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Translesion DNA Polymerases Remodel the Replisome and Alter the Speed of the Replicative Helicase

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Translesion DNA Polymerases Remodel the Replisome and Alter the Speed of the Replicative Helicase Translesion DNA polymerases remodel the replisome INAUGURAL ARTICLE and alter the speed of the replicative helicase Chiara Indiania, Lance D. Langstona, Olga Yurievaa, Myron F. Goodmanb, and Mike O’Donnella,1 aLaboratory of DNA Replication, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, Box 228, New York, NY 10065; and bDepartments of Biological Sciences and Chemistry, University of Southern California, Los Angeles, CA 90089 This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2006. Contributed by Mike O’Donnell, February 11, 2009 (sent for review January 22, 2009) All cells contain specialized translesion DNA polymerases that very slow to rise, peaking 45 min after SOS induction (4, 9, 11, replicate past sites of DNA damage. We find that Escherichia coli 12). In vitro studies have shown that these DNA polymerases translesion DNA polymerase II (Pol II) and polymerase IV (Pol IV) function with the ␤-clamp for lesion bypass and trade places on function with DnaB helicase and regulate its rate of unwinding, ␤ with a stalled Poll III replicase, as originally demonstrated in slowing it to as little as 1 bp/s. Furthermore, Pol II and Pol IV freely bacteriophage T4 (14–17). Once the lesion is bypassed, the exchange with the polymerase III (Pol III) replicase on the ␤-clamp high-fidelity Pol III can switch back on the ␤-clamp to reestablish and function with DnaB helicase to form alternative replisomes, the fast moving E. coli replisome (650 nt per s) (14, 17). In this even before Pol III stalls at a lesion. DNA damage-induced levels of view of translesion synthesis, the TLS polymerase only occupies Pol II and Pol IV dominate the clamp, slowing the helicase and the ␤-clamp during the short time interval of lesion bypass. stably maintaining the architecture of the replication machinery It seems unlikely that TLS polymerases would be able to while keeping the fork moving. We propose that these dynamic replace Pol III in the context of a moving replisome, because actions provide additional time for normal excision repair of TLS polymerases are very slow and have low fidelity. These lesions before the replication fork reaches them and also enable characteristics are inconsistent with the rapid speed and high the appropriate translesion polymerase to sample each lesion as it fidelity required for chromosome replication. It is possible that is encountered. TLS polymerases are excluded from replisomes by the tight BIOCHEMISTRY connection between Pol III and the DnaB helicase via the DNA repair ͉ DNA replication ͉ replication fork ͉ replisome sliding clamp ␶-subunit. Additional insurance against TLS polymerase entry into the replisome is the known action of DnaB helicase when it hromosomes are duplicated by replisome machines contain- becomes uncoupled from Pol III, whereupon it continues for Ϸ Cing helicase, primase, and DNA polymerase activites (1). only 1 kb before DnaB stops unwinding (18–20). Presumably, Replicative DNA polymerases are tethered to DNA by ring- DnaB dissociates from DNA when it is uncoupled from leading- shaped sliding clamp proteins. The Escherichia coli polymerase strand Pol III action. Once DnaB dissociates, it cannot get back III (Pol III) holoenzyme replicase contains 10 different subunits onto DNA without specialized helicase-loading factors, and consisting of a clamp loader that binds 2 molecules of Pol III, replication fork progression is halted. Therefore, even if a TLS each tethered to DNA by the ␤-sliding clamp for processive polymerase enters the replisome, the subsequent loss of Pol III leading and lagging strand synthesis (2). Pol III also binds tightly interaction with DnaB may result in DnaB dissociation, prevent- to the hexameric DnaB helicase that encircles the lagging strand ing further progression of the fork. template. The Pol III-to-DnaB interaction is mediated by the Fork breakdown upon takeover by Pol IV is supported by ␶-subunit within Pol III (3). This tight connection couples Pol III recent studies that show that overexpression of Pol IV in the motion to DnaB unwinding and increases the rate of unwinding absence of DNA damage inhibits cell growth in Bacillus subtilis by DnaB helicase from a basal level of 35 bp/s to a rate in excess and E. coli (21, 22). The authors of the E. coli study (21) note that of 500 bp/s (3). These tight connections of Pol III to DnaB and induction of Pol IV stops replication abruptly and kills the cell, the ␤-clamp provide the replisome with sufficient stability, in suggesting that Pol IV entry into the replisome is catastrophic. principle, to replicate the entire genome. In practice, however, Interestingly, these observations did not require the clamp