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Targeting CDC20 can enhance the radiosensitivity of tumor cells, but the function and mechanism of CDC20 on DNA damage repair response remains vague. To examine that issue, tumor cell lines, including KYSE200, KYSE450, and HCT116, were utilized to detect the expression, function, and underlying mechanism of CDC20 in radio-chemoresistance. Western blot and immunofluorescence staining were employed to confirm CDC20 expression and location, and radiation could upregulate the expression of CDC20 in the cell nucleus. The homologous recombination (HR) and non-homologous end joining (NHEJ) reporter gene systems were utilized to explore the impact of CDC20 on DNA damage repair, indicating that CDC20 could promote HR repair and radio/chemo-resistance. In the early stages of DNA damage, CDC20 stabilizes the RPA1 protein through protein-protein interactions, activating the ATR-mediated signaling cascade, thereby aiding in genomic repair. In the later stages, CDC20 assists in the subsequent steps of damage repair by the ubiquitin-mediated degradation of RPA1. CCK-8 and colony formation assay were used to detect the function of CDC20 in cell vitality and proliferation, and targeting CDC20 can exacerbate the increase in DNA damage levels caused by cisplatin or etoposide. A tumor xenograft model was conducted in BALB/c-nu/nu mice to confirm the function of CDC20 in vivo, confirming the in vitro results. In conclusion, this study provides further validation of the potential clinical significance of CDC20 as a strategy to overcome radio-chemoresistance via uncovering a novel role of CDC20 in regulating RPA1 during DNA damage repair.

The helicase MCM and the ribonucleotide reductase RNR are the complexes that provide the substrates (ssDNA templates and dNTPs, respectively) for DNA replication. Here, we demonstrate that MCM interacts physically with RNR and some of its regulators, including the kinase Dun1. These physical interactions encompass small subpopulations of MCM and RNR, are independent of the major subcellular locations of these two complexes, augment in response to DNA damage and, in the case of the Rnr2 and Rnr4 subunits of RNR, depend on Dun1. Partial disruption of the MCM/RNR interactions impairs the release of Rad52 -but not RPA-from the DNA repair centers despite the lesions are repaired, a phenotype that is associated with hypermutagenesis but not with alterations in the levels of dNTPs. These results suggest that a specifically regulated pool of MCM and RNR complexes plays non-canonical roles in genetic stability preventing persistent Rad52 centers and hypermutagenesis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Yáñez-Vilches et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The repair of DNA double-strand breaks (DSBs) occurs in chromatin, and several histone posttranslational modifications have been implicated in the process. Modifications of the histone H2A N-terminal tail have also been linked to DNA damage response, through acetylation or ubiquitination of lysine residues that regulate repair pathway choice. Here, we characterize a new DNA damage-induced phosphorylation on chromatin, at serine 15 of H2A in yeast. We show that this SQ motif functions independently of the classical S129 C-terminal site (γ-H2A) and that mutant-mimicking constitutive phosphorylation increases cell sensitivity to DNA damage. H2AS129ph is induced by Tel1 ATM and Mec1 ATR , and the loss of Lcd1 ATRIP or Mec1 signaling decreases γ-H2A spreading distal to the DSB. In contrast, H2AS15ph is completely dependent on Lcd1 ATRIP , indicating that this modification only happens when end resection is engaged. This is supported by an increase in replication protein A (RPA) and a decrease in DNA signal near the DSB in H2A-S15E phosphomimic mutants, indicating higher resection. In mammals, this serine is replaced by a lysine (H2AK15) which undergoes an acetyl-monoubiquityl switch to regulate binding of 53BP1 and resection. This regulation seems functionally conserved with budding yeast H2AS15 and 53BP1-homolog Rad9, using different posttranslational modifications between organisms but achieving the same function.

Proper activation of DNA repair pathways in response to DNA replication stress is critical for maintaining genomic integrity. Due to the complex nature of the replication fork (RF), problems at the RF require multiple proteins, some of which remain unidentified, for resolution. In this study, we identified the N-methyl-D-aspartate receptor synaptonuclear signaling and neuronal migration factor (NSMF) as a key replication stress response factor that is important for ataxia telangiectasia and Rad3-related protein (ATR) activation. NSMF localizes rapidly to stalled RFs and acts as a scaffold to modulate replication protein A (RPA) complex formation with cell division cycle 5-like (CDC5L) and ATR/ATR-interacting protein (ATRIP). Depletion of NSMF compromised phosphorylation and ubiquitination of RPA2 and the ATR signaling cascade, resulting in genomic instability at RFs under DNA replication stress. Consistently, NSMF knockout mice exhibited increased genomic instability and hypersensitivity to genotoxic stress. NSMF deficiency in human and mouse cells also caused increased chromosomal instability. Collectively, these findings demonstrate that NSMF regulates the ATR pathway and the replication stress response network for genome maintenance and cell survival., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The DNA damage checkpoint induces many cellular changes to cope with genotoxic stress. However, persistent checkpoint signaling can be detrimental to growth partly due to blockage of cell cycle resumption. Checkpoint dampening is essential to counter such harmful effects, but its mechanisms remain to be understood. Here, we show that the DNA helicase Srs2 removes a key checkpoint sensor complex, RPA, from chromatin to down-regulate checkpoint signaling in budding yeast. The Srs2 and RPA antagonism is supported by their numerous suppressive genetic interactions. Importantly, moderate reduction of RPA binding to single-strand DNA (ssDNA) rescues hypercheckpoint signaling caused by the loss of Srs2 or its helicase activity. This rescue correlates with a reduction in the accumulated RPA and the associated checkpoint kinase on chromatin in srs2 mutants. Moreover, our data suggest that Srs2 regulation of RPA is separable from its roles in recombinational repair and critically contributes to genotoxin resistance. We conclude that dampening checkpoint by Srs2-mediated RPA recycling from chromatin aids cellular survival of genotoxic stress and has potential implications in other types of DNA transactions., Competing Interests: The authors declare no competing interest.

