformational changes and change in binding energy resulting from the removal of each DNA-binding residue as the helicase progresses from either face: a zipper model (131
). This could be a good analogy because a zipper is itself inherently polar.
FIG. 15. A simple two-step model of Tus-Ter and DnaB interactions. (Left) DnaB approaching the permissive face of the Tus-Ter complex promotes the formation of the open, nonspecifically bound form of Tus, which may dissociate directly or slide along the DNA. If (more ...)
A variant of this model involves a progressive change in the affinity of Tus for the DNA as a result of the presence or action of the helicase. Direct helicase-Tus interaction would be one way of accomplishing this. Another would be for Tus to bind with different affinity to an intermediate forked DNA structure engineered by helicase action at either the nonpermissive or the permissive end. As we have noted earlier, there are several experimental data that support the latter possibility. It is also notable that amino acid interactions seen in the crystal structure at the permissive face are almost entirely with the strand that would pass through the central channel of the helicase (85
), and there is a cluster of basic residues (i.e., Lys119, His163, Lys245, Lys249, and His253) positioned just out of reach of the duplex DNA in the structure such that they might interact with the displaced strand at the permissive face, progressively driving further destabilization of the duplex DNA (85
). An alternate explanation comes from examination of basic residues similarly placed at the nonpermissive face (i.e., Arg145, Lys192, Lys195, and Arg205). Strand separation by the helicase could bring DNA phosphate groups into close proximity with these residues, thereby simply strengthening the Tus-Ter
The fundamental requirement of this form of model is that the pathway for dissociation of Tus from the Ter
DNA is limited. That is, there is an intermediate in the dissociation pathway that is accessible only when the helicase approaches from the permissive (or the nonpermissive) direction. A simple explanation for this behavior could be that the helicase, sitting as a cup over the fork-blocking face of Tus, physically prevents the removal of residue contacts that would ordinarily be disrupted early in the dissociation pathway. However, this would not so simply explain the polarity of action of the Tus-TerB
complex against the dimeric helicases, such as Rep (106
While the dissociation pathway is difficult to probe directly, by examining the association pathway in detail it may be possible to define intermediates that are disfavored when the replisome approaches from the nonpermissive side. Neylon et al. (131
) proposed a multistep (zipper) model for the binding of Tus to Ter
DNA based on SPR studies of Tus and Tus mutants. Minimally, the protein first binds nonspecifically to the DNA before specific interactions come into play, closing the structure and leading to deformation of the DNA (Fig. ). This is suggested by the observation that Arg198 plays an important part in nonspecific DNA binding but has a relatively minor role in determining specificity, whereas Lys89, Ala173, and Gln250 appear to be important for specific, but not nonspecific, binding. That is, some residues involved in a general nonspecific association with DNA appear to be separate from those involved in determining the sequence specificity of the interaction. In addition, the strong salt dependence of the association rate suggests that protein conformational changes take place after the initial collision step (142
The solution structure of free Tus is significantly different from the bound structure. This is suggested by circular dichroism spectroscopic data indicating a smaller proportion of β-sheet structure in the free protein (31
) than in the crystal structure (85
). Basic residues, including Arg198, are involved in the initial stages of DNA binding, forming an open nonspecific complex presumably capable of scanning DNA in search of Ter
sites. On finding a Ter
site, residues involved in sequence-specific binding, including but by no means limited to Lys89, Ala173, and Gln250, are in position to bind specifically to their ligand sites. This leads sequentially to the formation of the bound protein structure, closing the complex and deforming the DNA. This process would be expected to be highly salt dependent, resulting in extensive charge neutralization and burial of a large portion of the solvent-exposed protein and DNA surfaces.
This model can be used to explain some of the outstanding data that appear to contradict a clamp model. The presence of protection sites outside the apparent reach of the protein (Fig. ) is now predicted for a complex that is in equilibrium between a specifically bound form and a nonspecifically bound form. Furthermore, this suggests that such sites could spread farther in cases where the specific binding is weaker, such as with TerR2
, as is observed (54
). This can also explain an apparent discrepancy between results on the effects of the R198A mutation. Neylon et al. (131
) reported only a minor effect of mutagenesis on the rate of dissociation of Tus from TerB
, whereas Henderson et al. (59
) reported a 75-fold increase. This may be attributed to the difference in DNA fragments used in each case. Henderson et al. used a significantly longer DNA fragment in their measurements, and this should increase the contribution of nonspecific binding to dissociation. Neylon et al. (131
) nevertheless reported a large effect of the R198A mutation on nonspecific binding, leading to an immeasurably high dissociation rate constant in their assay (in 0.1 M KCl). The DNA length dependence of kinetic and thermodynamic parameters for site-specific DNA-protein interactions has been used in several instances to comment on the importance of nonspecific interactions (16
). These studies have been especially useful in dissecting the stepwise assembly or disassembly of site-specific DNA complexes with protein oligomers, but similar studies with Tus or other monomeric DNA-binding proteins have not been reported.
Amino acid and nucleotide substitution data can also be explained by the zipper model. Those residues that have a greater or different effect on replication arrest than is expected from the change in binding energy play a role in the kinetics of binding or dissociation. A comprehensive study of the effects of mutations in DNA-binding residues will provide more details of how stepwise binding/unbinding takes place. The solution structure of unliganded Tus would also be very helpful.
If an inaccessible intermediate on the dissociation pathway is similar to the complex between Tus and nonspecific DNA sequences, then some of the discrepancies in the results of helicase assays can also be explained. One of the main differences between assays from different research groups is the use of TerR2
as opposed to TerB
. Binding to TerR2
is significantly weaker than binding to TerB
) and may be part way between an “open” nonspecific complex and a “closed” specific Tus-TerB
complex. If this intermediate is more accessible, then helicases with different modes of action, such as those involved in replication as opposed to repair, may be expected to displace Tus more or less efficiently.
