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Protein–DNA Complexes Are the Primary Sources of Replication Fork Pausing in Escherichia Coli

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Protein–DNA Complexes Are the Primary Sources of Replication Fork Pausing in Escherichia Coli Protein–DNA complexes are the primary sources of replication fork pausing in Escherichia coli Milind K. Guptaa, Colin P. Guya, Joseph T. P. Yeelesb, John Atkinsona, Hazel Bella, Robert G. Lloydc, Kenneth J. Mariansb, and Peter McGlynnd,1 aSchool of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom; bMolecular Biology Program, Memorial Sloan–Kettering Cancer Center, New York, NY 10065; cCentre for Genetics and Genomics, Queen’s Medical Centre, University of Nottingham, Nottingham NG7 2UH, United Kingdom; and dDepartment of Biology, University of York, York YO10 5DD, United Kingdom Edited by Mike E. O’Donnell, The Rockefeller University, Howard Hughes Medical Institute, New York, NY, and approved March 25, 2013 (received for review February 28, 2013) Replication fork pausing drives genome instability, because any pausing remain unknown. In particular, although both transcrip- loss of paused replisome activity creates a requirement for tion complexes and DNA damage present known challenges to reloading of the replication machinery, a potentially mutagenic fork movement in bacteria and eukaryotes (15, 19–21), the rel- process. Despite this importance, the relative contributions to ative frequencies with which they affect fork progression in vivo fork pausing of different replicative barriers remain unknown. is not known. This is in part because physical detection of fork We show here that Deinococcus radiodurans RecD2 helicase inac- pausing is only possible when such pausing occurs at specific tivates Escherichia coli replisomes that are paused but still func- locations in the genome and is sufficiently frequent and/or pro- tional in vitro, preventing continued fork movement upon barrier longed to facilitate detection. However, many pausing events removal or bypass, but does not inactivate elongating forks. Us- might not meet these criteria. Furthermore, if a paused fork ing RecD2 to probe replisome pausing in vivo, we demonstrate subsequently resumes translocation, either upon removal or by- that most pausing events do not lead to replisome inactivation, pass of the barrier, there would be no easily detectable phenotypic that transcription complexes are the primary sources of this consequences. pausing, and that an accessory replicative helicase is critical for Here we demonstrate that Deinococcus radiodurans RecD2 minimizing the frequency and/or duration of replisome pauses. helicase inactivates paused but not elongating Escherichia coli BIOCHEMISTRY These findings reveal the hidden potential for replisome inacti- replisomes in vitro. The basis of this inactivation is unknown, but vation, and hence genome instability, inside cells. They also this specificity provides a tool to probe the relative frequencies of demonstrate that efficient chromosome duplication requires replisome pausing in vivo. Wild-type Escherichia coli can survive mechanisms that aid resumption of replication by paused repli- expression of RecD2 but chromosomal DNA content is per- somes, especially those halted by protein–DNA barriers such as turbed significantly, indicating that replisomes do pause fre- transcription complexes. quently in vivo. Cells lacking a helicase, Rep, that clears protein– DNA barriers ahead of forks (10, 22) are hypersensitive to DNA repair | genome stability | Rep | RNA polymerase | recombination RecD2 expression. In contrast, defects in base or nucleotide excision repair do not render RecD2 toxic. These data indicate aithful duplication of the genome is a key challenge to all that protein–DNA complexes, not template damage, are the Forganisms, and overcoming barriers to the timely progression primary sources of replisome pausing in nonstressed cells and of replication forks is a major part of this challenge. Template that most replicative barriers result in fork pausing but not in- damage, proteins bound to the DNA, and non–B-form DNA activation. Thus, although there is a very considerable potential structures all have the potential to halt movement of the repli- for triggering genomic instability during every cell cycle, this cation machinery (1–3). If forks pause at such barriers and lose potential is only rarely realized because of the intrinsic stability function, then replisome reloading, often via blocked fork pro- of the replisome and the ability of a secondary motor to help cessing by recombination enzymes, is required to resume genome drive forks along protein-bound DNA. Together, these two duplication (4), an error-prone process associated with gross factors minimize the likelihood of fork pausing leading to chromosomal rearrangements (5–7). However, pausing of repli- replisome inactivation and the need to restart replication. somes does not necessarily lead to fork breakdown, because paused replisomes can continue duplication upon removal or Results bypass of the block (8–11). The balance between resumption of RecD2 Inhibits Resumption