Repair Of Replication Forks Research Articles

Defects in the central metabolism prevent thymineless death in Escherichia coli, while still allowing significant protein synthesis.

Starvation of E. coli thyA auxotrophs for the required thymine or thymidine leads to cessation of DNA synthesis and, unexpectedly, to thymineless death (TLD). Previously, TLD-alleviating defects were identified by the candidate gene approach, for their contribution to replication initiation, fork repair, or SOS induction. However, no TLD-blocking mutations were ever found, suggesting a multifactorial nature of TLD. Since (until recently) no unbiased isolation of TLD suppressors was reported, we used enrichment after insertional mutagenesis to systematically isolate TLD suppressors. Our approach was validated by isolation of known TLD-alleviating mutants in recombinational repair. At the same time, and unexpectedly for the current TLD models, most of the isolated suppressors affected general metabolism, while the strongest suppressors impacted the central metabolism. Several temperature-sensitive (Ts)-mutants in important/essential functions, like nadA, ribB or coaA, almost completely suppressed TLD at 42°C. Since blocking protein synthesis completely by chloramphenicol prevents TLD, while reducing protein synthesis to 10% alleviates TLD only slightly, we measured the level of protein synthesis in these mutants at 42°C and found it to be 20-70% of the WT, not enough reduction to explain TLD prevention. We conclude that the isolated central metabolism mutants prevent TLD by affecting specific TLD-promoting functions.

Comparative analysis of structural dynamics and allosteric mechanisms of RecA/Rad51 family proteins: Integrated atomistic MD simulation and network-based analysis

Homologous recombination plays a key role in double-strand break repair, stalled replication fork repair, and meiosis. The RecA/Rad51 family recombinases catalyze the DNA strand invasion reaction that occurs during homologous recombination. However, the high sequence differences between homologous groups have hindered the thoroughly studies of this ancient protein family. The dynamic mechanisms of the family, particularly at the residual level, remain poorly understood. In this work, five representative RecA/Rad51 recombinase family members from all major kingdoms of living organisms: prokaryotes, eukaryotes, archaea, and viruses, were selected to explore the molecular mechanisms behind their conserved biological significance. A variety of techniques, including all-atom molecular dynamics simulation, perturbation response scanning, and protein structure network analysis, were used to examine the flexibility and correlation of protein domains, distribution of sensors and effectors and conserved hub residues. Furthermore, the potential communication routes between the ATP-binding region and the DNA-binding region of each recombinase were identified. Our results demonstrate the conserved molecular dynamics of these recombinases in the early stage of homologous recombination, including cooperative motions between regions, conserved sensing and effecting functional residue distribution, and conserved hub residues. Meanwhile, the unique ATP-DNA communication routes of each recombinase was also revealed. These results provide new insights into the mechanism of RecA/Rad51 family proteins, and provide new theoretical guidance for the development of allosteric inhibitors and the application of RecA/Rad51 family proteins.

Regulation of Rad52-dependent replication fork recovery through serine ADP-ribosylation of PolD3

Although Poly(ADP-ribose)-polymerases (PARPs) are key regulators of genome stability, how site-specific ADP-ribosylation regulates DNA repair is unclear. Here, we describe a novel role for PARP1 and PARP2 in regulating Rad52-dependent replication fork repair to maintain cell viability when homologous recombination is dysfunctional, suppress replication-associated DNA damage, and maintain genome stability. Mechanistically, Mre11 and ATM are required for induction of PARP activity in response to replication stress that in turn promotes break-induced replication (BIR) through assembly of Rad52 at stalled/damaged replication forks. Further, by mapping ADP-ribosylation sites induced upon replication stress, we identify that PolD3 is a target for PARP1/PARP2 and that its site-specific ADP-ribosylation is required for BIR activity, replication fork recovery and genome stability. Overall, these data identify a critical role for Mre11-dependent PARP activation and site-specific ADP-ribosylation in regulating BIR to maintain genome integrity during DNA synthesis.

CDK2 regulates collapsed replication fork repair in CCNE1-amplified ovarian cancer cells via homologous recombination.

