Homology-directed DNA repair (HDR) is critical for genome stability and tumor suppression. HDR is initiated by the RAD51 single-stranded (ss)DNA nucleoprotein filament which conducts the homology search and invades a homologous DNA template, creating a displacement-loop (D-loop). The RAD51 filament is assisted in these processes by several proteins. One such protein is RAD51-Associated-Protein 1 (RAD51AP1) which binds DNA and RNA and directly interacts with RAD51. Of note, RAD51AP1 overexpression is associated with poor prognosis in several different cancer types. Here, we show that RAD51AP1 activity is regulated by phosphorylation. RAD51AP1 bearing S277/282A mutations is more proficient in the stimulation of D-loop formation than wild type RAD51AP1 or phosphomimetic RAD51AP1-S277/282D. In EMSAs, RAD51AP1 with S277/282A mutations more avidly binds ssDNA, double-stranded (ds)DNA, and the nucleosome core particle than wild type RAD51AP1 or RAD51AP1-S277/282D. In cells, RAD51AP1-S277/282A confers no rescue of RAD51AP1 deficiency in toxicity tests and DNA replication assays. In contrast, RAD51AP1-S277/282D fully rescues RAD51AP1 deficiency. We provide evidence that RAD51AP1-S277 is a CDK2 target and propose a model in which RAD51AP1-S277/282 phosphorylation ensures RAD51AP1 flexibility for dynamic engagement in consecutive steps of the HDR reaction. Our results provide new mechanistic insights into RAD51AP1 regulation by a CDK.

Senataxin (SETX), an RNA-DNA helicase, accumulates at transcription pause sites through the tumor suppressor BRCA1. Here, we provide mechanistic insight into how SETX-BRCA1 resolves transcription-associated R-loops to prevent deleterious outcomes. Specifically, we show that full-length SETX unwinds R-loops with broad specificity and that the complex of BRCA1 and its obligatory partner BARD1 binds R-loops and stimulates R-loop unwinding by SETX. BRCA1-BARD1 alleviates the inhibitory effect of RAD52 on SETX-mediated R-loop unwinding. We also demonstrate that phosphorylation of Ser642 in SETX promotes its interaction with BRCA1 through the tandem BRCT domain of the latter. Accordingly, mutations impacting the catalytic domain or Ser642 in SETX lead to R-loop accumulation, transcription-replication conflicts, replication fork stalling and DNA double-strand breaks in human cells. Thus, our results delineate the molecular basis for functional synergy between SETX and BRCA1-BARD1 in R-loop resolution and the mitigation of transcription-replication conflicts to preserve genome integrity.

Abstract R-loops are transcription-induced, three-stranded nucleic acid structures that, if not properly resolved, can disrupt DNA repair and compromise genome stability. BRCA2, a tumor suppressor vital for homologous recombination (HR), also contributes to R-loop regulation, though the underlying mechanisms remain poorly understood. Here, we identify HELZ as a direct BRCA2 interactor and characterize it as an ssRNA-specific R-loop resolvase. BRCA2 enhances HELZ helicase activity and promotes its recruitment to R-loops. Importantly, HELZ resolves R-loops at DNA double-strand breaks, enabling efficient DNA end resection and HR, particularly within transcriptionally active genomic regions. We further demonstrate that HELZ is critical for R-loop clearance in cancers with elevated transcriptional activity and R-loop accumulation, such as estrogen receptor-positive breast cancer, where it becomes essential for cell survival under estrogen-induced transcriptional stress. These findings establish HELZ as a BRCA2-dependent regulator of R-loop homeostasis and identify it as a potential biomarker and therapeutic target in R-loop-driven malignancies.

PDS5B (Precocious Dissociation of Sisters 5B) functions in sister chromatid cohesion and genome organization. Interestingly, PDS5B also associates with RAD51, the recombinase required for DNA damage repair by homologous recombination (HR) and the preservation of stressed DNA replication forks against nucleolytic attrition. We show that PDS5B binds dsDNA preferentially over ssDNA and that it enhances RAD51-mediated DNA strand exchange via the capture of dsDNA. PDS5B also acts synergistically with BRCA2-DSS1 to help overcome the interference of RPA in DNA strand exchange and works in conjunction with RAD51 to protect dsDNA against digestion by MRE11-RAD50-NBS1. DNA binding activity resides within the disordered C-terminal region of PDS5B, and testing of a DNA binding mutant provides evidence that this PDS5B attribute underpins protein functions in vitro and in HR and replication fork protection in cells. Our findings thus reveal distinct functions of PDS5B in genome repair and maintenance.

