Stalled DNA Replication Forks Research Articles

The Heritability of Replication Problems.

The major challenge of DNA replication is to provide daughter cells with intact and fully duplicated genetic material. However, various endogenous or environmental factors can slow down or stall DNA replication forks; these replication problems are known to fuel genomic instability and associated pathology, including cancer progression. Whereas the mechanisms emphasizing the source and the cellular responses of replicative problems have attracted much consideration over the past decade, the propagation through mitosis of genome modification and its heritability in daughter cells when the stress is not strong enough to provoke a checkpoint response in G2/M was much less documented. Some recent studies addressing whether low replication stress could impact the DNA replication program of the next generation of cells made the remarkable discovery that DNA damage can indeed be transmitted to daughter cells and can be processed in the subsequent S-phase, and that the replication timing program at a subset of chromosomal domains can also be impacted in the next generation of cells. Such a progression of replication problems into mitosis and daughter cells may appear counter-intuitive, but it could offer considerable advantages by alerting the next generation of cells of potentially risky loci and offering the possibility of an adaptive mechanism to anticipate a reiteration of problems, notably for cancer cells in the context of resistance to therapy.

Regulation of E. coli Rep helicase activity by PriC

Stalled DNA replication forks can result in incompletely replicated genomes and cell death. DNA replication restart pathways have evolved to deal with repair of stalled forks and E. coli Rep helicase functions in this capacity. Rep and an accessory protein, PriC, assemble at a stalled replication fork to facilitate loading of other replication proteins. A Rep monomer is a rapid and processive single stranded (ss) DNA translocase but needs to be activated to function as a helicase. Activation of Rep in vitro requires self-assembly to form a dimer, removal of its auto-inhibitory 2B sub-domain, or interactions with an accessory protein. Rep helicase activity has been shown to be stimulated by PriC, although the mechanism of activation is not clear. Using stopped flow kinetics, analytical sedimentation and single molecule fluorescence methods, we show that a PriC dimer activates the Rep monomer helicase and can also stimulate the Rep dimer helicase. We show that PriC can self-assemble to form dimers and tetramers and that Rep and PriC interact in the absence of DNA. We further show that PriC serves as a Rep processivity factor, presumably co-translocating with Rep during DNA unwinding. Activation is specific for Rep since PriC does not activate the UvrD helicase. Interaction of PriC with the C-terminal acidic tip of the ssDNA binding protein, SSB, eliminates Rep activation by stabilizing the PriC monomer. This suggests a likely mechanism for Rep activation by PriC at a stalled replication fork.

Dynamics of the PriA Helicase at Stalled DNA Replication Forks.

The DNA helicase PriA is a key protein for restarting stalled DNA replication forks in bacteria. With 3' to 5' helicase activity, PriA is important in primosome assembly. We used atomic force microscopy (AFM) and specifically employed time-lapse AFM to visualize the interaction of PriA with two DNA substrates. The results show that most of the PriA molecules are observed bound at the fork. However, PriA is capable of translocating over distances of about 400 bp. There is a preference for the long-range translocation of PriA depending on the fork type. For a fork with the nascent leading strand as single-stranded DNA (ssDNA; F4 substrate), PriA translocates preferentially on the parental arm of the fork. For the substrate F14, which contains an additional ssDNA segment between the parental and lagging arms (5 nt gap), PriA translocates on both the parental and lagging strand arms. These data suggest that transient formation of the single-stranded regions during the DNA replication can change the selection of the DNA duplex by PriA. Translocation of the helicase was directly visualized by time-lapse AFM imaging, which revealed that PriA can switch strands during translocation. These novel features of PriA shed new light on the mechanisms of PriA interaction with stalled replication forks.

DNA2 in Chromosome Stability and Cell Survival—Is It All about Replication Forks?

The conserved nuclease-helicase DNA2 has been linked to mitochondrial myopathy, Seckel syndrome, and cancer. Across species, the protein is indispensable for cell proliferation. On the molecular level, DNA2 has been implicated in DNA double-strand break (DSB) repair, checkpoint activation, Okazaki fragment processing (OFP), and telomere homeostasis. More recently, a critical contribution of DNA2 to the replication stress response and recovery of stalled DNA replication forks (RFs) has emerged. Here, we review the available functional and phenotypic data and propose that the major cellular defects associated with DNA2 dysfunction, and the links that exist with human disease, can be rationalized through the fundamental importance of DNA2-dependent RF recovery to genome duplication. Being a crucial player at stalled RFs, DNA2 is a promising target for anti-cancer therapy aimed at eliminating cancer cells by replication-stress overload.