Since it seems that under some experimental conditions, the Tus-Ter
complex is capable of arresting the progress of the replicative hexameric helicases in a polar manner, but not that of the monomeric or dimeric repair (or rolling-circle) helicases (14
), it may be illuminating to consider the differences in structure and mechanism between these two classes of enzymes. Structural studies, for example, with the rolling-circle helicases E. coli
Rep and Bacillus stearothermophilus
PcrA show that the site of DNA strand separation is within a channel in the protein structures (99
). In contrast, given that the hexameric enzymes are believed to work by a strand exclusion mechanism, strand separation may occur right at the face of the oncoming helicase. The functional interactions of the two classes of helicases with Tus-Ter
might therefore be quite different: only with the hexameric helicases might strand separation influence the Tus-Ter
interaction before the progress of the helicase is physically blocked by direct collision of the proteins. Polar replication fork arrest by the hexameric helicases could then be explained by differential effects of helicase-mediated strand separation on the rate of dissociation of the Tus-Ter
complex, depending on whether strands are being separated at the permissive or the nonpermissive face. If strand separation was important in determining polarity, then polarity should not be observed in assays that measure translocation of helicases rather than authentic DNA unwinding. Such assays are technically challenging.
It is thus possible in several ways to explain the polarity of replication fork arrest in terms of a mechanism that does not necessarily involve any direct physical interaction between replisomal components and the termination complex. Nevertheless, there are other studies that suggest that such specific protein-protein interactions exist. The primary functional evidence is from Andersen et al. (6
), who showed that the Tus-Ter
complex is a much more efficient block to the replication fork in E. coli
than it is in B. subtilis
and that the converse is true, to a lesser extent, of the B. subtilis
replication termination system. This suggests that an element of the replication arrest process is specific to the Tus-Ter
complex and the E. coli
Moreover, as described above, there are other recent reports that provide both direct (127
) and indirect (59
) evidence for protein-protein interactions. The effects of two L1 mutations, E47Q and E49K, on DNA binding, replication arrest, and binding to DnaB are consistent with a role for Tus-helicase interactions, and the preferential evolutionary conservation of residues on the fork-blocking face of Tus is suggestive of interactions between Tus and the replisome. Further studies on the nature and strength of the Tus-DnaB interaction are required. We note that the conserved GC6 base pair of Ter
sites, which when mutated affects replication fork arrest more profoundly than DNA binding (30
), is positioned at the nonpermissive face of the complex close to residues in the L1 loop of Tus (including Glu49). This may signal the existence of a new kind of interaction of GC6 and L1 at some stage of a process of helicase-promoted dissociation of the Tus-Ter
There are also other replisomal components that could be involved. The τ subunit of DNA polymerase III holoenzyme is the organizational center of the replicase, coordinating and physically linking the actions of the two replication fork polymerases and DnaB (50
). In the absence of these interactions, the progress of both the helicase and the replicase is retarded (93
). It is tempting, simply on grounds of elegance, to suggest that Tus could disrupt the interaction of τ and DnaB to destabilize the replisome, leading to dissociation of DnaB. The polymerase could then continue to extend the leading strand, halting when it comes into contact with Tus. In fact, Tus might even compete with τ for its binding site on DnaB. Further interactions of Tus with replisomal components, if they were to exist, would also go some way towards explaining the discrepancies both among assays and between in vivo and in vitro results, as well as the species specificity observed by Andersen et al. (6
The issue of measurements of DNA binding is an important one. Various research groups have measured equilibrium dissociation constants and association and dissociation rate constants in different experimental systems. In most cases only binding to specific Ter DNA fragments has been examined. The lengths and sequences of these fragments also vary among laboratories. If, as suggested, binding to nonspecific and Ter DNA involves different groups of residues, and if these play different roles in the replication arrest process, then the differences among these assays create a significant problem in interpretation of data.
More work clearly needs to be done. The tools to examine and dissect protein-protein interactions or DNA secondary structure are available and should be brought to bear on the problem. Detailed kinetic studies of the competition between Tus and DnaB for the DNA, combined with cross-linking experiments, should give insights into the process of replication arrest. Detailed and comprehensive examination of the effects of mutations on the association and dissociation processes will provide further clues to the events preceding the removal of Tus or the helicase from the DNA. Attention should also be refocused on the approach of DnaB from the permissive end of the complex. The process by which DnaB removes Tus from the DNA has received little, if any attention despite the fact that understanding the polarity of antihelicase action depends critically on understanding how the helicase overcomes the barrier when translocating in this direction.
Structural studies of the free protein and of the open protein-DNA and closed full-size Tus-Ter complexes would provide a helicase-eye view of the complex as it approaches. The dynamics of the Tus-TerB complex versus the Tus-TerR2 complex may provide important clues to the factors that lead to the experimentally observed differences between them. Simulations could provide clues to the molecular dynamics that occur within the various complexes. Another important requirement is a detailed examination of the effects of the protein complement on in vitro replication and helicase assays. It is clear that some of the elements of the in vivo process may be missing from the in vitro assays.
Further molecular dissection of the Tus-Ter
complex, the helicase-DNA complex, the replisome, and the interactions among them can be expected ultimately to unravel the details of this fascinating process. Finally, we note in passing that polar binding protein-mediated replication fork arrest is not restricted to prokaryotic replicons. Study of similar processes in yeast and mammalian systems is under way (see, for example, references 52
, and 124
and references therein).