CCNE1 amplification is a common alteration in high-grade serous ovarian cancer and occurs in 15-20% of these tumors. These amplifications are mutually exclusive with homologous recombination deficiency, and, as they have intact homologous recombination, are intrinsically resistant to poly (ADP-ribose) polymerase inhibitors or chemotherapy agents. Understanding the molecular mechanisms that lead to this mutual exclusivity may reveal therapeutic vulnerabilities that could be leveraged in the clinic in this still underserved patient population. Here, we demonstrate that CCNE1-amplified high-grade serous ovarian cancer cells rely on homologous recombination to repair collapsed replication forks. Cyclin-dependent kinase 2, the canonical partner of cyclin E1, uniquely regulates homologous recombination in this genetic context, and as such cyclin-dependent kinase 2 inhibition synergizes with DNA damaging agents in vitro and in vivo. We demonstrate that combining a selective cyclin-dependent kinase 2 inhibitor with a DNA damaging agent could be a powerful tool in the clinic for high-grade serous ovarian cancer.

EEPD1 promotes repair of oxidatively-stressed replication forks.

Unrepaired oxidatively-stressed replication forks can lead to chromosomal instability and neoplastic transformation or cell death. To meet these challenges cells have evolved a robust mechanism to repair oxidative genomic DNA damage through the base excision repair (BER) pathway, but less is known about repair of oxidative damage at replication forks. We found that depletion or genetic deletion of EEPD1 decreases clonogenic cell survival after oxidative DNA damage. We demonstrate that EEPD1 is recruited to replication forks stressed by oxidative damage induced by H2O2 and that EEPD1 promotes replication fork repair and restart and decreases chromosomal abnormalities after such damage. EEPD1 binds to abasic DNA structures and promotes resolution of genomic abasic sites after oxidative stress. We further observed that restoration of expression of EEPD1 via expression vector transfection restores cell survival and suppresses chromosomal abnormalities induced by oxidative stress in EEPD1-depleted cells. Consistent with this, we found that EEPD1 preserves replication fork integrity by preventing oxidatively-stressed unrepaired fork fusion, thereby decreasing chromosome instability and mitotic abnormalities. Our results indicate a novel role for EEPD1 in replication fork preservation and maintenance of chromosomal stability during oxidative stress.

A moving target for drug discovery: Structure activity relationship and many genome (de)stabilizing functions of the RAD52 protein

BRCA-ness phenotype, a signature of many breast and ovarian cancers, manifests as deficiency in homologous recombination, and as defects in protection and repair of damaged DNA replication forks. A dependence of such cancers on DNA repair factors less important for survival of BRCA-proficient cells, offers opportunities for development of novel chemotherapeutic interventions. The first drugs targeting BRCA-deficient cancers, poly-ADP-ribose polymerase (PARP) inhibitors have been approved for the treatment of advanced, chemotherapy resistant cancers in patients with BRCA1/2 germline mutations. Nine additional proteins that can be targeted to selectively kill BRCA-deficient cancer cells have been identified. Among them, a DNA repair protein RAD52 is an especially attractive target due to general tolerance of the RAD52 loss of function, and protective role of an inactivating mutation. Yet, the effective pharmacological inhibitors of RAD52 have not been forthcoming. In this review, we discuss advances in the state of our knowledge of the RAD52 structure, activities and cellular functions, with a specific focus on the features that make RAD52 an attractive, but difficult drug target.

Challenging endings: How telomeres prevent fragility.

It has become apparent that difficulties to replicate telomeres concern not only the very ends of eukaryotic chromosomes. The challenges already start when the replication fork enters the telomeric repeats. The obstacles encountered consist mainly of noncanonical nucleic acid structures that interfere with replication if not resolved. Replication stress at telomeres promotes the formation of so-called fragile telomeres displaying an abnormal appearance in metaphase chromosomes though their exact molecular nature remains to be elucidated. A substantial number of factors is required to counteract fragility. In this review we promote the hypothesis that telomere fragility is not caused directly by an initial insult during replication but it results as a secondary consequence of DNA repair of damaged replication forks by the homologous DNA recombination machinery. Incomplete DNA synthesis at repair sites or partial chromatin condensation may become apparent as telomere fragility. Fragility and DNA repair during telomere replication emerges as a common phenomenon which exacerbates in multiple disease conditions.

