T4 Replication: What Does ''Processivity'' Really Mean?

COMMENTARY

T4 replication: What does ‘‘processivity’’ really mean?

Catherine M. Joyce* Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520

NA polymerases are full of surprises. It is widely ac- cepted that replicative polymerases are highly processive, thanks,D in large part, to accessory subunits that act as sliding clamps and tether the polymerase to its DNA template. The bacteriophage T4 replication system is typical in that processivity measurements indicate that a substantial fraction of replication complexes should remain associated with DNA for the Ϸ15 min required to copy an entire genome length (1). However, an elegant study by Yang et al. (2) in this issue of PNAS challenges our notion of proces- Fig. 1. Simpliﬁed representation of the proteins at the T4 replication fork. The replisome contains two sivity by showing that, within the highly DNA polymerase subunits (gp43), one for the leading and one for the lagging strand, each with its own processive T4 replisome, individual sliding clamp (gp45), and with a clamp loader (gp44͞62 complex) associated with the lagging strand DNA polymerase molecules are ex- polymerase. Helicase (gp41) drives the replication fork and is associated with primase (gp61). Not shown changing rapidly, maybe 90 times per are gp59, which functions to load helicase onto the fork, and gp32, which binds to single-stranded DNA genome length under the conditions generated during replication. The RNA primers that initiate DNA synthesis on the lagging strand are prevailing in vivo. Thus, if the replisome shown in red (not to scale). The components and assembly pathway of the T4 replisome have been is visualized as a molecular factory, the established from work in many laboratories, exempliﬁed by refs. 3 and 4. workers are making frequent shift changes. which are catalytically inactive but oth- minute, implying that, despite their Processivity Measurements erwise unchanged in their ability to bind apparent high processivity, both poly- to DNA or other replisome components. merases were available to exchange with The bacteriophage T4 replisome, on Such mutant proteins can bind to their the inactive mutant. Control experi- which these studies were carried out, normal target and then inhibit the ments ruled out indirect effects caused serves as a simple model incorporating replisome. This approach confirmed the by the mutant polymerase interfering the essential features of multisubunit results from dilution experiments by with other components of the replisome. replication systems. The components of showing a rapid shutdown of lagging the T4 system are illustrated in cartoon Polymerase Exchange Mechanisms form in Fig. 1. The high overall proces- strand replication when inactive forms sivity of the replisome does not neces- of the primase or clamp loader were The paradoxical aspect of the trapping sarily mean that all subunits are equally introduced (6). experiment is that the onset of inhibi- processive, and two approaches have In this issue of PNAS, Yang et al. (2) tion by the inactive polymerase was been informative in measuring the life- describe the surprising results obtained faster than dissociation of polymerase times of individual components within when investigating the lifetime of the from the replisome (as determined by an actively synthesizing replisome. The DNA polymerase (gp43) in the T4 repli- the dilution experiment). The kinetics of simplest is to determine the sensitivity some. In a dilution experiment, where trapping can be explained by an ‘‘active of DNA synthesis to dilution of individ- the final concentration of the polymer- exchange’’ mechanism in which the trap ual components. This approach has ase was too low to permit rebinding af- polymerase in some way assists the dis- demonstrated that the helicase (gp41) is ter dissociation, there was only a very placement of the replicating polymerase. very stably associated with the replica- gradual decrease in both leading and This interpretation makes biological tion fork (1). By contrast, lagging strand lagging strand synthesis, indicating that sense because it ensures that a replicat- synthesis was sensitive to dilution of the the two polymerase subunits, like heli- ing polymerase will not dissociate from clamp (gp45), clamp loader (gp44͞62 case, apparently remain associated with the replisome unless a replacement poly- complex), and primase (gp61) (5, 6), the replisome for synthesis of all or merase is ready to take over, avoiding reflecting the remodeling of the lagging most of the phage genome. A reason- spontaneous loss of the replicating strand machinery that must take place able prediction from this result is that polymerase and the need to recruit a as each new Okazaki fragment is replication should be inhibited very little replacement from bulk solution. started. Significantly, dilution of the by an inactive polymerase mutant acting A possible mechanism for the binding polymerase (gp43) did not affect lagging as a protein trap, because dissociation of additional polymerase molecules to strand synthesis, implying that it remains of the replicating polymerase, and the replisome is suggested by a key ob- associated with the replisome through- exchange with the inactive trap, should servation of Yang et al. (2) that a T4 out the synthesis of multiple Okazaki be very infrequent. Surprisingly, the op- DNA polymerase mutant with a small fragments. posite result was obtained. Addition of a A second approach to investigate the gp43 active site mutant to an ongoing processivity of components of the repli- replication reaction caused rapid inhibi- See companion article on page 8289. some is the use of protein traps, mutant tion of both leading and lagging strand *E-mail: [email protected]. forms of the subunit under investigation, synthesis within approximately one © 2004 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0402850101 