Rescue Of Stalled Replication Forks Research Articles

Restriction of RecG translocation by DNA mispairing

BackgroundThe RecG DNA helicase plays a crucial role in stalled replication fork rescue. We have recently discovered that interaction of RecG with single-strand DNA binding protein (SSB) remodels RecG, allowing it to spontaneously translocate upstream of the fork. Based on these findings, we hypothesized that mispairing of DNA could limit such translocation of RecG. MethodsHere, we used atomic force microscopy (AFM) to directly test this hypothesis and investigate how sensitive RecG translocation is to different types of mispairing. ResultsWe found that a CC mispairing, at a distance of 30 bp from the fork position, prevents translocation of RecG over this mispairing. A G-bulge, placed at the same distance, also has a similar blocking efficiency. However, a CC mispairing, 10 bp away from the fork, does not prevent RecG translocation beyond 10 bp distance, but decreases complex yield. Modeling of RecG-DNA complexes show that 10 bp distance from the fork is within the binding footprint of RecG on DNA. ConclusionsOur results suggest that the RecG translocation upstream of the replication fork is limited by mispairings in the parental arm of the replication fork.General significanceThese findings led us to propose dual functions for RecG, in which the thermally driven translocation of RecG can be a mechanism for the additional control of the DNA paring in which RecG can detect the lesions in front of the replication fork, adding to the fidelity of the DNA replication machinery.

Coordinated Cut and Bypass: Replication of Interstrand Crosslink-Containing DNA.

DNA interstrand crosslinks (ICLs) are covalently bound DNA lesions, which are commonly induced by chemotherapeutic drugs, such as cisplatin and mitomycin C or endogenous byproducts of metabolic processes. This type of DNA lesion can block ongoing RNA transcription and DNA replication and thus cause genome instability and cancer. Several cellular defense mechanism, such as the Fanconi anemia pathway have developed to ensure accurate repair and DNA replication when ICLs are present. Various structure-specific nucleases and translesion synthesis (TLS) polymerases have come into focus in relation to ICL bypass. Current models propose that a structure-specific nuclease incision is needed to unhook the ICL from the replication fork, followed by the activity of a low-fidelity TLS polymerase enabling replication through the unhooked ICL adduct. This review focuses on how, in parallel with the Fanconi anemia pathway, PCNA interactions and ICL-induced PCNA ubiquitylation regulate the recruitment, substrate specificity, activity, and coordinated action of certain nucleases and TLS polymerases in the execution of stalled replication fork rescue via ICL bypass.

Single-molecule insight into stalled replication fork rescue in Escherichia coli.

DNA replication forks stall at least once per cell cycle in Escherichia coli. DNA replication must be restarted if the cell is to survive. Restart is a multi-step process requiring the sequential action of several proteins whose actions are dictated by the nature of the impediment to fork progression. When fork progress is impeded, the sequential actions of SSB, RecG and the RuvABC complex are required for rescue. In contrast, when a template discontinuity results in the forked DNA breaking apart, the actions of the RecBCD pathway enzymes are required to resurrect the fork so that replication can resume. In this review, we focus primarily on the significant insight gained from single-molecule studies of individual proteins, protein complexes, and also, partially reconstituted regression and RecBCD pathways. This insight is related to the bulk-phase biochemical data to provide a comprehensive review of each protein or protein complex as it relates to stalled DNA replication fork rescue.

Remodeling of RecG Helicase at the DNA Replication Fork by SSB Protein.

The RecG DNA helicase a key player in stalled replication fork rescue. The single-stranded DNA binding protein (SSB) participates in this process, but its role in the interaction of RecG with the fork remains unclear. We used atomic force microscopy (AFM) to visualize the interaction of RecG with a fork DNA in the presence of SSB. We discovered that SSB enhances RecG loading efficiency onto the DNA fork by threefold. Additionally, SSB interacts with RecG leading to the RecG remodeling. As a result, RecG separates from the fork, but remains bound to the DNA duplex. Moreover, in this new binding mode RecG is capable of translocation along the parental duplex DNA. We propose a model of RecG interaction with the replication fork involving two RecG binding modes. SSB plays the role of a remodeling factor defining the mode of RecG binding to the fork mediated by the SSB C-terminus. In the translocating mode, RecG remains in the vicinity of the fork and is capable of initiating the fork regression. Our results afford novel mechanistic insights into RecG interaction with the replication fork and provide the basis for further structural studies.

RecG Interaction with the DNA Replication Fork. The Role of E. coli SSB Protein

The RecG DNA helicase is a guardian of the bacterial genome. It binds to a variety of forked DNA structures thereby minimizing pathological DNA replication and facilitating stalled replication fork rescue. It is becomingly increasingly clear that SSB plays a critical role in the function of RecG, but the mechanisms of its interaction with SSB remain unclear. Here we use the atomic force microscope (AFM) to image the structure of RecG with a model fork substrate in the presence or absence of the single strand DNA binding protein (SSB). The DNA substrate has a 3′-end 69nt single stranded DNA (ssDNA) segment inserted between the two DNA duplexes. This design mimics a stalled fork with an ssDNA gap in the leading strand. The SSB proteins bind very specifically to the ssDNA segment with a yield of ∼90%. RecG protein also binds to the fork but the yield was ∼10%. However, the RecG loading on the same substrate increases three-fold in the presence of SSB suggesting that SSB facilitates RecG loading onto the fork. Moreover, in the presence of SSB, RecG becomes capable of translocating along the DNA duplex in an ATP hydrolysis-independent manner. No such mobility of RecG was observed in the absence of SSB. The preferable translocation direction is moving of RecG along parental arm which implies RecG could clear obstacles bound ahead of the fork. This novel activity of SSB requires the SSB C-terminus as a truncated SSB mutant does not substitute for wild type. SSB loading of RecG and subsequent translocation are unaffected by ATP, ADP or the non-hydrolyzable analog ATP-γ-S. Overall the results obtained reveal novel properties of RecG and highlight a new chaperone-type role of SSB in the DNA repair process.