Abstract SP151: BRCA and homologous recombination deficiency mediated mutagenesis in breast cancer

Abstract The genes BRCA1, BRCA2 and PALB2 genes are the major high penetrance risk alleles in breast cancer have function in a DNA repair mechanism called homologous recombination (HR). These and a wider group of other genes including the RAD51 paralogues and kinase regulators of HR can also be subject to somatic mutation or epigenetic modification in cancer and, usually in the setting of bi-allelic inactivation, effect the use of HR. The dysregulation of BRCA1 by methylation of its regulatory region is frequent occurring, in approximately 30% of sporadic basal-like breast cancers. While there is evidence that BRCA1 silencing causes functional HR deficiency, there is also evidence suggesting this may be more “plastic” and subject to change to maintain cell fitness and DNA replication capacity under the evolutionary pressures that cancer cells are subjected to during progression and therapyexposure.HR deficient cancers change to use of other DNA break and DNA replication fork repair mechanisms that are mutagenic. A number of methods based on tumor genome sequencing have been developed to assesssomatic mutational signatures. These methods have revealed pathognomonic “scars” or “tattoos” indicating exposure to HR deficiency during breast cancer evolution and the variable performance of mutational signatures in the contexts of genetic and epigenetic loss of HR function. HR deficiency induced mutagenesis can also inactivate genes that negatively regulate HR, or re-activate (reversion mutation) the previously inactivated HR gene, driving a reversal of HR deficiency. As this changes a cell’s DNA repair phenotype and its ability to handle to therapeutic damage, these are increasingly found to be relevant to the development of resistance to DNA interacting drugs such as platinums and PARP inhibitors as well as to emerging DNA damage response (DDR) targeting therapeutics. In the setting of resistance both the residual HR deficiency induced genome landscape or the altered use of the DNA damage response in breast cancer cells may offer new vulnerabilities, enhanced in tumor cells, that are subject to targeted DDR directed or immunotherapy therapy and inform combination or sequential therapy approaches and associated patient selection biomarkers now being assessed in the clinic. Citation Format: A Tutt. BRCA and homologous recombination deficiency mediated mutagenesis in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr SP151.

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.

Mechanism and significance of chromosome damage repair by homologous recombination.

Homologous recombination (HR) is a major, conserved pathway of chromosome damage repair. It not only fulfills key functions in the removal of deleterious lesions such as DNA double-strand breaks (DSBs) and interstrand cross-links (ICLs), but also in replication fork repair and protection. Several familial and acquired cancer predisposition syndromes stem from defects in HR. In particular, individuals with mutations in HR genes exhibit predisposition to breast, ovarian, pancreatic, and prostate cancers, and they also show signs of accelerated aging. However, aberrant and untimely HR events can lead to the loss of heterozygosity, genomic rearrangements, and cytotoxic nucleoprotein intermediates. Thus, it is critically important that HR be tightly regulated. In addition to DNA repair, HR is also involved in meiotic chromosome segregation and telomere maintenance in cells that lack telomerase. In this review, we focus on the role of HR in DSB repair (DSBR) and summarize the current state of the field.

RecQ Family Helicases in Replication Fork Remodeling and Repair: Opening New Avenues towards the Identification of Potential Targets for Cancer Chemotherapy.

Replication fork reversal and restart has gained immense interest as a central response mechanism to replication stress following DNA damage. Although the exact mechanism of fork reversal has not been elucidated precisely, the involvement of diverse pathways and different factors has been demonstrated, which are central to this phenomenon. RecQ helicases known for their vital role in DNA repair and maintaining genome stability has recently been implicated in the restart of regressed replication forks. Through interaction with vital proteins like Poly (ADP) ribose polymerase 1 (PARP1), these helicases participate in the replication fork reversal and restart phenomenon. Most therapeutic agents used for cancer chemotherapy act by causing DNA damage in replicating cells and subsequent cell death. These DNA damages can be repaired by mechanisms involving fork reversal as the key phenomenon eventually reducing the efficacy of the therapeutic agent. Hence the factors contributing to this repair process can be good selective targets for developing more efficient chemotherapeutic agents. In this review, we have discussed in detail the role of various proteins in replication fork reversal and restart with special emphasis on RecQ helicases. Involvement of other proteins like PARP1, recombinase rad51, SWI/SNF complex has also been discussed. Since RecQ helicases play a central role in the DNA damage response following chemotherapeutic treatment, we propose that targeting these helicases can emerge as an alternative to available intervention strategies. We have also summarized the current research status of available RecQ inhibitors and siRNA based therapeutic approaches that targets RecQ helicases. In summary, our review gives an overview of the DNA damage responses involving replication fork reversal and provides new directions for the development of more efficient and sustainable chemotherapeutic approaches.