Abstract IA-026: Gaps alone kill BRCA deficient cancer cells

Abstract Defects in DNA repair and/or the protection of stalled DNA replication forks are thought to underlie the chemosensitivity of tumors deficient in the hereditary breast cancer genes, BRCA1 and BRCA2 (BRCA). Challenging this assumption, recent findings indicate that chemotherapies such as cisplatin used to treat BRCA-deficient tumors do not initially cause DNA double-strand-breaks (DSBs). Here, we show that single stranded DNA (ssDNA) replication gaps underlie the hypersensitivity of BRCA-deficient cancer, and we propose that defects in homologous recombination (HR) or fork protection (FP) do not. Specifically, in BRCA-deficient cells, ssDNA gaps develop because replication is not effectively restrained in response to stress. Gap suppression by either restoration of fork restraint or gap filling confers therapy resistance in tissue culture and BRCA patient tumors. In contrast, restored FP and HR can be uncoupled from therapy resistance when gaps are present. Moreover, we find that DSBs are not detected after therapy when apoptosis is inhibited, supporting a framework in which DSBs are not directly induced by genotoxic agents, but rather from cell death nucleases. Together, this data indicate that ssDNA replication gaps underlie the BRCA cancer phenotype, “BRCAness,” and we propose are fundamental to the mechanism-of-action of genotoxic chemotherapies. Citation Format: Sharon B. Cantor, Nicholas Panzarino. Gaps alone kill BRCA deficient cancer cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-026.

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.

Replication Gaps Underlie BRCA Deficiency and Therapy Response.

Defects in DNA repair and the protection of stalled DNA replication forks are thought to underlie the chemosensitivity of tumors deficient in the hereditary breast cancer genes BRCA1 and BRCA2 (BRCA). Challenging this assumption are recent findings that indicate chemotherapies, such as cisplatin used to treat BRCA-deficient tumors, do not initially cause DNA double-strand breaks (DSB). Here, we show that ssDNA replication gaps underlie the hypersensitivity of BRCA-deficient cancer and that defects in homologous recombination (HR) or fork protection (FP) do not. In BRCA-deficient cells, ssDNA gaps developed because replication was not effectively restrained in response to stress. Gap suppression by either restoration of fork restraint or gap filling conferred therapy resistance in tissue culture and BRCA patient tumors. In contrast, restored FP and HR could be uncoupled from therapy resistance when gaps were present. Moreover, DSBs were not detected after therapy when apoptosis was inhibited, supporting a framework in which DSBs are not directly induced by genotoxic agents, but rather are induced from cell death nucleases and are not fundamental to the mechanism of action of genotoxic agents. Together, these data indicate that ssDNA replication gaps underlie the BRCA cancer phenotype, "BRCAness," and we propose they are fundamental to the mechanism of action of genotoxic chemotherapies. SIGNIFICANCE: This study suggests that ssDNA replication gaps are fundamental to the toxicity of genotoxic agents and underlie the BRCA-cancer phenotype "BRCAness," yielding promising biomarkers, targets, and opportunities to resensitize refractory disease.See related commentary by Canman, p. 1214.

Characterize the Interaction of the DNA Helicase PriA with the Stalled DNA Replication Fork Using Atomic Force Microscopy.

In bacteria, the restart of stalled DNA replication forks requires the DNA helicase PriA. PriA can recognize and remodel abandoned DNA replication forks, unwind DNA in the 3'-to-5' direction, and facilitate the loading of the helicase DnaB onto the DNA to restart replication. ssDNA-binding protein (SSB) is typically present at the abandoned forks, protecting the ssDNA from nucleases. Research that is based on the assays for junction dissociation, surface plasmon resonance, single-molecule FRET, and x-ray crystal structure has revealed the helicase activity of PriA, the SSB-PriA interaction, and structural information of PriA helicase. Here, we used Atomic Force Microscopy (AFM) to visualize the interaction between PriA and DNA substrates with or without SSB in the absence of ATP to delineate the substrate recognition pattern of PriA before its ATP-catalyzed DNA-unwinding reaction. The protocol describes the steps to obtain high-resolution AFM images and the details of data analysis and presentation.