The histone chaperone FACT is upregulated during mammary tumorigenesis and necessary for the viability and growth of breast tumor cells. We established that only proliferating tumor cells are sensitive to FACT knockdown, suggesting that FACT functions during DNA replication in tumor cells but not in normal cells. We hypothesized that the basal level of replication stress defines the FACT dependence of cells. Using genetic and chemical tools, we demonstrated that FACT is needed to overcome replication stress. In the absence of FACT during replication stress, the MCM2-7 helicase dissociates from chromatin, resulting in the absence of ssDNA accumulation, RPA binding, and activation of the ATR/CHK1 checkpoint response. Without this response, stalled replication forks are not stabilized, and new origin firing cannot be prevented, leading to the accumulation of DNA damage and cell death. Thus, we propose a novel role for FACT as a factor preventing helicase dissociation from chromatin during replication stress.

Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maintain genomic stability. Here we characterize C17orf53/MCM8IP, an OB-fold containing protein that binds ssDNA, as a DNA repair factor involved in HR. MCM8IP-deficient cells exhibit HR defects, especially in long-tract gene conversion, occurring downstream of RAD51 loading, consistent with a role for MCM8IP in HR-dependent DNA synthesis. Moreover, loss of MCM8IP confers cellular sensitivity to crosslinking agents and PARP inhibition. Importantly, we report that MCM8IP directly associates with MCM8-9, a helicase complex mutated in primary ovarian insufficiency, and RPA1. We additionally show that the interactions of MCM8IP with MCM8-9 and RPA facilitate HR and promote replication fork progression and cellular viability in response to treatment with crosslinking agents. Mechanistically, MCM8IP stimulates the helicase activity of MCM8-9. Collectively, our work identifies MCM8IP as a key regulator of MCM8-9-dependent DNA synthesis during DNA recombination and replication.

BRCA1/2 help maintain genomic integrity by stabilizing stalled forks. Here, we identify the E3 ligase RFWD3 as an essential modulator of stalled fork stability in BRCA2-deficient cells and show that codepletion of RFWD3 rescues fork degradation, collapse, and cell sensitivity upon replication stress. Stalled forks in BRCA2-deficient cells accumulate phosphorylated and ubiquitinated replication protein A (ubq-pRPA), the latter of which is mediated by RFWD3. Generation of this intermediate requires SMARCAL1, suggesting that it depends on stalled fork reversal. We show that in BRCA2-deficient cells, rescuing fork degradation might not be sufficient to ensure fork repair. Depleting MRE11 in BRCA2-deficient cells does block fork degradation, but it does not prevent fork collapse and cell sensitivity in the presence of replication stress. No such ubq-pRPA intermediate is formed in BRCA1-deficient cells, and our results suggest that BRCA1 may function upstream of BRCA2 in the stalled fork repair pathway. Collectively, our data uncover a novel mechanism by which RFWD3 destabilizes forks in BRCA2-deficient cells., (© 2020 Duan et al.)

Chromosomes are constantly damaged by exogenous and endogenous factors. To cope with DNA damage, eukaryotic cells are equipped with three phosphatidylinositol 3-kinase-related kinases (PIKKs), such as ATM, ATR, and DNA-PK. PIKKs are structurally related to phosphatidylinositol 3-kinase (lipid kinase), however possess protein kinase activities. The Mre11-Rad50-Nbs1 and the Ku complex interact with and activate ATM and DNA-PKcs at double-stranded DNA breaks (DSBs), respectively. In contrast, ATR responds to various types of DNA lesions by interacting with replication protein A (RPA)-covered single-stranded DNA (ssDNA). Several lines of evidence have established a model in which ATR is activated by interacting with ATR activating proteins including TopBP1 and ETAA1 at DNA lesions in humans, yet the interaction of ATR with RPA-covered ssDNA does not result in ATR activation. In budding yeast, the Mec1-Ddc2 complex (Mec1-Ddc2) corresponds to ATR-ATRIP. Similar to ATR, Mec1 activation is accomplished by interactions with Mec1 activating proteins, which are Ddc1, Dpb11 (TopBP1 homolog) and Dna2. However, recent studies provide results supporting the idea that Mec1 ATR is also activated by interacting with RPA-covered ssDNA tracts. These observations suggest that all the ATM, ATR, DNA-PK family proteins can be activated immediately upon DNA damage recognition.

Cellular processes, such as DNA replication, recombination and transcription, require DNA strands separation and single-stranded DNA is formation. The single-stranded DNA is promptly wrapped by human single-stranded DNA binding proteins, replication protein A (RPA) complex. RPA binding not only prevent nuclease degradation and annealing, but it also coordinates cell-cycle checkpoint activation and DNA repair. However, RPA binding offers little protection against the chemical modification of DNA bases. This review focuses on the type of DNA base damage that occurs in single-stranded DNA and how the damage is rectified in human cells. The discovery of DNA repair proteins, such as ALKBH3, AGT, UNG2, NEIL3, being able to repair the damaged base in the single-stranded DNA, renewed the interest to study single-stranded DNA repair. These mechanistically different proteins work independently from each other with the overarching goal of increasing fidelity of recombination and promoting error-free replication., Competing Interests: Declaration of Competing Interest The author declares that there are no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Severe side effects often restrict clinical application of the widely used chemotherapeutic drug doxorubicin. In order to decrease required substance concentrations, new concepts for successful combination therapy are needed. Since doxorubicin causes DNA damage, combination with compounds that modulate DNA repair could be a promising strategy. Very recently, a role of nuclear actin for DNA damage repair has been proposed, making actin a potential target for cancer therapy in combination with DNA-damaging therapeutics. This is of special interest, since actin-binding compounds have not yet found their way into clinics. We find that low-dose combination treatment of doxorubicin with the actin polymerizer chondramide B (ChB) synergistically inhibits tumor growth in vivo. On the cellular level we demonstrate that actin binders inhibit distinctive double strand break (DSB) repair pathways. Actin manipulation impairs the recruitment of replication factor A (RPA) to the site of damage, a process crucial for homologous recombination. In addition, actin binders reduce autophosphorylation of DNA-dependent protein kinase (DNA-PK) during nonhomologous end joining. Our findings substantiate a direct involvement of actin in nuclear DSB repair pathways, and propose actin as a therapeutic target for combination therapy with DNA-damaging agents such as doxorubicin.