Function of Mte1/ZGRF1 in Homologous Recombination

Fanconi anemia (FA) is a human genetic disorder that causes organ defects, physical abnormalities, bone marrow failure, and increased risk of malignancy. These phenotypes result from an impaired response to DNA damage due to the dysfunction of a cluster of FA proteins. Among these, human FANCM (budding yeast ortholog, Mph1) functions in DNA replication fork (RF) repair and DNA double‐strand break (DSB) repair by homologous recombination (HR). In a recent study, Mte1 in Saccharomyces cerevisiae was found to protect the Rad51‐made D‐loop, essential for HR, from disassociation by Mph1 (Xue et al. 2016). Our preliminary data showed that Mte1 interacts with Rad51 in vitro. However, the biological role of this interaction remains unknown. The goal of this project is to examine the effects of 2 different binding site mutants of Mte1, where mutations are hypothesized to affect binding of Rad51. Quick change site directed mutagenesis will be used to create the 3 mutants by changing 2 different phenylalanine side groups to alanine at positions 156, 169, and 156/169 combined. The Rad51 interaction of each mutant will be evaluated via in vitro pull down assay. The D‐loop binding activity of each mutant will be evaluated via electrophoresis mobility shift assays (EMSA). Mutation effects on Rad51‐made D‐loop formations can direct further studies investigating proteins required for HR. Evidence suggest that human ZGRF1 is functionally related to Mte1 due to N‐terminus conservation between the two. However, little is known about the role and function that ZGRF1 plays in HR in humans.Support or Funding InformationResearch supported by NIH R21 #ES028792 awarded to XX. JD supported by NIH R25 Bridge to Doctorate Program (GM102783)

Cohesin Is Out for Stalled Replication Fork Restart.

Replication stress underlies many genomic alterations in cancer cells. In this issue of Developmental Cell, Benedict etal. show that WAPL-dependent cohesin removal is needed to restart DNA synthesis at stalled forks and promote survival following replication stress, uncovering an unexpected link between stress and sister chromatid cohesion loss.

WAPL-Dependent Repair of Damaged DNA Replication Forks Underlies Oncogene-Induced Loss of Sister Chromatid Cohesion.

Premature loss of sister chromatid cohesion at metaphase is a diagnostic marker for different cohesinopathies. Here, we report that metaphase spreads of many cancer cell lines also show premature loss of sister chromatid cohesion. Cohesion loss occurs independently of mutations in cohesion factors including SA2, a cohesin subunit frequently inactivated in cancer. In untransformed cells, induction of DNA replication stress by activation of oncogenes or inhibition of DNA replication is sufficient to trigger sister chromatid cohesion loss. Importantly, cell growth under conditions of replication stress requires the cohesin remover WAPL. WAPL promotes rapid RAD51-dependent repair and restart of broken replication forks. We propose that active removal ofcohesin allows cancer cells to overcome DNA replication stress. This leads to oncogene-induced cohesion loss from newly synthesized sister chromatids that may contribute to genomic instability and likely represents a targetable cancer cell vulnerability.

The oncoprotein DEK affects the outcome of PARP1/2 inhibition during mild replication stress.

DNA replication stress is a major source of genomic instability and is closely linked to tumor formation and progression. Poly(ADP-ribose)polymerases1/2 (PARP1/2) enzymes are activated in response to replication stress resulting in poly(ADP-ribose) (PAR) synthesis. PARylation plays an important role in the remodelling and repair of impaired replication forks, providing a rationale for targeting highly replicative cancer cells with PARP1/2 inhibitors. The human oncoprotein DEK is a unique, non-histone chromatin architectural protein whose deregulated expression is associated with the development of a wide variety of human cancers. Recently, we showed that DEK is a high-affinity target of PARylation and that it promotes the progression of impaired replication forks. Here, we investigated a potential functional link between PAR and DEK in the context of replication stress. Under conditions of mild replication stress induced either by topoisomerase1 inhibition with camptothecin or nucleotide depletion by hydroxyurea, we found that the effect of acute PARP1/2 inhibition on replication fork progression is dependent on DEK expression. Reducing DEK protein levels also overcomes the restart impairment of stalled forks provoked by blocking PARylation. Non-covalent DEK-PAR interaction via the central PAR-binding domain of DEK is crucial for counteracting PARP1/2 inhibition as shown for the formation of RPA positive foci in hydroxyurea treated cells. Finally, we show by iPOND and super resolved microscopy that DEK is not directly associated with the replisome since it binds to DNA at the stage of chromatin formation. Our report sheds new light on the still enigmatic molecular functions of DEK and suggests that DEK expression levels may influence the sensitivity of cancer cells to PARP1/2 inhibitors.