Flap Endonuclease 1

First discovered as a structure-specific endonuclease that evolved to cut at the base of single-stranded flaps, flap endonuclease (FEN1) is now recognized as a central component of cellular DNA metabolism. Substrate specificity allows FEN1 to process intermediates of Okazaki fragment maturation, long-patch base excision repair, telomere maintenance, and stalled replication fork rescue. For Okazaki fragments, the RNA primer is displaced into a 5' flap and then cleaved off. FEN1 binds to the flap base and then threads the 5' end of the flap through its helical arch and active site to create a configuration for cleavage. The threading requirement prevents this active nuclease from cutting the single-stranded template between Okazaki fragments. FEN1 efficiency and specificity are critical to the maintenance of genome fidelity. Overall, recent advances in our knowledge of FEN1 suggest that it was an ancient protein that has been fine-tuned over eons to coordinate many essential DNA transactions.

DNA2 and EXO1 in replication-coupled, homology-directed repair and in the interplay between HDR and the FA/BRCA network

During DNA replication, stalled replication forks and DSBs arise when the replication fork encounters ICLs (interstrand crosslinks), covalent protein/DNA intermediates or other discontinuities in the template. Recently, homologous recombination proteins have been shown to function in replication-coupled repair of ICLs in conjunction with the Fanconi anemia (FA) regulatory factors FANCD2-FANCI, and, conversely, the FA gene products have been shown to play roles in stalled replication fork rescue even in the absence of ICLs, suggesting a broader role for the FA network than previously appreciated. Here we show that DNA2 helicase/nuclease participates in resection during replication-coupled repair of ICLs and other replication fork stresses. DNA2 knockdowns are deficient in HDR (homology-directed repair) and the S phase checkpoint and exhibit genome instability and sensitivity to agents that cause replication stress. DNA2 is partially redundant with EXO1 in these roles. DNA2 interacts with FANCD2, and cisplatin induces FANCD2 ubiquitylation even in the absence of DNA2. DNA2 and EXO1 deficiency leads to ICL sensitivity but does not increase ICL sensitivity in the absence of FANCD2. This is the first demonstration of the redundancy of human resection nucleases in the HDR step in replication-coupled repair, and suggests that DNA2 may represent a new mediator of the interplay between HDR and the FA/BRCA pathway.

Rescue of Stalled Replication Forks by the DNA Translocase FANCM

Fanconi anemia (FA) is a chromosomal instability syndrome that manifests with congenital abnormalities, bone marrow failure and cancer predisposition. To date, fourteen FA genes have been identified and implicated in a ubiquitin signaling pathway, in S phase checkpoint activation, in lesion bypass and in DNA double‐strand break (DSB) repair by homologous recombination.FANCM is a DNA translocase that acts as a molecular scaffold involved in multiple DNA damage signaling and processing reactions. FANCM interacts with : 1) the checkpoint protein HCLK2 to facilitate DNA damage signaling; 2) with the Bloom's complex at stalled forks; 3) and with the FA ubiquitin ligase complex, promoting its recruitment to chromatin and the monoubiquitination of FANCD2 and FANCI.Our biochemical studies revealed that FANCM binds and translocates the branch point of DNA structures that mimic replication and repair intermediates, suggesting that FANCM may remodel replication intermediates to promote recovery. Using a molecular combing approach, we have observed that FANCM facilitates the restart of replication forks stalled by DNA topoisomerase I inhibitors. Our recent efforts have focused on studying the role new DNA damage response proteins that interact with FANCM.Research support: ARC Association pour la Recherche sur le Cancer, ANR Agence Nationale de la Recherche, SNSF Swiss National Science Foundation.

Functional regulation of FEN1 nuclease and its link to cancer

Flap endonuclease-1 (FEN1) is a member of the Rad2 structure-specific nuclease family. FEN1 possesses FEN, 5′-exonuclease and gap-endonuclease activities. The multiple nuclease activities of FEN1 allow it to participate in numerous DNA metabolic pathways, including Okazaki fragment maturation, stalled replication fork rescue, telomere maintenance, long-patch base excision repair and apoptotic DNA fragmentation. Here, we summarize the distinct roles of the different nuclease activities of FEN1 in these pathways. Recent biochemical and genetic studies indicate that FEN1 interacts with more than 30 proteins and undergoes post-translational modifications. We discuss how FEN1 is regulated via these mechanisms. Moreover, FEN1 interacts with five distinct groups of DNA metabolic proteins, allowing the nuclease to be recruited to a specific DNA metabolic complex, such as the DNA replication machinery for RNA primer removal or the DNA degradosome for apoptotic DNA fragmentation. Some FEN1 interaction partners also stimulate FEN1 nuclease activities to further ensure efficient action in processing of different DNA structures. Post-translational modifications, on the other hand, may be critical to regulate protein–protein interactions and cellular localizations of FEN1. Lastly, we also review the biological significance of FEN1 as a tumor suppressor, with an emphasis on studies of human mutations and mouse models.