Understanding the molecular dynamics of DNA replication in vivo has been a formidable challenge requiring the development of advanced technologies. Over the past 50 years or so, studies involving DNA autoradiography in bacterial cells have led to sophisticated DNA tract analyses in human cells to characterize replication dynamics at the single-molecule level. Our own lab has used DNA fiber analysis to characterize replication in helicase-deficient human cells. This work led us to propose a model in which the human DNA helicase RECQ1 acts as a governor of the single-stranded DNA binding protein RPA and regulates its bioavailability for DNA synthesis. We have also used the DNA fiber approach to investigate the interactive role of DDX11 helicase with a replication fork protection protein (Timeless) in human cells when they are under pharmacologically induced stress. In this methods chapter, we present a step-by-step protocol for the single-molecule DNA fiber assay. We describe experimental designs to study replication stress and staining patterns from pulse-chase labeling experiments to address the dynamics of replication forks in stressed cells.

ATR kinase is activated by RPA-coated single-stranded DNA (ssDNA) to orchestrate DNA damage responses. Here we show that ATR inhibition differs from ATR loss. Mouse model expressing kinase-dead ATR (Atr +/KD ), but not loss of ATR (Atr +/- ), displays ssDNA-dependent defects at the non-homologous region of X-Y chromosomes during male meiosis leading to sterility, and at telomeres, rDNA, and fragile sites during mitosis leading to lymphocytopenia. Mechanistically, we find that ATR kinase activity is necessary for the rapid exchange of ATR at DNA-damage-sites, which in turn promotes CHK1-phosphorylation. ATR-KD, but not loss of ATR, traps a subset of ATR and RPA on chromatin, where RPA is hyper-phosphorylated by ATM/DNA-PKcs and prevents downstream repair. Consequently, Atr +/KD cells have shorter inter-origin distances and are vulnerable to induced fork collapses, genome instability and mitotic catastrophe. These results reveal mechanistic differences between ATR inhibition and ATR loss, with implications for ATR signaling and cancer therapy.

Translesion DNA synthesis (TLS) is one mode of DNA damage tolerance that uses specialized DNA polymerases to replicate damaged DNA. DNA polymerase η (Polη) is well known to facilitate TLS across ultraviolet (UV) irradiation and mutations in POLH are implicated in skin carcinogenesis. However, the basis for recruitment of Polη to stalled replication forks is not completely understood. In this study, we used an affinity purification approach to isolate a Polη-containing complex and have identified SART3, a pre-mRNA splicing factor, as a critical regulator to modulate the recruitment of Polη and its partner RAD18 after UV exposure. We show that SART3 interacts with Polη and RAD18 via its C-terminus. Moreover, SART3 can form homodimers to promote the Polη/RAD18 interaction and PCNA monoubiquitination, a key event in TLS. Depletion of SART3 also impairs UV-induced single-stranded DNA (ssDNA) generation and RPA focus formation, resulting in an impaired Polη recruitment and a higher mutation frequency and hypersensitivity after UV treatment. Notably, we found that several SART3 missense mutations in cancer samples lessen its stimulatory effect on PCNA monoubiquitination. Collectively, our findings establish SART3 as a novel Polη/RAD18 association regulator that protects cells from UV-induced DNA damage, which functions in a RNA binding-independent fashion.

RAD51 recombinase assembles on single-stranded (ss)DNA substrates exposed by DNA end-resection to initiate homologous recombination (HR), a process fundamental to genome integrity. RAD51 assembly has been characterized using purified proteins, but its ultrastructural topography in the cell nucleus is unexplored. Here, we combine cell genetics with single-molecule localization microscopy and a palette of bespoke analytical tools, to visualize molecular transactions during RAD51 assembly in the cellular milieu at resolutions approaching 30-40 nm. In several human cell types, RAD51 focalizes in clusters that progressively extend into long filaments, which abut-but do not overlap-with globular bundles of replication protein A (RPA). Extended filaments alter topographically over time, suggestive of succeeding steps in HR. In cells depleted of the tumor suppressor protein BRCA2, or overexpressing its RAD51-binding BRC repeats, RAD51 fails to assemble at damage sites, although RPA accumulates unhindered. By contrast, in cells lacking a BRCA2 carboxyl (C)-terminal region targeted by cancer-causing mutations, damage-induced RAD51 assemblies initiate but do not extend into filaments. We suggest a model wherein RAD51 assembly proceeds concurrently with end-resection at adjacent sites, via an initiation step dependent on the BRC repeats, followed by filament extension through the C-terminal region of BRCA2.

Background: Viral protein R (Vpr) is an accessory protein of HIV-1, which is potentially involved in the infection of macrophages and the induction of the ataxia-telangiectasia and Rad3-related protein (ATR)-mediated DNA damage response (DDR). It was recently proposed that the SLX4 complex of structure-specific endonuclease is involved in Vpr-induced DDR, which implies that aberrant DNA structures are responsible for this phenomenon. However, the mechanism by which Vpr alters the DNA structures remains unclear., Results: We found that Vpr unwinds double-stranded DNA (dsDNA) and invokes the loading of RPA70, which is a single-stranded DNA-binding subunit of RPA that activates the ATR-dependent DDR. We demonstrated that Vpr influenced RPA70 to accumulate in the corresponding region utilizing the LacO/LacR system, in which Vpr can be tethered to the LacO locus. Interestingly, RPA70 recruitment required chromatin remodelling via Vpr-mediated ubiquitination of histone H2B. On the contrary, Q65R mutant of Vpr, which lacks ubiquitination activity, was deficient in both chromatin remodelling and RPA70 loading on to the chromatin. Moreover, Vpr-induced unwinding of dsDNA coincidently resulted in the accumulation of negatively supercoiled DNA and covalent complexes of topoisomerase 1 and DNA, which caused DNA double-strand breaks (DSBs) and DSB-directed integration of proviral DNA. Lastly, we noted the dependence of Vpr-promoted HIV-1 infection in resting macrophages on topoisomerase 1., Conclusions: The findings of this study indicate that Vpr-induced structural alteration of DNA is a primary event that triggers both DDR and DSB, which ultimately contributes to HIV-1 infection.