MiR223-3p promotes synthetic lethality in BRCA1-deficient cancers

Defects in DNA repair give rise to genomic instability, leading to neoplasia. Cancer cells defective in one DNA repair pathway can become reliant on remaining repair pathways for survival and proliferation. This attribute of cancer cells can be exploited therapeutically, by inhibiting the remaining repair pathway, a process termed synthetic lethality. This process underlies the mechanism of the Poly-ADP ribose polymerase-1 (PARP1) inhibitors in clinical use, which target BRCA1 deficient cancers, which is indispensable for homologous recombination (HR) DNA repair. HR is the major repair pathway for stressed replication forks, but when BRCA1 is deficient, stressed forks are repaired by back-up pathways such as alternative nonhomologous end-joining (aNHEJ). Unlike HR, aNHEJ is nonconservative, and can mediate chromosomal translocations. In this study we have found that miR223-3p decreases expression of PARP1, CtIP, and Pso4, each of which are aNHEJ components. In most cells, high levels of microRNA (miR) 223-3p repress aNHEJ, decreasing the risk of chromosomal translocations. Deletion of the miR223 locus in mice increases PARP1 levels in hematopoietic cells and enhances their risk of unprovoked chromosomal translocations. We also discovered that cancer cells deficient in BRCA1 or its obligate partner BRCA1-Associated Protein-1 (BAP1) routinely repress miR223-3p to permit repair of stressed replication forks via aNHEJ. Reconstituting the expression of miR223-3p in BRCA1- and BAP1-deficient cancer cells results in reduced repair of stressed replication forks and synthetic lethality. Thus, miR223-3p is a negative regulator of the aNHEJ DNA repair and represents a therapeutic pathway for BRCA1- or BAP1-deficient cancers.

Expression and Characterization of YoaA, a Putative Helicase in Bacteria, Involved in Repairing Blocks to DNA Replication

While cells have efficient pathways for repairing damage to DNA, some DNA damage avoids repair and is found in DNA replication. Because the DNA polymerases that replicate the genome are high fidelity enzymes, DNA damage blocks DNA synthesis and ultimately progression of the replication fork. It is known that cells would not be able to survive without mechanisms to repair these replication blocks and to restart replication. With a genetic screen, our collaborators at Lovett laboratory, using 3’azidothymidine (AZT) as a tool to inhibit replication, identified a novel gene in Escherichia coli, yoaA, which was required along with holC to provide cells with tolerance to AZT. HolC is a protein subunit of the E.coli DNA polymerase III holoenzyme that does the bulk of synthesis during DNA replication, and yoaA binds HolC. Based on sequence, the yoaA gene encodes an iron-sulfur (Fe-S) helicases. E.coli contains a second Fe-S helicase, DinG, and human cells contain four Fe-S helicases, XPD, FANCJ, RTEL1, and ChlR1, that are involved in DNA repair. The overall goal of this project is to express YoaA protein in soluble form and characterize its biochemical activities to determine how YoaA aids in DNA replication fork repair. In our initial studies, we made fluorescent labeled DNA for helicase assays and tested them which allowed us to work towards our long term goals of determining: 1) whether YoaA is DNA helicase and what are the best substrates for YoaA, and 2) how HolC affects YoaA activities. We have subcloned the yoaA gene into different expression vectors to express YoaA with and without affinity tags, and to co-express YoaA with HolC. We are developing strategies to purify YoaA alone and in a complex with HolC. We have subcloned the yoaA gene into different expression vectors to express yoaA with pCOLADuet. We are developing strategies to purify YoaA alone. The initial results working towards this are presented.

