Break-induced Replication Research Articles

Tracking break-induced replication shows that it stalls at roadblocks

Break-induced replication (BIR) repairs one-ended double strand breaks (DSBs) similar to those formed by replication collapse or telomere erosion, and it has been implicated in the initiation of genome instability in cancer and other human disease1,2. Previous studies have defined the enzymes required for BIR1–5; however, understanding of initial and extended BIR synthesis as well as how the migrating D-loop proceeds through known replication roadblocks has been precluded by technical limitations. Here, using a newly developed assay, we demonstrate that BIR synthesis initiates soon after strand invasion and proceeds slower than S-phase replication. Without primase, leading strand synthesis is initiated efficiently, but fails to proceed beyond 30 kb, suggesting that primase is needed for stabilization of the nascent leading strand. DNA synthesis can initiate in the absence of Pif1 or Pol32 but does not proceed efficiently. We demonstrate that interstitial telomeric DNA disrupts and terminates BIR progression. Also, BIR initiation is suppressed by transcription proportionally to the transcription level. Collisions between BIR and transcription lead to mutagenesis and chromosome rearrangements at levels that exceed instabilities induced by transcription during normal replication. Together, these results provide fundamental insights into the mechanism of BIR and on how BIR contributes to genome instability.

Alternative lengthening of telomeres is a self-perpetuating process in ALT-associated PML bodies

Alternative lengthening of telomeres (ALT) is mediated by break-induced replication (BIR), but how BIR is regulated at telomeres is poorly understood. Here, we show that telomeric BIR is a self-perpetuating process. By tethering PML-IV to telomeres, we induced telomere clustering in ALT-associated PML bodies (APBs) and a POLD3-dependent ATR response at telomeres, showing that BIR generates replication stress. Ablation of BLM helicase activity in APBs abolishes telomere synthesis but causes multiple chromosome bridges between telomeres, revealing a function of BLM in processing inter-telomere BIR intermediates. Interestingly, the accumulation of BLM in APBs requires its own helicase activity and POLD3, suggesting that BIR triggers a feedforward loop to further recruit BLM. Enhancing BIR induces PIAS4-mediated TRF2 SUMOylation, and PIAS4 loss deprives APBs of repair proteins and compromises ALT telomere synthesis. Thus, a BLM-driven and PIAS4-mediated feedforward loop operates in APBs to perpetuate BIR, providing a critical mechanism to extend ALT telomeres.

Determining the kinetics of break-induced replication (BIR) by the assay for monitoring BIR elongation rate (AMBER).

A detailed understanding of how homologous recombination proceeds at the molecular level in vivo requires the ability to detect in real time the appearance of specific intermediates of DNA repair. The most detailed analysis of double-strand break (DSB) repair in eukaryotes has come from the study of budding yeast, using an inducible site-specific HO endonuclease to initiate recombination synchronously in nearly all cells of the population. Polymerase chain reaction (PCR) and chromatin immunoprecipitation (ChIP) methods have been used to visualize the timing of the DSB, its resection by 5' to 3' exonucleases, the binding of the Rad51 recombinase and the pairing of the Rad51 filament with a homologous donor sequence. PCR has also been used to identify the next key step: the initiation of new DNA synthesis to extend the invading stand and copy the donor template. In break-induced replication (BIR), there appears to be a very long delay between strand invasion and this primer extension step. Here we describe an alternative method, an assay for monitoring BIR elongation rate (AMBER) based on digital droplet PCR that yields a much earlier time of initial DNA synthesis. We suggest that previous methods have failed to recover the initial long, single-stranded primer extension product that is readily detected by AMBER.

Recombinational repair in the absence of holliday junction resolvases in E. coli.