Background and Purpose: Neoadjuvant concurrent chemoradiotherapy (CCRT) is a standard treatment of locally advanced colon cancer cell (CRC). In order to maximize efficacy and minimize toxicity, new drugs have been developed and used in combination with CCRT. Recently, it has been shown that G9a plays a role in mediating phenotypes of cancer stem cells (CSCs). This study aimed to characterize G9a as a biomarker in predicting therapy response to prevent overtreatment and adverse effects in CRC patients., Experimental Design: The primary tumors from 39 patients who received CCRT for rectal cancer were selected. In vivo tumor xenograft models for tumorigenic properties in immunodeficient mice were developed. In vitro stemness ability was performed by tumor-sphere assays, cell response to anti-cancer agents and stemness-related genes analysis., Results: Cells survived from radiation treatment, and displayed high levels of G9a. A significantly positive correlation was shown between G9a and CSCs marker CD133 in locally advanced rectal cancer patients with CCRT. Knockdown of G9a increased the sensitivity of cells to radiation treatment and sensitized cells to DNA damage agents through PP2A-RPA axis., Conclusions: Our study theorized that G9a might serve as a novel target in colon cancer, which offers exciting potential in prediction of response to preoperative chemoradiotherapy in patients with advanced CRC., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In response to DNA damage during S phase, cells slow DNA replication. This slowing is orchestrated by the intra-S checkpoint and involves inhibition of origin firing and reduction of replication fork speed. Slowing of replication allows for tolerance of DNA damage and suppresses genomic instability. Although the mechanisms of origin inhibition by the intra-S checkpoint are understood, major questions remain about how the checkpoint regulates replication forks: Does the checkpoint regulate the rate of fork progression? Does the checkpoint affect all forks, or only those encountering damage? Does the checkpoint facilitate the replication of polymerase-blocking lesions? To address these questions, we have analyzed the checkpoint in the fission yeast Schizosaccharomyces pombe using a single-molecule DNA combing assay, which allows us to unambiguously separate the contribution of origin and fork regulation towards replication slowing, and allows us to investigate the behavior of individual forks. Moreover, we have interrogated the role of forks interacting with individual sites of damage by using three damaging agents-MMS, 4NQO and bleomycin-that cause similar levels of replication slowing with very different frequency of DNA lesions. We find that the checkpoint slows replication by inhibiting origin firing, but not by decreasing fork rates. However, the checkpoint appears to facilitate replication of damaged templates, allowing forks to more quickly pass lesions. Finally, using a novel analytic approach, we rigorously identify fork stalling events in our combing data and show that they play a previously unappreciated role in shaping replication kinetics in response to DNA damage.

Proliferating cells rely on the so-called DNA replication checkpoint to ensure orderly completion of genome duplication, and its malfunction may lead to catastrophic genome disruption, including unscheduled firing of replication origins, stalling and collapse of replication forks, massive DNA breakage, and, ultimately, cell death. Despite many years of intensive research into the molecular underpinnings of the eukaryotic replication checkpoint, the mechanisms underlying the dismal consequences of its failure remain enigmatic. A recent development offers a unifying model in which the replication checkpoint guards against global exhaustion of rate-limiting replication regulators. Here we discuss how such a mechanism can prevent catastrophic genome disruption and suggest how to harness this knowledge to advance therapeutic strategies to eliminate cancer cells that inherently proliferate under increased DNA replication stress., (Copyright © 2017 Elsevier Inc. All rights reserved.)

RFWD3 is a recently identified Fanconi anemia protein FANCW whose E3 ligase activity toward RPA is essential in homologous recombination (HR) repair. However, how RPA ubiquitination promotes HR remained unknown. Here, we identified RAD51, the central HR protein, as another target of RFWD3. We show that RFWD3 polyubiquitinates both RPA and RAD51 in vitro and in vivo. Phosphorylation by ATR and ATM kinases is required for this activity in vivo. RFWD3 inhibits persistent mitomycin C (MMC)-induced RAD51 and RPA foci by promoting VCP/p97-mediated protein dynamics and subsequent degradation. Furthermore, MMC-induced chromatin loading of MCM8 and RAD54 is defective in cells with inactivated RFWD3 or expressing a ubiquitination-deficient mutant RAD51. Collectively, our data reveal a mechanism that facilitates timely removal of RPA and RAD51 from DNA damage sites, which is crucial for progression to the late-phase HR and suppression of the FA phenotype., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Defects in the repair of DNA interstrand crosslinks (ICLs) are associated with the genome instability syndrome Fanconi anemia (FA). Here we report that cells with mutations in RFWD3, an E3 ubiquitin ligase that interacts with and ubiquitylates replication protein A (RPA), show profound defects in ICL repair. An amino acid substitution in the WD40 repeats of RFWD3 (I639K) found in a new FA subtype abolishes interaction of RFWD3 with RPA, thereby preventing RFWD3 recruitment to sites of ICL-induced replication fork stalling. Moreover, single point mutations in the RPA32 subunit of RPA that abolish interaction with RFWD3 also inhibit ICL repair, demonstrating that RPA-mediated RFWD3 recruitment to stalled replication forks is important for ICL repair. We also report that unloading of RPA from sites of ICL induction is perturbed in RFWD3-deficient cells. These data reveal important roles for RFWD3 localization in protecting genome stability and preserving human health., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Drosophila telomeres are sequence-independent structures maintained by transposition to chromosome ends of three specialized retroelements rather than by telomerase activity. Fly telomeres are protected by the terminin complex that includes the HOAP, HipHop, Moi and Ver proteins. These are fast evolving, non-conserved proteins that localize and function exclusively at telomeres, protecting them from fusion events. We have previously suggested that terminin is the functional analogue of shelterin, the multi-protein complex that protects human telomeres. Here, we use electrophoretic mobility shift assay (EMSA) and atomic force microscopy (AFM) to show that Ver preferentially binds single-stranded DNA (ssDNA) with no sequence specificity. We also show that Moi and Ver form a complex in vivo. Although these two proteins are mutually dependent for their localization at telomeres, Moi neither binds ssDNA nor facilitates Ver binding to ssDNA. Consistent with these results, we found that Ver-depleted telomeres form RPA and γH2AX foci, like the human telomeres lacking the ssDNA-binding POT1 protein. Collectively, our findings suggest that Drosophila telomeres possess a ssDNA overhang like the other eukaryotes, and that the terminin complex is architecturally and functionally similar to shelterin., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Nicks are the most common form of DNA damage. The mechanisms of their repair are fundamental to genomic stability and of practical importance for genome engineering. We define two pathways that support homology-directed repair by single-stranded DNA donors. One depends upon annealing-driven strand synthesis and acts at both nicks and double-strand breaks. The other depends upon annealing-driven heteroduplex correction and acts at nicks. Homology-directed repair via these pathways, as well as mutagenic end joining, are inhibited by RAD51 at nicks but largely independent of RAD51 at double-strand breaks. Guidelines for coordinated design of targets and donors for gene correction emerge from definition of these pathways. This analysis further suggests that naturally occurring nicks may have significant recombinogenic and mutagenic potential that is normally inhibited by RAD51 loading onto DNA, thereby identifying a function for RAD51 in maintenance of genomic stability., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonuclease-deficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linker-induced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress.