The NuA4 acetyltransferase and histone H4 acetylation promote replication recovery after topoisomerase I-poisoning

BackgroundHistone acetylation plays an important role in DNA replication and repair because replicating chromatin is subject to dynamic changes in its structures. However, its precise mechanism remains elusive. In this report, we describe roles of the NuA4 acetyltransferase and histone H4 acetylation in replication fork protection in the fission yeast Schizosaccharomyces pombe.ResultsDownregulation of NuA4 subunits renders cells highly sensitive to camptothecin, a compound that induces replication fork breakage. Defects in NuA4 function or mutations in histone H4 acetylation sites lead to impaired recovery of collapsed replication forks and elevated levels of Rad52 DNA repair foci, indicating the role of histone H4 acetylation in DNA replication and fork repair. We also show that Vid21 interacts with the Swi1–Swi3 replication fork protection complex and that Swi1 stabilizes Vid21 and promotes efficient histone H4 acetylation. Furthermore, our genetic analysis demonstrates that loss of Swi1 further sensitizes NuA4 and histone H4 mutant cells to replication fork breakage.ConclusionConsidering that Swi1 plays a critical role in replication fork protection, our results indicate that NuA4 and histone H4 acetylation promote repair of broken DNA replication forks.

Abstract 3363: PUM3 is a triple-negative breast cancer dependency gene that functions in replication fork restart and repair

Abstract Triple-Negative Breast Cancers (TNBC) are characterized by high levels of structural chromosome alterations and harbor mutational scars of defects in homologous recombination. Importantly, by acting either as drivers, or as tumor specific dependencies mutated genes in these regions represent potential therapeutic targets In search of such genes we identified that knockdown of the PUM3 gene, which is contained in a 9p24 recurrent ampliicon, impaired the growth of a subset of breast cancer but not mammary epithelial cell lines. Furthermore, the growth defects observed upon PUM3 knockdown in SUM149, a BRCA1 deficient cell line, were reversed by reactivation of BRCA1's function suggesting a BRCA1-deficiency associated dependency. PUM3 knockdown led to an increase in gH2Ax and 53BP1 nuclear foci, impaired restart of stalled replication forks, and decreased RAD51 mediated repair of collapsed replication forks. As a result PUM3 depleted cells have reduced DNA replication rate and slowed cell cycle progression. Knockdown of PUM3 also impaired replication stress induced PARylation and PAR foci formation and PUM3 and PARP1 knockdown showed phenotypic epistasis with respect to cell growth suggesting a potential mechanism for PUM3s replication fork related functions. PUM3 forms a complex with gH2Ax and the replication stalling agent hyroxyurea and PARP-inhibitors increases the nuclear concentration of PUM3. Furthermore PUM3 knockdown increased the sensitivity of breast cancer cells to these agents indicating a role for PUM3 in the repair of lesions generating by these DNA damaging agents. By studying PUM3 we have identified a novel player in replication fork restart and repair whose activity may be particularly relevant in homologous recombination deficient TNBCs. A greater understanding of PUM3's function and the pathways that regulate it may identify novel therapeutic targets for TNBCs. Importantly, direct or indirect modulation of PUM's functions may potentiate the effects of replication stress inducing therapies such as PARP-inhibitors. Citation Format: Daniel Weekes, Elodie Noel, Callum Walker, Nick Balan, Vandna Shah, Bhavna Sidhu, Anna Pardix, Stephen Pettitt, Christopher J. Lord, Anita Grigoriadis, Tutt N. Andrew. PUM3 is a triple-negative breast cancer dependency gene that functions in replication fork restart and repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3363.

RNase H eliminates R-loops that disrupt DNA replication but is nonessential for efficient DSB repair.

In Saccharomyces cerevisiae, genome stability depends on RNases H1 and H2, which remove ribonucleotides from DNA and eliminate RNA-DNA hybrids (R-loops). In Schizosaccharomyces pombe, RNase H enzymes were reported to process RNA-DNA hybrids produced at a double-strand break (DSB) generated by I-PpoI meganuclease. However, it is unclear if RNase H is generally required for efficient DSB repair in fission yeast, or whether it has other genome protection roles. Here, we show that S. pombe rnh1∆ rnh201∆ cells, which lack the RNase H enzymes, accumulate R-loops and activate DNA damage checkpoints. Their viability requires critical DSB repair proteins and Mus81, which resolves DNA junctions formed during repair of broken replication forks. "Dirty" DSBs generated by ionizing radiation, as well as a "clean" DSB at a broken replication fork, are efficiently repaired in the absence of RNase H. RNA-DNA hybrids are not detected at a reparable DSB formed by fork collapse. We conclude that unprocessed R-loops collapse replication forks in rnh1∆ rnh201∆ cells, but RNase H is not generally required for efficient DSB repair.