Loss of the DNA Fork Remodeling Protein Smarcal1 Impairs the Replication Stress Response in Proliferating Hematopoietic Cells

Rapidly proliferating hematopoietic cells are particularly prone to threats to genomic integrity through replication stress. The response to replication stress involves complex molecular machinery to prevent replication fork collapse, avoid accumulation of DNA damage, and ensure the completion of DNA synthesis. Smarcal1, a DNA remodeling enzyme, has been linked to the replication stress response through stabilization, repair, and restart of stalled DNA replication forks. In humans, complete loss of function of Smarcal1 leads to the pleiotropic disorder Schimke immuno-osseous dysplasia (SIOD), which is characterized, in part, by a severe lymphoid immunodeficiency that leaves patients susceptible to opportunistic infections. Currently, the mechanisms driving the clinical immunodeficient phenotype of SIOD and the in vivo functions of Smarcal1 in hematopoietic cells remain largely unexplored. We evaluated the contribution of Smarcal1 to normal and stressed/emergency hematopoiesis, using a Smarcal1 knockout mouse model and hematopoietic cell replication stresses such as low-dose radiation, 5-fluorouracil, bone marrow transplantation, and oncogene- driven proliferation. We determined that, while loss of Smarcal1 does not affect hematopoietic stem and progenitor cells (HSPCs) or mature hematopoietic cell populations at a normal "resting" state, Smarcal1 is required in these cells undergoing proliferative stress. Following DNA replication stress, we detected defects in multiple Smarcal1-deficient hematopoietic cell populations. We also observed that loss of Smarcal1 led to significantly increased levels of replication stress, DNA damage, and apoptosis in proliferating cells. Through bone marrow transplantation, we determined that the deficiencies observed were not due to a defect in the bone marrow environment or an inability of the HSPCs to home to the bone marrow. Thus, our results reveal that Smarcal1 is critical to maintaining hematopoietic cell survival and function under conditions of DNA replication stress. Our data also suggest that the immunodeficiency of patients with Smarcal1 mutations may be due to an inability of hematopoietic cells, especially HSPCs, to properly respond to replication stress and proliferate. Since wild-type bone marrow is able to fully reconstitute and function in a Smarcal1-deficient environment, bone marrow transplantation may be an effective therapy for immunodeficient SIOD patients. Disclosures Eischen: AbbVie Inc.: Research Funding.

Combining PARP and DNA-PK Inhibitors With Irradiation Inhibits HPV-Negative Head and Neck Cancer Squamous Carcinoma Growth.

Novel targeted agents to inhibit DNA repair pathways to sensitize tumors to irradiation (IR) are being investigated as an alternative to chemoradiation for locally advanced human papilloma virus negative (HPV-negative) head and neck squamous cell carcinoma (HNSCC). Two well-characterized targets that, when inhibited, exhibit potent IR sensitization are PARP1 and DNA-PKcs. However, their cooperation in sensitizing HPV-negative HNSCC to IR remains to be explored given that PARP1 and DNA-PkCS bind to unresected stalled DNA replication forks and cooperate to recruit XRCC1 to facilitate double-strand break repair. Here, we show that the combination of the DNA-PK inhibitor NU7441 and the PARP inhibitor olaparib significantly decrease proliferation (61–78%) compared to no reduction with either agent alone (p < 0.001) in both SCC1 and SCC6 cell lines. Adding IR to the combination further decreased cell proliferation (91–92%, p < 0.001) in SCC1 and SCC6. Similar results were observed using long-term colony formation assays [dose enhancement ratio (DER) 2.3–3.2 at 4Gy, p < 0.05]. Reduced cell survival was attributed to increased apoptosis and G2/M cell cycle arrest. Kinomic analysis using tyrosine (PTK) and serine/threonine (STK) arrays reveals that combination treatment results in the most potent inhibition of kinases involved in the CDK and ERK pathways compared to either agent alone. In vivo, a significant delay of tumor growth was observed in UM-SCC1 xenografts receiving IR with olaparib and/or NU7441, which was similar to the cisplatin-IR group. Both regimens were less toxic than cisplatin-IR as assessed by loss of mouse body weight. Taken together, these results demonstrate that the combination of NU7441 and olaparib with IR enhances HPV-negative HNSCC inhibition in both cell culture and in mice, suggesting a potential innovative combination for effectively treating patients with HPV-negative HNSCC.

Succinylation at a key residue of FEN1 is involved in the DNA damage response to maintain genome stability.