Cells possess two major DNA damage tolerance pathways that allow them to duplicate their genomes despite the presence of replication blocking lesions: translesion synthesis (TLS) and daughter strand gap repair (DSGR). The TLS pathway involves specialized DNA polymerases that are able to synthesize past DNA lesions while DSGR relies on Recombinational Repair (RR). At least two mechanisms are associated with RR: Homologous Recombination (HR) and RecA Mediated Excision Repair (RAMER). While HR and RAMER both depend on RecFOR and RecA, only the HR mechanism should involve Holliday Junctions (HJs) resolvase reactions. In this study we investigated the role of HJ resolvases, RuvC, TopIII and RusA on the balance between RAMER and HR in E. coli MG1655 derivatives. Using UV survival measurements, we first clearly establish that, in this genetic background, topB and ruvC define two distinct pathways of HJ resolution. We observed that a recA mutant is much more sensitive to UV than the ruvC topB double mutant which is deficient in HR because of its failure to resolve HJs. This difference is independent of RAMER, the SOS system, RusA, and the three TLS DNA polymerases, and may be accounted for by Double Strand Break repair mechanisms such as Synthesis Dependent Strand Annealing, Single Strand Annealing, or Break Induced Replication, which are independent of HJ resolvases. We then used a plasmid-based assay, in which RR is triggered by a single blocking lesion present on a plasmid molecule, to establish that while HR requires topB, ruvC or rusA, RAMER is independent of these genes and, as expected, requires a functional UvrABC excinuclease. Surprisingly, analysis of the RR events in a strain devoid of HJ resolvases reveals that the UvrABC dependent repair of the single lesion present on the plasmid molecule can generate an excision track potentially extending to dozens of nucleotides.

Interstitial telomere sequences disrupt break-induced replication and drive formation of ectopic telomeres.

Break-induced replication (BIR) is a mechanism used to heal one-ended DNA double-strand breaks, such as those formed at collapsed replication forks or eroded telomeres. Instead of utilizing a canonical replication fork, BIR is driven by a migrating D-loop and is associated with a high frequency of mutagenesis. Here we show that when BIR encounters an interstitial telomere sequence (ITS), the machinery frequently terminates, resulting in the formation of an ectopic telomere. The primary mechanism to convert the ITS to a functional telomere is by telomerase-catalyzed addition of telomeric repeats with homology-directed repair serving as a back-up mechanism. Termination of BIR and creation of an ectopic telomere is promoted by Mph1/FANCM helicase, which has the capacity to disassemble D-loops. Other sequences that have the potential to seed new telomeres but lack the unique features of a natural telomere sequence, do not terminate BIR at a significant frequency in wild-type cells. However, these sequences can form ectopic telomeres if BIR is made less processive. Our results support a model in which features of the ITS itself, such as the propensity to form secondary structures and telomeric protein binding, pose a challenge to BIR and increase the vulnerability of the D-loop to dissociation by helicases, thereby promoting ectopic telomere formation.

Replication stress at microsatellites causes DNA double-strand breaks and break-induced replication

Short tandemly repeated DNA sequences, termed microsatellites, are abundant in the human genome. These microsatellites exhibit length instability and susceptibility to DNA double-strand breaks (DSBs) due to their tendency to form stable non-B DNA structures. Replication-dependent microsatellite DSBs are linked to genome instability signatures in human developmental diseases and cancers. To probe the causes and consequences of microsatellite DSBs, we designed a dual-fluorescence reporter system to detect DSBs at expanded (CTG/CAG) n and polypurine/polypyrimidine (Pu/Py) mirror repeat structures alongside the c-myc replication origin integrated at a single ectopic chromosomal site. Restriction cleavage near the (CTG/CAG)100 microsatellite leads to homology-directed single-strand annealing between flanking AluY elements and reporter gene deletion that can be detected by flow cytometry. However, in the absence of restriction cleavage, endogenous and exogenous replication stressors induce DSBs at the (CTG/CAG)100 and Pu/Py microsatellites. DSBs map to a narrow region at the downstream edge of the (CTG)100 lagging-strand template. (CTG/CAG) n chromosome fragility is repeat length-dependent, whereas instability at the (Pu/Py) microsatellites depends on replication polarity. Strikingly, restriction-generated DSBs and replication-dependent DSBs are not repaired by the same mechanism. Knockdown of DNA damage response proteins increases (Rad18, polymerase (Pol) η, Pol κ) or decreases (Mus81) the sensitivity of the (CTG/CAG)100 microsatellites to replication stress. Replication stress and DSBs at the ectopic (CTG/CAG)100 microsatellite lead to break-induced replication and high-frequency mutagenesis at a flanking thymidine kinase gene. Our results show that non-B structure-prone microsatellites are susceptible to replication-dependent DSBs that cause genome instability.