of Replication by Paused Replisomes. ′ ′ replication versus breakdown of paused forks is therefore a crit- Superfamily 1 helicases that translocate 3 -5 along ssDNA E. coli Bacillus stearothermophilus ical factor in the maintenance of genome stability. ( Rep and UvrD and PcrA) E. coli Forks halted by nucleoprotein complexes can resume repli- promote movement of replisomes through nucleoprotein D. radiodurans cation upon spontaneous dissociation of the nucleoprotein complexes, whereas RecD2 and bacteriophage T4 ′ ′ complex or active removal of the block by the replisome itself or Dda, 5 -3 superfamily 1 helicases do not (10). Indeed, addition accessory motors acting at the fork (9, 10, 12–14). Similarly, of RecD2 results in an apparent increase in the degree of rep- – a replisome encountering a lesion that inhibits synthesis by one lication blockage at protein DNA complexes in vitro (10). We of the polymerases may continue replication downstream of the lesion by repriming DNA synthesis. Such repriming occurs re- gardless of whether the damage is on the leading or the lagging Author contributions: C.P.G., J.T.P.Y., K.J.M., and P.M. designed research; M.K.G., C.P.G., J.T.P.Y., J.A., H.B., and P.M. performed research; R.G.L. and K.J.M. contributed new re- strand template, although recombination may be triggered at the agents/analytic tools; M.K.G., C.P.G., J.T.P.Y., J.A., K.J.M., and P.M. analyzed data; and gap left in the nascent strand (11, 15, 16). P.M. wrote the paper. Whether a replisome pauses at a barrier before resuming The authors declare no conflict of interest. replication or loses activity is determined by the rates of barrier This article is a PNAS Direct Submission. clearance/bypass and blocked replisome stability (17, 18). How- 1To whom correspondence should be addressed. E-mail: [email protected]. ever, despite the critical importance of fork pausing for genome This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. stability, the contributions of different types of barrier to replisome 1073/pnas.1303890110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1303890110 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 investigated the basis for this apparent increase in fork blockage a general feature of this class of helicases rather than an activity by RecD2. We used a system in which replisomes could be associated specifically with RecD2. blocked completely by a large array of lac repressor–operator complexes and then the block relieved by subsequent addition of RecD2 Inactivates Paused but Not Elongating Replisomes. Inhibition isopropyl β-D-1-thiogalactopyranoside (IPTG), allowing moni- of resumption of fork movement upon dissociation of the pro- toring of the ability of blocked replisomes to resume replication tein–DNA block could be explained by RecD2-catalyzed in- (18)(Fig. 1A and Fig. S1). Replisomes were reconstituted on activation of paused replisomes, of elongating replisomes, or plasmid templates bearing oriC and 22 tandem lac operators. In both. To distinguish between these possibilities we assessed the the absence of a topoisomerase, replisomes could proceed only impact of RecD2 on the activity of elongating and of paused a limited distance along the template owing to accumulation of forks in two parallel experiments. First, the effects of wild-type positive supercoiling. Continued fork movement depended on and pinless RecD2 were analyzed on elongating replication forks cleavage of the template by a restriction enzyme to relieve the whose movement around a supercoiled plasmid template lacking A i topological strain (17) (Fig. 1A, ii and iii and Fig. S1) with the engineered barriers was sustained by DNA gyrase (Fig. 2 , ). site of cleavage allowing only one of the two forks to progress Addition of either wild-type or pinless RecD2 alongside the lac A iii iv replication initiator DnaA had no significant impact on levels of toward the operators (Fig. 1 , and ). A ii In the absence of lac repressor, replication generated a pop- DNA synthesis by elongating forks (Fig. 2 , ). Second, repli- ulation of lagging strands of ∼0.5 kb and leading strands of 1.3 cation of the same supercoiled plasmid template was initiated by A vi B DnaA in the absence of a topoisomerase as in Fig. 1, resulting in and 5.2 kb (Fig. 1 , and , lane 1). In the presence of re- B i pressor, 3.5-kb, rather than 5.2-kb, leading strands were gener- fork stalling owing to topological strain (Fig. 2 , ). Subsequent addition of a restriction enzyme relieved the strain and allowed ated (Fig. 1A, vi and B, lane 2 and Fig. S1), as expected if paused forks that retained function to continue (Fig. S1). movement of replisomes was inhibited within the repressor– However, addition of wild-type, but not pinless, RecD2 alongside operator array. Subsequent addition of IPTG to these blocked the restriction enzyme inhibited subsequent DNA synthesis (Fig. forks relieved this inhibition (Fig. 1B, lane 3), reflecting the 2 B, ii). RecD2 helicase activity results, therefore, in inactivation initial retention of activity of replisomes blocked at lac repressor– of forks paused by positive supercoiling.
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