Human flap endonuclease 1 (FEN1) is a structure-specific, multifunctional endonuclease essential for DNA replication and repair. Our previous study showed that in response to DNA damage, FEN1 interacts with the PCNA-like Rad9-Rad1-Hus1 complex instead of PCNA to engage in DNA repair activities, such as stalled DNA replication fork repair, and undergoes SUMOylation by SUMO-1. Here, we report that succinylation of FEN1 was stimulated in response to DNA replication fork-stalling agents, such as ultraviolet (UV) irradiation, hydroxyurea, camptothecin, and mitomycin C. K200 is a key succinylation site of FEN1 that is essential for gap endonuclease activity and could be suppressed by methylation and stimulated by phosphorylation to promote SUMO-1 modification. Succinylation at K200 of FEN1 promoted the interaction of FEN1 with the Rad9-Rad1-Hus1 complex to rescue stalled replication forks. Impairment of FEN1 succinylation led to the accumulation of DNA damage and heightened sensitivity to fork-stalling agents. Altogether, our findings suggest an important role of FEN1 succinylation in regulating its roles in DNA replication and repair, thus maintaining genome stability.

Nuclear IGF1R interact with PCNA to preserve DNA replication after DNA-damage in a variety of human cancers.

Nuclear IGF1R has been linked to poor outcome in cancer. We recently showed that nuclear IGF1R phosphorylates PCNA and increases DNA damage tolerance. In this paper we aimed to describe this mechanism in cancer tissue as well as in cancer cell lines. In situ proximity ligation assay identified frequent IGF1R and PCNA colocalization in many cancer types. IGF1R/PCNA colocalization was more frequently increased in tumor cells than in adjacent normal, and more prominent in areas with dysplasia and invasion. However, the interaction was often lost in tumors with poor response to neoadjuvant treatment and most metastatic lesions. In two independent cohorts of serous ovarian carcinomas and oropharyngeal squamous cell carcinomas, stronger IGF1R/PCNA colocalization was significantly associated with a higher overall survival. Ex vivo irradiation of ovarian cancer tissue acutely induced IGF1R/PCNA colocalization together with γH2AX-foci formations. In vitro, RAD18 mediated mono-ubiquitination of PCNA during replication stress was dependent on IGF1R kinase activity. DNA fiber analysis revealed that IGF1R activation could rescue stalled DNA replication forks, but only in cancer cells with baseline IGF1R/PCNA interaction. We believe that the IGF1R/PCNA interaction is a basic cellular mechanism to increase DNA stress tolerance during proliferation, but that this mechanism is lost with tumor progression in conjunction with accumulated DNA damage and aberrant strategies to tolerate genomic instability. To exploit this mechanism in IGF1R targeted therapy, IGF1R inhibitors should be explored in the context of concomitant induction of DNA replication stress as well as in earlier clinical stages than previously tried.

Roles of Chk2/CHEK2 in guarding against environmentally induced DNA damage and replication-stress.

Checkpoint kinase 2 (human CHEK2; murine Chk2) is a critical mediator of the DNA damage response and has established roles in DNA double strand break (DSB)-induced apoptosis and cell cycle arrest. DSBs may be invoked directly by ionizing radiation but may also arise indirectly from environmental exposures such as solar ultraviolet (UV) radiation. The primary forms of DNA damage induced by UV are DNA photolesions (such as cyclobutane pyrimidine dimers CPD and 6-4 photoproducts) which interfere with DNA synthesis and lead to DNA replication fork stalling. Persistently stalled and unresolved DNA replication forks can "collapse" to generate DSBs that induce signaling via Chk2 and its upstream activator the ataxia telangiectasia-mutated (ATM) protein kinase. This review focuses on recently defined roles of Chk2 in protecting against DNA replication-associated genotoxicity. Several DNA damage response factors such as Rad18, Nbs1 and Chk1 suppress stalling and collapse of DNA replication forks. Defects in the primary responders to DNA replication fork stalling lead to generation of DSB and reveal "back-up" roles for Chk2 in S-phase progression and genomic stability. In humans, there are numerous variants of the CHEK2 gene, including CHEK2*1100delC. Individuals with the CHEK2*1100delC germline alteration have an increased risk of developing breast cancer and malignant melanoma. DNA replication fork-stalling at estrogen-DNA adducts and UV-induced photolesions are implicated in the etiology of breast cancer and melanoma, respectively. It is likely therefore that the Chk2/CHEK2-deficiency is associated with elevated risk for tumorigenesis caused by replication-associated genotoxicities that are exacerbated by environmental genotoxins and intrinsic DNA-damaging agents.