PNAS ͉ June 1, 2004 ͉ vol. 101 ͉ no. 22 ͉ 8255–8256 Downloaded by guest on October 3, 2021 C-terminal deletion did not act as a ase to a specialized lesion bypass Dynamic Processivity trap. The C-terminal deletion, which polymerase. One can envisage a An important message from the article prevents binding to the sliding clamp straightforward targeting process. Stall- by Yang et al. (2) is the need to adopt a but does not otherwise affect polymer- ing of the replicative polymerase at a more nuanced view of the concepts of ase-DNA binding (7), removes a con- site of DNA damage could enhance re- dissociation and processivity and to rec- served sequence, present on several lease of the primer terminus for capture ognize that different experimental ap- other polymerases, that has been shown by a specialized lesion bypass polymer- proaches may yield apparently contra- to interact with the clamp (8–10). Yang dictory answers because they interrogate et al. therefore infer that the exchange different steps in the process of dissocia- of polymerase subunits in the T4 repli- The exchange of tion. The loss of a subunit from a large some is mediated by means of the trim- assembly is likely to be a multistep pro- eric gp45 sliding clamp, and they present polymerase subunits cess, with early steps involving the re- two structural models to explain how lease from contacts with immediate the sliding clamp may bind both the rep- in the T4 replisome is neighbors and leading eventually to licating polymerase and a ‘‘spare’’ poly- complete escape of the subunit into the merase that is available to replace it. mediated by the gp45 surrounding medium. Dilution experi- Regardless of the precise details of the ments address dissociation in a macro- binding geometry, sliding clamps, being sliding clamp. scopic sense because they measure cap- multimeric, clearly have the potential to ture of the target subunit from the bulk provide additional binding sites for asso- solution. On the other hand, protein- ciated proteins. The active exchange in- ase. If the bypass polymerase has a trapping experiments may probe an ear- ferred from the kinetics of inhibition in lower intrinsic affinity for the primer lier stage provided that, as in the the trapping experiment could result terminus than does the replicative poly- present case, the protein trap has access from an allosteric destabilization of the merase, this scenario would facilitate to the macromolecular complex. Thus, replicating polymerase caused by bind- exchange subsequent to lesion bypass, the T4 polymerase trapping experiment ing of a polymerase to a second site on thus avoiding the synthesis of long tracts reveals not only the dynamics of the the sliding clamp. Alternatively, the rep- of DNA by the less accurate bypass DNA polymerase subunits within the T4 licating polymerase may transiently let polymerase. It also has been suggested replisome but also the availability of go of the DNA in the normal course of that transient release of DNA by a binding sites for additional polymerase molecules. synthesis, and the spare polymerase, by replicating polymerase could relieve How dynamic then is the T4 repli- virtue of its proximity, would be well torsional stress at the replication fork some? It is well established that lagging placed to take advantage of this. Tran- and could facilitate transfer of a mis- strand components, other than the poly- sient release of the primer terminus by paired primer terminus to the editing merase, are recruited continuously from the polymerase is to be expected based site (9, 12). solution. The current study by Yang et on the rather low processivity of T4 Recent work suggests that the Esche- al. (2) shows that the polymerase is ex- gp43 (and other replicative polymerases) richia coli sliding clamp can bind a large changeable within the replisome com- in the absence of accessory factors (11). number of replication proteins, includ- plex but only rarely leaves the complex The enhanced processivity conferred by ing all five bacterial polymerases, the if a replacement polymerase is not avail- the sliding clamp derives not from pre- clamp loader, and DNA ligase (8). All able. The helicase appears to be the venting the initial release of the primer of these interactions use the same site most solidly fixed component and has terminus but from tethering the poly- on the clamp, and many, although not been suggested to be the ‘‘cornerstone’’ merase and thus increasing the probabil- all, involve the conserved C-terminal of the replisome (1). Although the high ity of rebinding to the primer terminus. peptide mentioned earlier. These find- processivity of the helicase has not ings reinforce the idea of the sliding been challenged by a protein-trapping Biological Implications clamp as a ‘‘tool belt’’ carrying addi- approach, it is difficult to imagine a bio- A dynamic relationship between the tional replication proteins whose activi- logical rationale for helicase exchange polymerase subunit and the replisome ties may be required under specific or a mechanism by which it could take may be advantageous in a variety of bio- circumstances (13). Given the limited place. Finally, the many analogies be- logical situations. The most obvious is in number of potential binding sites on tween the T4 replication system and the replication past a blocking lesion on the the sliding clamp, frequent exchange of more complex bacterial and eukaryotic template. The exchange mechanism pro- the tools (or multiple tool belts) may be systems suggest that the dynamic notion posed by Yang et al. (2) provides a necessary to ensure the availability of all of polymerase processivity put forward means of transferring the primer termi- of the appropriate enzymatic activities by Yang et al. should be considered in nus from a stalled replicative polymer- when needed. these systems too.

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