Pif1, RPA, and FEN1 modulate the ability of DNA polymerase δ to overcome protein barriers during DNA synthesis

Successful DNA replication requires carefully regulated mechanisms to overcome numerous obstacles that naturally occur throughout chromosomal DNA. Scattered across the genome are tightly bound proteins, such as transcription factors and nucleosomes, that are necessary for cell function, but that also have the potential to impede timely DNA replication. Using biochemically reconstituted systems, we show that two transcription factors, yeast Reb1 and Tbf1, and a tightly positioned nucleosome, are strong blocks to the strand displacement DNA synthesis activity of DNA polymerase δ. Although the block imparted by Tbf1 can be overcome by the DNA-binding activity of the single-stranded DNA-binding protein RPA, efficient DNA replication through either a Reb1 or a nucleosome block occurs only in the presence of the 5'-3' DNA helicase Pif1. The Pif1-dependent stimulation of DNA synthesis across strong protein barriers may be beneficial during break-induced replication where barriers are expected to pose a problem to efficient DNA bubble migration. However, in the context of lagging strand DNA synthesis, the efficient disruption of a nucleosome barrier by Pif1 could lead to the futile re-replication of newly synthetized DNA. In the presence of FEN1 endonuclease, the major driver of nick translation during lagging strand replication, Pif1-dependent stimulation of DNA synthesis through a nucleosome or Reb1 barrier is prevented. By cleaving the short 5' tails generated during strand displacement, FEN1 eliminates the entry point for Pif1. We propose that this activity would protect the cell from potential DNA re-replication caused by unwarranted Pif1 interference during lagging strand replication.

Cytogenetically visible inversions are formed by multiple molecular mechanisms

Cytogenetically detected inversions are generally assumed to be copy number and phenotypically neutral events. While nonallelic homologous recombination is thought to play a major role, recent data suggest the involvement of other molecular mechanisms in inversion formation. Using a combination of short‐read whole‐genome sequencing (WGS), 10X Genomics Chromium WGS, droplet digital polymerase chain reaction and array comparative genomic hybridization we investigated the genomic structure of 18 large unique cytogenetically detected chromosomal inversions and achieved nucleotide resolution of at least one chromosomal inversion junction for 13/18 (72%). Surprisingly, we observed that seemingly copy number neutral inversions can be accompanied by a copy‐number gain of up to 350 kb and local genomic complexities (3/18, 17%). In the resolved inversions, the mutational signatures are consistent with nonhomologous end‐joining (8/13, 62%) or microhomology‐mediated break‐induced replication (5/13, 38%). Our study indicates that short‐read 30x coverage WGS can detect a substantial fraction of chromosomal inversions. Moreover, replication‐based mechanisms are responsible for approximately 38% of those events leading to a significant proportion of inversions that are actually accompanied by additional copy‐number variation potentially contributing to the overall phenotypic presentation of those patients.

Break-induced replication promotes fragile telomere formation.