Role of HP1β during spermatogenesis and DNA replication.

Heterochromatin protein 1β (HP1β), encoded by the Cbx1 gene, has been functionally linked to chromatin condensation, transcriptional regulation, and DNA damage repair. Here we report that testis-specific Cbx1 conditional knockout (Cbx1 cKO) impairs male germ cell development in mice. Depletion of HP1β negatively affected sperm maturation and increased seminiferous tubule degeneration in Cbx1 cKO mice. In addition, the spermatogonia have elevated γ-H2AX foci levels as do Cbx1 deficient mouse embryonic fibroblasts (MEFs) as compared to wild-type (WT) control MEFs. The increase in γ-H2AX foci in proliferating Cbx1 cKO cells indicates defective replication-dependent DNA damage repair. Depletion or loss of HP1β from human cells and MEFs increased DNA replication fork stalling and firing of new origins of replication, indicating defective DNA synthesis. Taken together, these results suggest that loss of HP1β in proliferating cells leads to DNA replication defects with associated DNA damage that impact spermatogenesis.

Disease-associated DNA2 nuclease-helicase protects cells from lethal chromosome under-replication.

DNA2 is an essential nuclease–helicase implicated in DNA repair, lagging-strand DNA synthesis, and the recovery of stalled DNA replication forks (RFs). In Saccharomyces cerevisiae, dna2Δ inviability is reversed by deletion of the conserved helicase PIF1 and/or DNA damage checkpoint-mediator RAD9. It has been suggested that Pif1 drives the formation of long 5′-flaps during Okazaki fragment maturation, and that the essential function of Dna2 is to remove these intermediates. In the absence of Dna2, 5′-flaps are thought to accumulate on the lagging strand, resulting in DNA damage-checkpoint arrest and cell death. In line with Dna2’s role in RF recovery, we find that the loss of Dna2 results in severe chromosome under-replication downstream of endogenous and exogenous RF-stalling. Importantly, unfaithful chromosome replication in Dna2-mutant cells is exacerbated by Pif1, which triggers the DNA damage checkpoint along a pathway involving Pif1’s ability to promote homologous recombination-coupled replication. We propose that Dna2 fulfils its essential function by promoting RF recovery, facilitating replication completion while suppressing excessive RF restart by recombination-dependent replication (RDR) and checkpoint activation. The critical nature of Dna2’s role in controlling the fate of stalled RFs provides a framework to rationalize the involvement of DNA2 in Seckel syndrome and cancer.

Productive herpesvirus lytic replication in primary effusion lymphoma cells requires S-phase entry.

Gammaherpesviruses establish lifelong latent infection in B lymphocytes and are the causative agent of several B-cell malignancies and lymphoproliferative disorders. While a quiescent latent infection allows these pathogens to evade immune detection, initiation of an alternative lifecycle stage, known as lytic replication, is an essential step in the production and dissemination of infectious progeny. Although cessation of cellular proliferation is an eventual consequence of lytic induction, exactly how gammaherpesviruses manipulate the cell cycle prior to amplification of viral DNA remains under debate. Here we show that the onset of Kaposi's sarcoma-associated herpesvirus (KSHV) lytic reactivation in B cells leads to S-phase accumulation and that exit from G1 is required for efficient viral DNA replication. We also show that lytic replication leads to an S-phase-specific activation of the DNA damage response (DDR) that is abrogated when lytic replication is restricted to G0/G1. Finally, we observe that expression of early lytic viral genes results in cellular replication stress with increased stalling of DNA replication forks. Overall, we demonstrate that S-phase entry is important for optimal KSHV replication, that G1 arresting compounds are effective inhibitors of viral propagation, and that lytic-induced cell-cycle arrest could occur through the obstruction of cellular replication forks and subsequent activation of the DDR.

