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Fate of the Replisome Following Arrest by UV-Induced DNA Damage in Escherichia Coli

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Fate of the Replisome Following Arrest by UV-Induced DNA Damage in Escherichia Coli Fate of the replisome following arrest by UV-induced DNA damage in Escherichia coli H. Arthur Jeiranian, Brandy J. Schalow, Charmain T. Courcelle, and Justin Courcelle1 Department of Biology, Portland State University, Portland, OR 97201 Edited by Mike E. O’Donnell, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, and approved May 24, 2013 (received for review January 18, 2013) Accurate replication in the presence of DNA damage is essential to allowing the machinery to overcome specific challenges such as genome stability and viability in all cells. In Escherichia coli, DNA collisions with the transcription apparatus or DNA-bound pro- replication forks blocked by UV-induced damage undergo a partial teins (1, 12, 13). In this study, we used thermosensitive replication resection and RecF-catalyzed regression before synthesis resumes. mutants to characterize how the composition of the replisome These processing events generate distinct structural intermediates changes following encounters with UV-induced photoproducts, a on the DNA that can be visualized in vivo using 2D agarose gels. biologically relevant lesion that is known to block the progression However, the fate and behavior of the stalled replisome remains of the replisome when located in the leading strand template (6, a central uncharacterized question. Here, we use thermosensitive 14–16). The results demonstrate that the DNA polymerases can mutants to show that the replisome’s polymerases uncouple and dissociate from the replisome in a modular manner without transiently dissociate from the DNA in vivo. Inactivation of α, β,or compromising the integrity of the replication fork. Dissociation of τ subunits within the replisome is sufficient to signal and induce the DNA polymerase from the replisome is sufficient and can the RecF-mediated processing events observed following UV dam- serve to initiate the processing of the replication fork DNA via the age. By contrast, the helicase–primase complex (DnaB and DnaG) RecF pathway, similar to that seen when replication is arrested remains critically associated with the fork, leading to a loss of fork by UV-induced damage. By comparison, the helicase complex integrity, degradation, and aberrant intermediates when disrupted. remains associated with the replication fork throughout the re- The results reveal a dynamic replisome, capable of partial disassem- covery process. If the helicase is disrupted, aberrant intermediates, bly to allow access to the obstruction, while retaining subunits that degradation, and loss of fork integrity ensue. We propose that the CELL BIOLOGY maintain fork licensing and direct reassembly to the appropriate retention of the helicase is needed to maintain licensing for the location after processing has occurred. replication fork and direct reassembly to the appropriate location after processing has occurred. replication fork processing | RecF pathway Results he replisome consists of several, multisubunit protein com- Dissociation of the Polymerase from the Replisome Is Sufficient to Tplexes and is responsible for duplicating the genome. In Induce Processing Events Similar to Those Observed After UV-Induced Escherichia coli, it is comprised of three DNA polymerase com- Arrest. A schematic of each of the components of the replisome A plexes tethered to the DNA template by dimeric processivity tested in this study and their function is presented in Fig. 1 . factors, a τ complex that couples leading and lagging strand Temperature-sensitive mutants exist in subunits from each of – replisome’s complexes for which viability or functionality is sup- synthesis, and a helicase primase complex that separates the B duplex DNA and primes lagging strand synthesis (1–3). ported at 30 °C, but not at 42 °C (Fig. 1 ). Although replica- When the replisome encounters DNA damage that blocks its tion proceeds normally at the permissive temperature, it rapidly progression, the potential for mutagenesis, rearrangements, and decreases following inactivation of the thermosensitive protein at fi the restrictive temperature, similar to that seen after UV irradi- lethality increases signi cantly. Replication in the presence of C DNA damage can generate mutations if the wrong base is in- ation (Fig. 1 ). The exception to this is in the proofreading sub- unit e, encoded by dnaQts, which is mutagenic at the restrictive corporated, rearrangements if it resumes from the wrong site, temperature, but is not essential for viability or replication (17). or lethality if the obstructing lesion cannot be overcome. Follow- To determine how the replisome behaves or is modified fol- ing the arrest of replication at UV-induced damage, the nascent lowing encounters with DNA damage in vivo, these thermo- lagging strand is partially resected by the combined action of the sensitive mutants were used to deliberately disrupt the specific RecQ helicase and RecJ nuclease (4, 5). RecF-O-R, along with components of the replisome. We then compared the