replication of long chromosomal DNA is an uneven path with a binding residues of Pol IV, suggesting that Pol IV blocks variety of obstacles along the way, including protein blocks and replication by a process distinct from TLS polymerase switching damaged templates. on a clamp. However, Pol IV was overexpressed to abnormally In the presence of high levels of DNA damage, Ͼ40 gene high levels in that study. Thus, the mechanism of the replication products are induced by the SOS response that act to restore block by Pol IV was suggested to be separate from the physio- genomic integrity and assure cell survival (4). Among the logical action of the enzyme. Lower levels of Pol IV expression SOS-induced gene products are 3 translesion DNA polymerases, appeared to decrease replication without cell killing, suggesting Pols II, IV, and V (encoded by polB, dinB, and umuCD, Pol IV may slow a replication fork, but this hypothesis could not respectively), that perform potentially mutagenic DNA synthesis be confirmed and was not tested in vitro. across template lesions (5). Pols IV and V are members of the In the current study we examine the effects of normal and Y family of error-prone DNA polymerases; they lack a proof- induced levels of Pol II and Pol IV on moving replisomes reading 3Ј–5Ј exonuclease (6, 7). Pol II is a B-family polymerase; it has high fidelity and contains proofreading activity (8). Pols II Author contributions: C.I., L.D.L., and M.O.D. designed research; C.I., L.D.L., and O.Y. and IV are present during normal cell growth and may be performed research; M.F.G. contributed new reagents/analytic tools; C.I., L.D.L., M.F.G., involved in repairing low levels of DNA damage that occur and M.O.D. analyzed data; and C.I., L.D.L., and M.O.D. wrote the paper. routinely in the cell (9–11). Pol V, however, is closely associated The authors declare no conflict of interest. with mutagenesis and is not detectable in cells under normal Freely available online through the PNAS open access option. conditions (7, 12, 13). 1To whom correspondence should be addressed. E-mail: [email protected]. Pol II and Pol IV are among the first genes that are up- This article contains supporting information online at www.pnas.org/cgi/content/full/ regulated in the SOS response, whereas the levels of Pol V are 0901403106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0901403106 PNAS Early Edition ͉ 1of8 Downloaded by guest on September 25, 2021 Fig. 1. Pol II and Pol IV form alternative replisomes with DnaB helicase and the ␤ clamp. (A) Minicircle DNA was incubated with DnaB, then TLS polymerase (5 pmol), clamp loader, clamp and dCTP, dGTP for 5 min. Replication was initiated by addition of SSB, DnaG primase, dATP, dTTP, [␣32P]dTTP, and the 4 rNTPs. Newly-synthesized leading strand DNA is in red, and lagging strand is in blue. As controls, either DnaB or clamp/clamp loader were excluded from the reaction. (B and C) Time courses of Pol II (B) or Pol IV (C) in the presence of DnaB, the ␤-clamp, and the clamp loader (lanes 1–6), in the absence of clamp and clamp loader (lanes 7–12), and in the absence of DnaB (lanes 13–18). Products were analyzed in alkaline agarose gels. (D) Pol II (15 pmol) and Pol IV [5 pmol (Center) or 7 pmol (Right)] were added simultaneously to the reaction. (Left) The gel shows synthesis by Pol II in the absence of Pol IV for comparison. reconstituted in vitro. We find that these TLS polymerases can addition of Pol II or Pol IV in the presence of only dCTP and gain access to the moving Pol III replisome despite the strong Pol dGTP to prevent fork movement. After 5 min, replication was III–␶–DnaB connection. Surprisingly, the fork does not break initiated upon adding dATP, dTTP, SSB, DnaG primase, and down. Instead the TLS polymerases function with DnaB to form the 4 rNTPs (see Fig. 1A). In these assays, dissociation of DnaB replisomes that move more slowly than the intrinsic rate of from DNA will halt the fork because SSB is present and the assay DnaB, with the rate of helicase unwinding adjusting to the speed does not contain DnaB-reloading factors. Replication fork pro- of the polymerase at the fork. At the levels of Pol II and Pol IV gression is followed by analysis of timed aliquots in an alkaline present in undamaged cells, the Pol III replisome is unaffected, agarose gel. The results are compared with similar reactions whereas at levels of Pol II and Pol IV that correspond to their performed in the absence of DnaB helicase or the absence of the concentrations in DNA damage-induced cells, the TLS poly- clamp and clamp loader. merases dominate the replication fork and slow it down. Ex- Surprisingly, the analysis shows that Pol II and Pol IV function pression of Pol II and Pol IV in vivo in the absence of DNA with DnaB and the ␤-clamp, because the presence of both of damage also slows DNA replication and depends on TLS these factors is required to synthesize long DNA chains (Fig.
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