TRF1 facilitates the replication of telomeric DNA in part by recruiting the BLM helicase, which can resolve G-quadruplexes on the lagging-strand template. Lagging-strand telomeres lacking TRF1 or BLM form fragile telomeres-structures that resemble common fragile sites (CFSs)-but how they are formed is not known. We report that analogous to CFSs, fragile telomeres in BLM-deficient cells involved double-strand break (DSB) formation, in this case by the SLX4/SLX1 nuclease. The DSBs were repaired by POLD3/POLD4-dependent break-induced replication (BIR), resulting in fragile telomeres containing conservatively replicated DNA. BIR also promoted fragile telomere formation in cells with FokI-induced telomeric DSBs and in alternative lengthening of telomeres (ALT) cells, which have spontaneous telomeric damage. BIR of telomeric DSBs competed with PARP1-, LIG3-, and XPF-dependent alternative nonhomologous end joining (alt-NHEJ), which did not generate fragile telomeres. Collectively, these findings indicate that fragile telomeres can arise from BIR-mediated repair of telomeric DSBs.

Investigation of Break-Induced Replication in Yeast.

Repair of double-strand DNA breaks (DSBs) is important for preserving genomic integrity and stability. Break-induced replication (BIR) is a mechanism aimed to repair one-ended double-strand DNA breaks, similar to those formed by replication fork collapse or by telomere erosion. Unlike S-phase replication, BIR is carried out by a migrating DNA bubble and is associated with conservative inheritance of newly synthesized DNA. This unusual DNA synthesis leads to high level of mutagenesis and chromosomal rearrangements during BIR. Here, we focus on several genetic and molecular methods to investigate BIR using our system in yeast Saccharomyces cerevisiae where BIR is initiated by a site-specific DNA break, and the repair involves two copies of chromosome III.

High-resolution analysis of Merkel Cell Polyomavirus in Merkel Cell Carcinoma reveals distinct integration patterns and suggests NHEJ and MMBIR as underlying mechanisms.

Merkel Cell Polyomavirus (MCPyV) is the etiological agent of the majority of Merkel Cell Carcinomas (MCC). MCPyV positive MCCs harbor integrated, defective viral genomes that constitutively express viral oncogenes. Which molecular mechanisms promote viral integration, if distinct integration patterns exist, and if integration occurs preferentially at loci with specific chromatin states is unknown. We here combined short and long-read (nanopore) next-generation sequencing and present the first high-resolution analysis of integration site structure in MCC cell lines as well as primary tumor material. We find two main types of integration site structure: Linear patterns with chromosomal breakpoints that map closely together, and complex integration loci that exhibit local amplification of genomic sequences flanking the viral DNA. Sequence analysis suggests that linear patterns are produced during viral replication by integration of defective/linear genomes into host DNA double strand breaks via non-homologous end joining, NHEJ. In contrast, our data strongly suggest that complex integration patterns are mediated by microhomology-mediated break-induced replication, MMBIR. Furthermore, we show by ChIP-Seq and RNA-Seq analysis that MCPyV preferably integrates in open chromatin and provide evidence that viral oncogene expression is driven by the viral promoter region, rather than transcription from juxtaposed host promoters. Taken together, our data explain the characteristics of MCPyV integration and may also provide a model for integration of other oncogenic DNA viruses such as papillomaviruses.