Embryonic stem cells (ESCs) represent a transient biological state, where pluripotency is coupled with fast proliferation. ESCs display a constitutively active DNA damage response (DDR), but its molecular determinants have remained elusive. Here we show in cultured ESCs and mouse embryos that H2AX phosphorylation is dependent on Ataxia telangiectasia and Rad3 related (ATR) and is associated with chromatin loading of the ssDNA-binding proteins RPA and RAD51. Single-molecule analysis of replication intermediates reveals massive ssDNA gap accumulation, reduced fork speed and frequent fork reversal. All these marks of replication stress do not impair the mitotic process and are rapidly lost at differentiation onset. Delaying the G1/S transition in ESCs allows formation of 53BP1 nuclear bodies and suppresses ssDNA accumulation, fork slowing and reversal in the following S-phase. Genetic inactivation of fork slowing and reversal leads to chromosomal breakage in unperturbed ESCs. We propose that rapid cell cycle progression makes ESCs dependent on effective replication-coupled mechanisms to protect genome integrity.

High expression levels of SLFN11 correlate with the sensitivity of human cancer cells to DNA-damaging agents. However, little is known about the underlying mechanism. Here, we show that SLFN11 interacts directly with RPA1 and is recruited to sites of DNA damage in an RPA1-dependent manner. Furthermore, we establish that SLFN11 inhibits checkpoint maintenance and homologous recombination repair by promoting the destabilization of the RPA-ssDNA complex, thereby sensitizing cancer cell lines expressing high endogenous levels of SLFN11 to DNA-damaging agents. Finally, we demonstrate that the RPA1-binding ability of SLFN11 is required for its function in the DNA damage response. Our findings not only provide novel insight into the molecular mechanisms underlying the drug sensitivity of cancer cell lines expressing SLFN11 at high levels, but also suggest that SLFN11 expression can serve as a biomarker to predict responses to DNA-damaging therapeutic agents., (© 2015 The Authors.)

DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.

Following DNA double-strand breaks, poly(ADP-ribose) (PAR) is quickly and heavily synthesized to mediate fast and early recruitment of a number of DNA damage response factors to the sites of DNA lesions and facilitates DNA damage repair. Here, we found that EXO1, an exonuclease for DNA damage repair, is quickly recruited to the sites of DNA damage via PAR-binding. With further dissection of the functional domains of EXO1, we report that the PIN domain of EXO1 recognizes PAR both in vitro and in vivo and the interaction between the PIN domain and PAR is sufficient for the recruitment. We also found that the R93G variant of EXO1, generated by a single nucleotide polymorphism, abolishes the interaction and the early recruitment. Moreover, our study suggests that the PAR-mediated fast recruitment of EXO1 facilities early DNA end resection, the first step of homologous recombination repair. We observed that other PIN domains could also recognize DNA damage-induced PAR. Taken together, our study demonstrates a novel class of PAR-binding module that plays an important role in DNA damage response., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Topoisomerase 1 (Top1) is the intercellular target of camptothecins (CPTs). CPT blocks DNA religation in the Top1-DNA complex and induces Top1-attached nick DNA lesions. In this study, we demonstrate that excision repair cross complementing 1 protein-xeroderma pigmentosum group F (ERCC1-XPF) endonuclease and replication protein A (RPA) participate in the repair of Top1-attached nick DNA lesions together with other nucleotide excision repair (NER) factors. ERCC1-XPF shows nuclease activity in the presence of RPA on a 3'-phosphotyrosyl bond nick-containing DNA (Tyr-nick DNA) substrate, which mimics a Top1-attached nick DNA lesion. In addition, ERCC1-XPF and RPA form a DNA/protein complex on the nick DNA substrate in vitro, and co-localize in CPT-treated cells in vivo. Moreover, the DNA repair synthesis of Tyr-nick DNA lesions occurred in the presence of NER factors, including ERCC1-XPF, RPA, DNA polymerase delta, flap endonuclease 1 and DNA ligase 1. Therefore, some of the NER repair machinery might be an alternative repair pathway for Top1-attached nick DNA lesions. Clinically, these data provide insights into the potential of ERCC1 as a biomarker during CPT regimens., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Damage repair mechanisms at transcriptionally active sites during the G0/G1 phase are largely unknown. To elucidate these mechanisms, we introduced genome site-specific oxidative DNA damage and determined the role of transcription in repair factor assembly. We find that KU and NBS1 are recruited to damage sites independent of transcription. However, assembly of RPA1, RAD51C, RAD51, and RAD52 at such sites is strictly governed by active transcription and requires both wild-type Cockayne syndrome protein B (CSB) function and the presence of RNA in the G0/G1 phase. We show that the ATPase activity of CSB is indispensable for loading and binding of the recombination factors. CSB counters radiation-induced DNA damage in both cells and zebrafish models. Taken together, our results have uncovered a novel, RNA-based recombination mechanism by which CSB protects genome stability from strand breaks at transcriptionally active sites and may provide insight into the clinical manifestations of Cockayne syndrome.