effect that RecA, limit this degradation and promote a transient regression the loss of that component had on replication processing to that of the DNA branch point, which is thought to be important for seen when replication encounters DNA damage. Replication and restoring the damaged region to a form that can be acted on by – processing intermediates were visualized in vivo on replicating repair enzymes or translesion DNA polymerases (4 10). These fragments of the plasmid pBR322 (Fig. 2). This plasmid replicates processing events generate distinct structural intermediates on using the host’s replication machinery, it can be linearized at its the DNA that can be readily visualized using 2D agarose gel single origin of replication, and it maintains a moderate copy analysis, a technique that allows one to identify the shape and number, making it useful to detect rare events such as replication structure of DNA molecules (5, 11). through a defined fragment (18). In this type of analysis, actively Although the processing that occurs on the DNA is well char- acterized, little is known about the behavior or composition of the replisome itself during these events. If the replisome remains Author contributions: H.A.J., B.J.S., C.T.C., and J.C. designed research; H.A.J., B.J.S., C.T.C., bound to the arresting lesion, it may sterically obstruct repair or and J.C. performed research; H.A.J., B.J.S., C.T.C., and J.C. analyzed data; and H.A.J. wrote bypass from occurring. Conversely, complete dissociation of the the paper. replisome would likely abolish the licensing for the replication The authors declare no conflict of interest. fork and expose DNA ends that have the potential to recombine, This article is a PNAS Direct Submission. generating deletions, duplications, or rearrangements on the 1To whom correspondence should be addressed. E-mail: [email protected]. chromosome. Recent studies in vitro have suggested that dynamic This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. interactions between replisome components may play a role in 1073/pnas.1300624110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1300624110 PNAS Early Edition | 1of6 A B β are required to maintain DNA polymerase binding to the DNA template (19, 20). The processing intermediates were induced Polymerase and Clamp WT specifically by the disruption of the DNA polymerase III sub- Helicase- dnaX dnaB Primase C units, because no processing intermediates were induced in wild- dnaG Complex 30 dnaN type cells at the restrictive temperature or following inactivation of the nonessential proofreading subunit, e (DnaQ) (Fig. 2 and 6 dnaE dnaQ Fig. S1). Similar to the DNA polymerase subunits, UV-like processing DnaB WT intermediates were also induced following inactivation of τ DnaG dnaX dnaB (DnaX), which is responsible for coupling the polymerases to the C replisome, coordinating leading and lagging strand synthesis, and Coupling- 42 dnaN dnaG clamp loading repetitive cycling of the processivity factor onto the lagging complex dnaE dnaQ strand template (21–25). The observed induction of UV-like C intermediates following inactivation of the DNA polymerase or UV Irrad Shift Temperature to 42 C coupling factor indicates that the UV-induced processing of replication forks is likely to involve the transient dissociation of 1.0 the polymerase from the DNA. dnaQ WT WT The helicase–primase complex interacts with τ and the holo- Post-UV Post-TS dnaG 0.1 enzyme and tracks along the lagging strand template, serving to dnaE – α β dnaN unwind and prime it during replication (26 28). In contrast to , , dnaX τ Rate of DNA Synthesis dnaB or , inactivation of either the helicase (DnaB) or primase (DnaG) -20 0 20 40 60 80 -20 0 20 40 60 80 -20 0 20 40 60 80 -20 0 20 40 60 80 -20 0 20 40 60 80 led to the production of aberrant structural intermediates that Minutes Post Treatment were unlike any of those that are observed during the pro- Fig. 1. Replication is disrupted by UV-induced damage or following inac- cessing of UV-induced damage (Fig. 2 and Fig. S1), arguing that tivation of the DNA polymerase, τ complex, or helicase–primase complex. (A) disruption of the helicase complex does not normally occur A diagram of the replisome, indicating the subunits of each protein complex. during the recovery of replication after UV-induced arrest. (B) Thermosensitive mutants that inactivate the polymerase core, τ complex, or helicase complex are viable at 30 °C but fail to grow at the restrictive Similar to UV, the Integrity of the Replication Fork DNA Is Maintained temperature of 42 °C following overnight incubation. (C) The rate of DNA After Polymerase Dissociation, and Replication Fork Processing Is synthesis is inhibited following UV-induced damage or inactivation of the Catalyzed by RecF. The above results are consistent with the idea replisome’s essential subunits. Wild-type or mutant cultures, grown at 30 °C that the DNA polymerases dissociate from the replisome upon were pulse-labeled with 1 μCi per 10 μg/mL [3H]thymidine for 2 min at the 2 encounters with UV-induced damage whereas the helicase com- indicated times following mock treatment (open symbols), 50 J/m UV irra- plex remains associated with the DNA.
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