Abstract 626: Mechanistic differentiation of targeted DDR agent combinations

Abstract The DNA damage response (DDR) safeguards genome stability and promotes cellular survival by counteracting deleterious consequences of DNA damage. Deficiencies in DDR pathways contribute to genome instability, a hallmark of cancer development, and targeting the DDR is a promising anti-cancer therapy strategy. We describe here in vitro and in vivo studies allowing mechanistic understanding of the benefit of combining our DDR kinase inhibitors (DDRi) targeting ATM (AZD0156), ATR (AZD6738) and DNA-PK (AZD7648) with the PARP inhibitor, olaparib, in homologous recombination-deficient backgrounds. Using the break-induced replication (BIR) assay, which measures homologous-recombination repair (HRR) of DNA double-strand breaks resembling broken replication forks, we observed that ATRi decreases this type of homologous recombination repair by up to 50%, while inhibiting DNA-PK or ATM showed no effect. This provides a mechanistic rationale for combining ATRi with compounds that cause replication-fork collapse, such as PARPi. Indeed, combination of olaparib with the ATRi in BRCA1 mutant UWB1.289 cells enhanced olaparib sensitivity a thousand-fold (GI50 from 590 nM to 0.6 nM). This correlates with the complete tumour growth inhibitions (TGI) observed for the ATRi + olaparib combination in the BRCA2 mutant HBCx-10 triple negative breast cancer PDX model and is consistent with a synergistic increase of genome instability measured by metaphase spread analysis (total aberrations: olaparib; 11, ATRi; 8, combination; 35). DNA-PKi, however, did not synergise with olaparib, showing little enhancement of olaparib single-agent treatment (GI50 from 0.59 μM to 0.33 μM) and no significant impact on the total amount of chromosomal aberrations (total aberrations: olaparib; 11, DNA-PKi ; 3, combination; 14), suggesting that DNA-PKi alone or in combination with olaparib have little impact on BRCA1 mutated tumours. In ATM KO FaDu xenografts, ATRi or DNA-PKi single-agent treatment resulted in TGI, with complete tumour regressions when combined. Focusing on the olaparib combination, metaphase spread analysis of ATRi-treated FaDu ATM KO cells showed an enhancement of DNA replication-dependent chromatid breaks (1.3 vs 0.2 breaks/spread in ATM WT), while DNA-PKi treatment predominantly caused chromosome breaks (1.7 vs 0.2 breaks/spread in ATM WT) that are not dependent on replication. When combined with olaparib, ATRi acted synergistically and in a manner similar to that observed in the BRCA1 mutant cells, while the DNA-PKi + olaparib combination was additive. These data suggest that enhanced olaparib sensitisation of ATM KO cells by ATRi or DNA-PKi combinations occurs through different mechanisms. Together, these findings provide mechanistic differentiation of our DDRi in specific genetic backgrounds. This work informs how to clinically position these agents to benefit the right patients not only as single-agent treatments, but also in combination. Citation Format: Josep V. Forment, Paul Wijnhoven, Antonio Ramos-Montoya, Jacqueline Fok, Valeria Follia, Mercedes Vazquez-Chantada, Rajesh Oderda, Zena Wilson, Alan Lau, Elisabetta Leo, Stephen Durant, Elaine Cadogan, Mark J. O'Connor. Mechanistic differentiation of targeted DDR agent combinations [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 626.

Mechanisms underlying genome instability mediated by formation of foldback inversions in Saccharomyces cerevisiae.

Foldback inversions, also called inverted duplications, have been observed in human genetic diseases and cancers. Here, we used a Saccharomyces cerevisiae genetic system that generates gross chromosomal rearrangements (GCRs) mediated by foldback inversions combined with whole-genome sequencing to study their formation. Foldback inversions were mediated by formation of single-stranded DNA hairpins. Two types of hairpins were identified: small-loop hairpins that were suppressed by MRE11, SAE2, SLX1, and YKU80 and large-loop hairpins that were suppressed by YEN1, TEL1, SWR1, and MRC1. Analysis of CRISPR/Cas9-induced double strand breaks (DSBs) revealed that long-stem hairpin-forming sequences could form foldback inversions when proximal or distal to the DSB, whereas short-stem hairpin-forming sequences formed foldback inversions when proximal to the DSB. Finally, we found that foldback inversion GCRs were stabilized by secondary rearrangements, mostly mediated by different homologous recombination mechanisms including single-strand annealing; however, POL32-dependent break-induced replication did not appear to be involved forming secondary rearrangements.