In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56Ac) is present in newly synthesized histones deposited throughout the genome during DNA replication. The sirtuins Hst3 and Hst4 deacetylate H3K56 after S phase, and virtually all histone H3 molecules are K56 acetylated throughout the cell cycle in hst3∆ hst4∆ mutants. Failure to deacetylate H3K56 causes thermosensitivity, spontaneous DNA damage, and sensitivity to replicative stress via molecular mechanisms that remain unclear. Here we demonstrate that unlike wild-type cells, hst3∆ hst4∆ cells are unable to complete genome duplication and accumulate persistent foci containing the homologous recombination protein Rad52 after exposure to genotoxic drugs during S phase. In response to replicative stress, cells lacking Hst3 and Hst4 also displayed intense foci containing the Rfa1 subunit of the single-stranded DNA binding protein complex RPA, as well as persistent activation of DNA damage-induced kinases. To investigate the basis of these phenotypes, we identified histone point mutations that modulate the temperature and genotoxic drug sensitivity of hst3∆ hst4∆ cells. We found that reducing the levels of histone H4 lysine 16 acetylation or H3 lysine 79 methylation partially suppresses these sensitivities and reduces spontaneous and genotoxin-induced activation of the DNA damage-response kinase Rad53 in hst3∆ hst4∆ cells. Our data further suggest that elevated DNA damage-induced signaling significantly contributes to the phenotypes of hst3∆ hst4∆ cells. Overall, these results outline a novel interplay between H3K56Ac, H3K79 methylation, and H4K16 acetylation in the cellular response to DNA damage., (Copyright © 2015 by the Genetics Society of America.)

Brc1, which was first identified as a high-copy, allele-specific suppressor of a mutation impairing the Smc5-Smc6 holocomplex in Schizosaccharomyces pombe, protects genome integrity during normal DNA replication and when cells are exposed to toxic compounds that stall or collapse replication forks. The C-terminal tandem BRCT (BRCA1 C-terminus) domain of fission yeast Brc1 docks with phosphorylated histone H2A (γH2A)-marked chromatin formed by ATR/Rad3 checkpoint kinase at arrested and damaged replication forks; however, how Brc1 functions in relation to other genome protection modules remains unclear. Here, an epistatic mini-array profile reveals critical requirements for Brc1 in mutants that are defective in multiple DNA damage response pathways, including checkpoint signaling by Rad3-Rad26/ATR-ATRIP kinase, DNA repair by Smc5-Smc6 holocomplex, replication fork stabilization by Mrc1/claspin and Swi1-Swi3/Timeless-Tipin, and control of ubiquitin-regulated proteolysis by the COP9 signalosome (CSN). Exogenous genotoxins enhance these negative genetic interactions. Rad52 and RPA foci are increased in CSN-defective cells, and loss of γH2A increases genotoxin sensitivity, indicating a critical role for the γH2A-Brc1 module in stabilizing replication forks in CSN-defective cells. A negative genetic interaction with the Nse6 subunit of Smc5-Smc6 holocomplex indicates that the DNA repair functions of Brc1 and Smc5-Smc6 holocomplex are at least partially independent. Rtt107, the Brc1 homolog in Saccharomyces cerevisiae, has a very different pattern of genetic interactions, indicating evolutionary divergence of functions and DNA damage responses., (Copyright © 2015 Sánchez et al.)

The Replication Protein A (RPA) complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA (ssDNA) through multiple oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA complex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current understanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications.

The BRCA1 tumor suppressor plays an important role in homologous recombination (HR)-mediated DNA double-strand-break (DSB) repair. BRCA1 is phosphorylated by Chk2 kinase upon γ-irradiation, but the role of Chk2 phosphorylation is not understood. Here, we report that abrogation of Chk2 phosphorylation on BRCA1 delays end resection and the dispersion of BRCA1 from DSBs but does not affect the assembly of Mre11/Rad50/NBS1 (MRN) and CtIP at DSBs. Moreover, we show that BRCA1 is ubiquitinated by SCF(Skp2) and that abrogation of Chk2 phosphorylation impairs its ubiquitination. Our study suggests that BRCA1 is more than a scaffold protein to assemble HR repair proteins at DSBs, but that Chk2 phosphorylation of BRCA1 also serves as a built-in clock for HR repair of DSBs. BRCA1 is known to inhibit Mre11 nuclease activity. SCF(Skp2) activity appears at late G1 and peaks at S/G2, and is known to ubiquitinate phosphodegron motifs. The removal of BRCA1 from DSBs by SCF(Skp2)-mediated degradation terminates BRCA1-mediated inhibition of Mre11 nuclease activity, allowing for end resection and restricting the initiation of HR to the S/G2 phases of the cell cycle.

The causal metabolic pathway and the underlying mechanism between folate deficiency and neural tube defects (NTDs) remain obscure. Thymidylate (dTMP) is catalyzed by thymidylate synthase (TS) using the folate-derived one-carbon unit as the sole methyl donor. This study aims to examine the role of dTMP biosynthesis in the development of neural tube in mice by inhibition of TS via a specific inhibitor, raltitrexed (RTX). Pregnant mice were intraperitoneally injected with various doses of RTX on gestational day 7.5, and embryos were examined for the presence of NTDs on gestational day 11.5. TS activity and changes of dUMP and dTMP levels were measured following RTX treatment at the optimal dose. DNA damage was determined by detection of phosphorylated replication protein A2 (RPA2) and γ-H2AX in embryos with NTDs induced by RTX. Besides, apoptosis and proliferation were also analyzed in RTX-treated embryos with NTDs. We found that NTDs were highly occurred by the treatment of RTX at the optimal dose of 11.5 mg/kg b/w. RTX treatment significantly inhibited TS activity. Meanwhile, dTMP was decreased associated with the accumulation of dUMP in RTX-treated embryos. Phosphorylated RPA2 and γ-H2AX were significantly increased in RTX-treated embryos with NTDs compared to control. More apoptosis and decreased proliferation were also found in embryos with NTDs induced by RTX. These results indicate that impairment of dTMP biosynthesis caused by RTX led to the development of NTDs in mice. DNA damage and imbalance between apoptosis and proliferation may be potential mechanisms.