Atomic force microscopy–based characterization of the interaction of PriA helicase with stalled DNA replication forks

In bacteria, the restart of stalled DNA replication forks requires the DNA helicase PriA. PriA can recognize and remodel abandoned DNA replication forks, unwind DNA in the 3'-to-5' direction, and facilitate the loading of the helicase DnaB onto the DNA to restart replication. Single-stranded DNA-binding protein (SSB) is typically present at the abandoned forks, but it is unclear how SSB and PriA interact, although it has been shown that the two proteins interact both physically and functionally. Here, we used atomic force microscopy to visualize the interaction of PriA with DNA substrates with or without SSB. These experiments were done in the absence of ATP to delineate the substrate recognition pattern of PriA before its ATP-catalyzed DNA-unwinding reaction. These analyses revealed that in the absence of SSB, PriA binds preferentially to a fork substrate with a gap in the leading strand. Such a preference has not been observed for 5'- and 3'-tailed duplexes, suggesting that it is the fork structure that plays an essential role in PriA's selection of DNA substrates. Furthermore, we found that in the absence of SSB, PriA binds exclusively to the fork regions of the DNA substrates. In contrast, fork-bound SSB loads PriA onto the duplex DNA arms of forks, suggesting a remodeling of PriA by SSB. We also demonstrate that the remodeling of PriA requires a functional C-terminal domain of SSB. In summary, our atomic force microscopy analyses reveal key details in the interactions between PriA and stalled DNA replication forks with or without SSB.

DNA Helicase-SSB Interactions Critical to the Regression and Restart of Stalled DNA Replication forks in Escherichia coli.

In Escherichia coli, DNA replication forks stall on average once per cell cycle. When this occurs, replisome components disengage from the DNA, exposing an intact, or nearly intact fork. Consequently, the fork structure must be regressed away from the initial impediment so that repair can occur. Regression is catalyzed by the powerful, monomeric DNA helicase, RecG. During this reaction, the enzyme couples unwinding of fork arms to rewinding of duplex DNA resulting in the formation of a Holliday junction. RecG works against large opposing forces enabling it to clear the fork of bound proteins. Following subsequent processing of the extruded junction, the PriA helicase mediates reloading of the replicative helicase DnaB leading to the resumption of DNA replication. The single-strand binding protein (SSB) plays a key role in mediating PriA and RecG functions at forks. It binds to each enzyme via linker/OB-fold interactions and controls helicase-fork loading sites in a substrate-dependent manner that involves helicase remodeling. Finally, it is displaced by RecG during fork regression. The intimate and dynamic SSB-helicase interactions play key roles in ensuring fork regression and DNA replication restart.

Nanoscale interaction of RecG with mobile fork DNA.

RecG DNA helicase is a guardian of the bacterial genome where it dominates stalled DNA replication fork rescue. The single-stranded DNA binding protein (SSB) is involved in this process and promotes the binding of RecG to stalled replication forks. Atomic force microscopy (AFM) was used to investigate the interaction of RecG and SSB on a mobile fork substrate capable of being regressed. In the absence of proteins, the fork undergoes spontaneous dynamics between two states, defined by the length of the DNA complementarity at the fork. The binding of SSB does not affect these dynamics as it binds to single-stranded regions as expected. In contrast, RecG interacts with the two states quite differently. We demonstrate that RecG has two modes of interaction with fork DNA in the presence of SSB and ATP. In the first mode, RecG translocates over the duplex region and this activity is defined by SSB-mediated remodeling of helicase. In the second mode, RecG utilizes its helicase activity to regress the fork, in an ATP-dependent manner, displacing SSB on the ssDNA. Overall, our results highlight two functions of RecG that can be employed in the regulation of stalled DNA replication fork rescue.

Control of homologous recombination by the HROB-MCM8-MCM9 pathway.

DNA repair by homologous recombination (HR) is essential for genomic integrity, tumor suppression, and the formation of gametes. HR uses DNA synthesis to repair lesions such as DNA double-strand breaks and stalled DNA replication forks, but despite having a good understanding of the steps leading to homology search and strand invasion, we know much less of the mechanisms that establish recombination-associated DNA polymerization. Here, we report that C17orf53/HROB is an OB-fold-containing factor involved in HR that acts by recruiting the MCM8-MCM9 helicase to sites of DNA damage to promote DNA synthesis. Mice with targeted mutations in Hrob are infertile due to depletion of germ cells and display phenotypes consistent with a prophase I meiotic arrest. The HROB-MCM8-MCM9 pathway acts redundantly with the HELQ helicase, and cells lacking both HROB and HELQ have severely impaired HR, suggesting that they underpin two major routes for the completion of HR downstream from RAD51. The function of HROB in HR is reminiscent of that of gp59, which acts as the replicative helicase loader during bacteriophage T4 recombination-dependent DNA replication. We therefore propose that the loading of MCM8-MCM9 by HROB may similarly be a key step in the establishment of mammalian recombination-associated DNA synthesis.