Folate stress induces SLX1- and RAD51-dependent mitotic DNA synthesis at the fragile X locus in human cells

Folate deprivation drives the instability of a group of rare fragile sites (RFSs) characterized by CGG trinucleotide repeat (TNR) sequences. Pathological expansion of the TNR within the FRAXA locus perturbs DNA replication and is the major causative factor for fragile X syndrome, a sex-linked disorder associated with cognitive impairment. Although folate-sensitive RFSs share many features with common fragile sites (CFSs; which are found in all individuals), they are induced by different stresses and share no sequence similarity. It is known that a pathway (termed MiDAS) is employed to complete the replication of CFSs in early mitosis. This process requires RAD52 and is implicated in generating translocations and copy number changes at CFSs in cancers. However, it is unclear whether RFSs also utilize MiDAS and to what extent the fragility of CFSs and RFSs arises by shared or distinct mechanisms. Here, we demonstrate that MiDAS does occur at FRAXA following folate deprivation but proceeds via a pathway that shows some mechanistic differences from that at CFSs, being dependent on RAD51, SLX1, and POLD3. A failure to complete MiDAS at FRAXA leads to severe locus instability and missegregation in mitosis. We propose that break-induced DNA replication is required for the replication of FRAXA under folate stress and define a cellular function for human SLX1. These findings provide insights into how folate deprivation drives instability in the human genome.

Rad9/53BP1 promotes DNA repair via crossover recombination by limiting the Sgs1 and Mph1 helicases

The DNA damage checkpoint (DDC) is often robustly activated during the homologous recombination (HR) repair of DNA double strand breaks (DSBs). DDC activation controls several HR repair factors by phosphorylation, preventing premature segregation of entangled chromosomes formed during HR repair. The DDC mediator 53BP1/Rad9 limits the nucleolytic processing (resection) of a DSB, controlling the formation of the 3′ single-stranded DNA (ssDNA) filament needed for recombination, from yeast to human. Here we show that Rad9 promotes stable annealing between the recombinogenic filament and the donor template in yeast, limiting strand rejection by the Sgs1 and Mph1 helicases. This regulation allows repair by long tract gene conversion, crossover recombination and break-induced replication (BIR), only after DDC activation. These findings shed light on how cells couple DDC with the choice and effectiveness of HR sub-pathways, with implications for genome instability and cancer.

High-resolution mapping of mitotic DNA synthesis regions and common fragile sites in the human genome through direct sequencing

DNA replication stress, a feature of human cancers, often leads to instability at specific genomic loci, such as the common fragile sites (CFSs). Cells experiencing DNA replication stress may also exhibit mitotic DNA synthesis (MiDAS). To understand the physiological function of MiDAS and its relationship to CFSs, we mapped, at high resolution, the genomic sites of MiDAS in cells treated with the DNA polymerase inhibitor aphidicolin. Sites of MiDAS were evident as well-defined peaks that were largely conserved between cell lines and encompassed all known CFSs. The MiDAS peaks mapped within large, transcribed, origin-poor genomic regions. In cells that had been treated with aphidicolin, these regions remained unreplicated even in late S phase; MiDAS then served to complete their replication after the cells entered mitosis. Interestingly, leading and lagging strand synthesis were uncoupled in MiDAS, consistent with MiDAS being a form of break-induced replication, a repair mechanism for collapsed DNA replication forks. Our results provide a better understanding of the mechanisms leading to genomic instability at CFSs and in cancer cells.

MutSα deficiency increases tolerance to DNA damage in yeast lacking postreplication repair

By combining mutations in DNA repair genes, important and unexpected interactions between different repair pathways can be discovered. In this study, we identified a novel link between mismatch repair (MMR) genes and postreplication repair (PRR) in Saccharomyces cerevisiae. Strains lacking Rad5 (HLTF in mammals), a protein important for restarting stalled replication forks in the error-free PRR pathway, were supersensitive to the DNA methylating agent methyl methanesulfonate (MMS). Deletion of the mismatch repair genes, MSH2 or MSH6, which together constitutes the MutSα complex, partially suppressed the MMS super-sensitivity of the rad5Δ strain. Deletion of MSH2 also suppressed the MMS sensitivity of mms2Δ, which acts together with Rad5 in error-free PRR. However, inactivating the mismatch repair genes MSH3 and MLH1 did not suppress rad5Δ, showing that the suppression was specific for disabling MutSα. The partial suppression did not require translesion DNA synthesis (REV1, REV3 or RAD30), base excision repair (MAG1) or homologous recombination (RAD51). Instead, the underlying mechanism was dependent on RAD52 while independent of established pathways involving RAD52, like single-strand annealing and break-induced replication. We propose a Rad5- and Rad51-independent template switch pathway, capable of compensating for the loss of the error-free template-switch subpathway of postreplication repair, triggered by the loss of MutSα.