Analysis of accumulation of repair and checkpoint proteins at repair sites in yeast nuclei has conventionally used chemical agents, ionizing radiation or induction of endonucleases to inflict localized damage. In addition to these methods, similar studies in mammalian cells have used laser irradiation, which has the advantage that damage is inflicted at a specific nuclear region and at a precise time, and this allows accurate kinetic analysis of protein accumulation at DNA damage sites. We show here that it is feasible to use short pulses of near-infrared laser irradiation to inflict DNA damage in subnuclear regions of yeast nuclei by multiphoton absorption. In conjunction with use of fluorescently-tagged proteins, this allows quantitative analysis of protein accumulation at damage sites within seconds of damage induction. PCNA accumulated at damage sites rapidly, such that maximum accumulation was seen approximately 50 s after damage, then levels declined linearly over 200-1000 s after irradiation. RPA accumulated with slower kinetics such that hardly any accumulation was detected within 60 s of irradiation, and levels subsequently increased linearly over the next 900 s, after which levels were approximately constant (up to ca. 2700 s) at the damage site. This approach complements existing methodologies to allow analysis of key damage sensors and chromatin modification changes occurring within seconds of damage inception.

Successful pathogens must be able to swiftly respond to and repair DNA damages inflicted by the host defence. The replication protein A (RPA) complex plays multiple roles in DNA damage response and is regulated by phosphorylation. However, the regulators of RPA phosphorylation remain unclear. Here, we investigated Rfa2 phosphorylation in the pathogenic fungus Candida albicans. Rfa2, a RFA subunit, is phosphorylated when DNA replication is inhibited by hydroxyurea and dephosphorylated during the recovery. By screening a phosphatase mutant library, we found that Pph3 associates with different regulatory subunits to differentially control Rfa2 dephosphorylation in stressed and unstressed cells. Site-directed mutagenesis revealed T11, S18, S29, and S30 being critical for Rfa2 phosphorylation in response to genotoxic insult. We obtained evidence that the genome integrity checkpoint kinase Mec1 and the cyclin-dependent kinase Clb2-Cdc28 mediate Rfa2 phosphorylation. Although cells expressing either a phosphomimetic or a non-phosphorylatable version of Rfa2 had defects, the latter exhibited greater sensitivity to genotoxic challenge, failure to repair DNA damages and to deactivate Rad53-mediated checkpoint pathways in a dosage-dependent manner. These mutants were also less virulent in mice. Our results provide important new insights into the regulatory mechanism and biological significance of Rfa2 phosphorylation in C. albicans., (© 2014 John Wiley & Sons Ltd.)

Replication protein A (RPA) is the main human single-stranded DNA (ssDNA)-binding protein. It is essential for cellular DNA metabolism and has important functions in human cell cycle and DNA damage signaling. RPA is indispensable for accurate homologous recombination (HR)-based DNA double-strand break (DSB) repair and its activity is regulated by phosphorylation and other post-translational modifications. HR occurs only during S and G2 phases of the cell cycle. All three subunits of RPA contain phosphorylation sites but the exact set of HR-relevant phosphorylation sites on RPA is unknown. In this study, a high resolution capillary isoelectric focusing immunoassay, used under native conditions, revealed the isoforms of the RPA heterotrimer in control and damaged cell lysates in G2. Moreover, the phosphorylation sites of chromatin-bound and cytosolic RPA in S and G2 phases were identified by western and IEF analysis with all available phosphospecific antibodies for RPA2. Strikingly, most of the RPA heterotrimers in control G2 cells are phosphorylated with 5 isoforms containing up to 7 phosphates. These isoforms include RPA2 pSer23 and pSer33. DNA damaged cells in G2 had 9 isoforms with up to 14 phosphates. DNA damage isoforms contained pSer4/8, pSer12, pThr21, pSer23, and pSer33 on RPA2 and up to 8 unidentified phosphorylation sites., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Phosphorylation of replication protein A (RPA) by Cdk2 and the checkpoint kinase ATR (ATM and Rad3 related) during replication fork stalling stabilizes the replisome, but how these modifications safeguard the fork is not understood. To address this question, we used single-molecule fiber analysis in cells expressing a phosphorylation-defective RPA2 subunit or lacking phosphatase activity toward RPA2. Deregulation of RPA phosphorylation reduced synthesis at forks both during replication stress and recovery from stress. The ability of phosphorylated RPA to stimulate fork recovery is mediated through the PALB2 tumor suppressor protein. RPA phosphorylation increased localization of PALB2 and BRCA2 to RPA-bound nuclear foci in cells experiencing replication stress. Phosphorylated RPA also stimulated recruitment of PALB2 to single-strand deoxyribonucleic acid (DNA) in a cell-free system. Expression of mutant RPA2 or loss of PALB2 expression led to significant DNA damage after replication stress, a defect accentuated by poly-ADP (adenosine diphosphate) ribose polymerase inhibitors. These data demonstrate that phosphorylated RPA recruits repair factors to stalled forks, thereby enhancing fork integrity during replication stress., (© 2014 Murphy et al.)

DNA polymerase η (pol η) synthesizes across from damaged DNA templates in order to prevent deleterious consequences like replication fork collapse and double-strand breaks. This process, termed translesion synthesis (TLS), is an overall positive for the cell, as cells deficient in pol η display higher mutation rates. This outcome occurs despite the fact that the in vitro fidelity of bypass by pol η alone is moderate to low, depending on the lesion being copied. One possible means of increasing the fidelity of pol η is interaction with replication accessory proteins present at the replication fork. We have previously utilized a bacteriophage based screening system to measure the fidelity of bypass using purified proteins. Here we report on the fidelity effects of a single stranded binding protein, replication protein A (RPA), when copying the oxidative lesion 7,8-dihydro-8-oxo-guanine(8-oxoG) and the UV-induced cis-syn thymine-thymine cyclobutane pyrimidine dimer (T-T CPD). We observed no change in fidelity dependent on RPA when copying these damaged templates. This result is consistent in multiple position contexts. We previously identified single amino acid substitution mutants of pol η that have specific effects on fidelity when copying both damaged and undamaged templates. In order to confirm our results, we examined the Q38A and Y52E mutants in the same full-length construct. We again observed no difference when RPA was added to the bypass reaction, with the mutant forms of pol η displaying similar fidelity regardless of RPA status. We do, however, observe some slight effects when copying undamaged DNA, similar to those we have described previously. Our results indicate that RPA by itself does not affect pol η dependent lesion bypass fidelity when copying either 8-oxoG or T-T CPD lesions.

DNA repair and DNA damage checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA damage are coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5' to 3' exonuclease activity. Here we have used purified core nucleotide excision repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA damage checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and DNA damaged by a UV-mimetic agent to analyze the basic steps of DNA damage checkpoint response in a biochemically defined system. We find that checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the excision gap generated by the excision repair system by the 5' to 3' exonuclease activity of EXO1. We conclude that, in addition to damaged DNA, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of factors for ATR-mediated DNA damage checkpoint response.

DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination-mediated (error-free) and translesion synthesis-mediated (error-prone) DNA damage tolerance pathways. Crucial for error-free DNA damage tolerance is template switching, which depends on the formation and resolution of damage-bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication-associated lesions into the error-free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C-terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication-associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability.

Stalling of replication forks at unrepaired DNA lesions can result in discontinuities opposite the damage in the newly synthesized DNA strand. Translesion synthesis or facilitating the copy from the newly synthesized strand of the sister duplex by template switching can overcome such discontinuities. During template switch, a new primer-template junction has to be formed and two mechanisms, including replication fork reversal and D-loop formation have been suggested. Genetic evidence indicates a major role for yeast Rad5 in template switch and that both Rad5 and its human orthologue, Helicase-like transcription factor (HLTF), a potential tumour suppressor can facilitate replication fork reversal. This study demonstrates the ability of HLTF and Rad5 to form a D-loop without requiring ATP binding and/or hydrolysis. We also show that this strand-pairing activity is independent of RAD51 in vitro and is not mechanistically related to that of another member of the SWI/SNF family, RAD54. In addition, the 3'-end of the invading strand in the D-loop can serve as a primer and is extended by DNA polymerase. Our data indicate that HLTF is involved in a RAD51-independent D-loop branch of template switch pathway that can promote repair of gaps formed during replication of damaged DNA.

PRP19 is a ubiquitin ligase involved in pre-mRNA splicing and the DNA damage response (DDR). Although the role for PRP19 in splicing is well characterized, its role in the DDR remains elusive. Through a proteomic screen for proteins that interact with RPA-coated single-stranded DNA (RPA-ssDNA), we identified PRP19 as a sensor of DNA damage. PRP19 directly binds RPA and localizes to DNA damage sites via RPA, promoting RPA ubiquitylation in a DNA-damage-induced manner. PRP19 facilitates the accumulation of ATRIP, the regulatory partner of the ataxia telangiectasia mutated and Rad3-related (ATR) kinase, at DNA damage sites. Depletion of PRP19 compromised the phosphorylation of ATR substrates, recovery of stalled replication forks, and progression of replication forks on damaged DNA. Importantly, PRP19 mutants that cannot bind RPA or function as an E3 ligase failed to support the ATR response, revealing that PRP19 drives ATR activation by acting as an RPA-ssDNA-sensing ubiquitin ligase during the DDR., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Human cells lacking DNA polymerase η (polη) are sensitive to platinum-based cancer chemotherapeutic agents. Using DNA combing to directly investigate the role of polη in bypass of platinum-induced DNA lesions in vivo, we demonstrate that nascent DNA strands are up to 39% shorter in human cells lacking polη than in cells expressing polη. This provides the first direct evidence that polη modulates replication fork progression in vivo following cisplatin and carboplatin treatment. Severe replication inhibition in individual platinum-treated polη-deficient cells correlates with enhanced phosphorylation of the RPA2 subunit of replication protein A on serines 4 and 8, as determined using EdU labelling and immunofluorescence, consistent with formation of DNA strand breaks at arrested forks in the absence of polη. Polη-mediated bypass of platinum-induced DNA lesions may therefore represent one mechanism by which cancer cells can tolerate platinum-based chemotherapy.

Breast cancer gene 1 (BRCA1) deficient cells not only are hypersensitive to double-strand breaks but also are hypersensitive to UV irradiation and other agents that cause replication blockade; however, the molecular mechanisms behind these latter sensitivities are largely unknown. Here, we report that BRCA1 promotes cell survival by directly regulating the DNA damage tolerance pathway in response to agents that create cross-links in DNA. We show that BRCA1 not only promotes efficient mono- and polyubiquitination of proliferating cell nuclear antigen (PCNA) by regulating the recruitment of replication protein A, Rad18, and helicase-like transcription factor to chromatin but also directly recruits translesion polymerases, such as Polymerase eta and Rev1, to the lesions through protein-protein interactions. Our data suggest that BRCA1 plays a critical role in promoting translesion DNA synthesis as well as DNA template switching.

RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA-XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA-XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA-XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed.

A wide range of environmental and carcinogenic agents form bulky lesions on DNA that are removed from the human genome in the form of short, ∼30-nucleotide oligonucleotides by the process of nucleotide excision repair. Although significant insights have been made regarding the mechanisms of damage recognition, dual incisions, and repair resynthesis during nucleotide excision repair, the fate of the dual incision/excision product is unknown. Using excision assays with both mammalian cell-free extract and purified proteins, we unexpectedly discovered that lesion-containing oligonucleotides are released from duplex DNA in complex with the general transcription and repair factor, Transcription Factor IIH (TFIIH). Release of excision products from TFIIH requires ATP but not ATP hydrolysis, and release occurs slowly, with a t(1/2) of 3.3 h. Excised oligonucleotides released from TFIIH then become bound by the single-stranded binding protein Replication Protein A or are targeted by cellular nucleases. These results provide a mechanism for release and an understanding of the initial fate of excised oligonucleotides during nucleotide excision repair.

In response to DNA damage, many DNA damage factors, such as MDC1 and 53BP1, redistribute to sites of DNA damage. The mechanism governing the turnover of these factors at DNA damage sites, however, remains enigmatic. Here, we show that MDC1 is sumoylated following DNA damage, and the sumoylation of MDC1 at Lys1840 is required for MDC1 degradation and removal of MDC1 and 53BP1 from sites of DNA damage. Sumoylated MDC1 is recognized and ubiquitinated by the SUMO-targeted E3 ubiquitin ligase RNF4. Mutation of the MDC1 Lys 1840 (K1840R) results in impaired CtIP, replication protein A, and Rad51 accumulation at sites of DNA damage and defective homologous recombination (HR). The HR defect caused by MDC1K1840R mutation could be rescued by 53BP1 downregulation. These results reveal the intricate dynamics governing the assembly and disassembly of DNA damage factors at sites of DNA damage for prompt response to DNA damage.