The role of D‐loop synthesis and progression in trinucleotide repeat stability via a break induced replication (BIR) model.

Homologous recombination (HR) is a complex, multi‐step process, that has been linked to increasing instability of trinucleotide repeats in Saccharomyces cerevisiae. One step of HR, formation and progression of the D‐loop, involves the invasion of ssDNA into a homologous sequence in the genome. Due to the involvement of a variety of polymerases and accessory factors during D‐loop synthesis and progression, it is possible to hypothesize that there is a high risk of slippage and stalling which could lead to changes in length (instability) of a CAG repeat. Break induced replication (BIR) is a genomic repair process that involves the progression of a D‐loop over long tracts of DNA, and thus is a good model to analyze this structure. In this project an assay system, involving a galactose inducible HO double strand break, results in the formation of a D‐loop due to homology to the URA3 gene on both the left and right arms of chromosome V. Repair through this tract of DNA on the right arm of chromosome V has been characterized through measures of viability, PCR of trinucleotide repeat tract, as well as Southern and Western blots. The characterization of this repair currently leads to the ultimate goal of discerning the impact of different DNA repair mutants on tract length changes within the assay system.Support or Funding InformationTufts University, Freudenreich Lab

Ectopic RNF168 expression promotes break-induced replication-like DNA synthesis at stalled replication forks.

The RNF168 E3 ubiquitin ligase is activated in response to double stranded DNA breaks (DSBs) where it mono-ubiquitinates γH2AX (ub-H2AX). RNF168 protein expression and ubiquitin signaling are finely regulated during the sensing, repair and resolution of DNA damage in order to avoid excessive spreading of ubiquitinated chromatin. Supra-physiological RNF168 protein expression levels have been shown to block DNA end resection at DSBs and increase PARP inhibitor (PARPi) sensitivity. In this study, we examined the impact of ectopic RNF168 overexpression on hydroxyurea (HU)-induced stalled replication forks in the setting of BRCA1 deficiency. Surprisingly, RNF168 overexpression resulted in the extension of DNA fibers, despite the presence of HU, in BRCA1 deficient cells. Mechanistically, RNF168 overexpression recruited RAD18 to ub-H2AX at HU-induced DNA breaks. Subsequently, a RAD18-SLF1 axis was responsible for initiating DNA synthesis in a manner that also required the break-induced replication (BIR) factors RAD52 and POLD3. Strikingly, the presence of wild-type BRCA1 blocked RNF168-induced DNA synthesis. Notably, BIR-like repair has previously been linked with tandem duplication events found in BRCA1-mutated genomes. Thus, in the absence of BRCA1, excessive RNF168 expression may drive BIR, and contribute to the mutational signatures observed in BRCA1-mutated cancers.

Current Understanding of RAD52 Functions: Fundamental and Therapeutic Insights

In this Special Issue, we would like to focus on the various functions of the RAD52 helicase-like protein and the current implications of such findings for cancer treatment. Over the last few years, various laboratories have discovered particular activities of mammalian RAD52—both in S and M phase—that are distinct from the auxiliary role of yeast RAD52 in homologous recombination. At DNA double-strand breaks, RAD52 was demonstrated to spur alternative pathways to compensate for the loss of homologous recombination functions. At collapsed replication forks, RAD52 activates break-induced replication. In the M phase, RAD52 promotes the finalization of DNA replication. Its compensatory role in the resolution of DNA double-strand breaks has put RAD52 in the focus of synthetic lethal strategies, which is particularly relevant for cancer treatment.