ABSTRACT The breast and ovarian tumor suppressor BRCA1 heterodimerizes with BARD1 to promote DNA double-strand break repair by homologous recombination (HR) and to protect stressed DNA replication forks against nuclease attack. The large, intrinsically disordered central region of BRCA1 harbors binding sites for DNA and multiple repair factors, but its lack of stable structure has hindered mechanistic dissection of these activities. Using biochemical mapping and NMR spectroscopy, we delineate the DNA binding and RAD51 interaction interfaces within this region and construct separation-of-function mutants that selectively ablate each activity. Both DNA binding and RAD51 interaction are required for BRCA1-BARD1 to promote RAD51-mediated DNA strand invasion, and DNA binding also contributes to BLM-DNA2 end resection. These findings provide mechanistic insights into how individual ligand binding activities within BRCA1 contribute to genome maintenance.

Given their defect in homologous recombination, BRCA1-deficient cancers use polymerase theta (POLQ) microhomology-mediated end joining (MMEJ) for repair of stressed replication forks. We found that BRCA1-deficient cells repress microRNA (miR) 4485-3p, and reconstituting expression of this miR resulted in the death of BRCA1-deficient cells. In our interrogation of the mechanism of this synthetic lethality, we discovered that miR-4485-3p suppresses expression of RPRD1B protein. Depletion of RPRD1B led to selective BRCA1-deficient cell death, decreased MMEJ, and decreased replication fork repair after replication stress. RPRD1B promotes the recruitment of the MMEJ components 53BP1, PARP1, and POLQ to the stressed replication fork. However, RPRD1B does not have a canonical DNA-binding domain, and our search for the mechanism of its recruitment to stressed replication forks discovered that this is mediated by RECQL5 and ubiquitinated (Ub) PCNA. Thus, our results unveil an essential signaling cascade at the stressed replication fork in BRCA1-deficient cancer cells, in which Ub PCNA recruits RECQL5, which recruits RPRD1B to mediate the assembly of the MMEJ repair apparatus. These data suggest that this repair scaffold may be targeted in the development of a novel synthetic therapy of BRCA1-deficient cancers.

Break-induced DNA replication (BIR) is a highly mutagenic recombination pathway used by eukaryotic cells to repair single-ended DNA breaks, mediate mitotic DNA synthesis and promote telomerase-independent telomere maintenance in certain cancers. Leading-strand synthesis in BIR is mediated by a migrating D-loop driven by DNA polymerase δ and Pif1 helicase but the mechanism of second-strand synthesis has remained poorly understood. Here we demonstrate that in yeast cells lacking Cdc13-Stn1-Ten1 (CST complex), the early steps of BIR including 5' strand resection, D-loop formation and first-strand synthesis proceed normally. However, second-strand synthesis is impaired, implicating CST in this critical BIR step. The function of CST in BIR is conserved in human cells. Using biochemical reconstitution with DNA substrates mimicking BIR intermediates, we demonstrate that yeast CST promotes second-strand synthesis by enhancing DNA polymerase α-primase activity. Our findings provide mechanistic insight into how BIR supports long-tract DNA synthesis independently of the S-phase replisome.

Homologous recombination (HR) repairs DNA double-strand breaks and stabilizes stressed replication forks, and HR deficiency promotes genome instability and cancer. HR requires assembly of RAD51 nucleoprotein filaments on single-stranded DNA (ssDNA), a process regulated by the human RAD51 paralogs RAD51C, XRCC3, RAD51D and XRCC2. Here, using cryo-electron microscopy, we find that the RAD51-XRCC3-RAD51C complex (RAD51-X3C) assembles into an octamer in which XRCC3 engages the RAD51 DNA-binding surface and RAD51 subunits adopt a misaligned configuration incompatible with filament formation. These features define an autoinhibited RAD51-X3C state that limits nonproductive RAD51 binding to double-stranded DNA or RNA-DNA hybrids while preserving RAD51 availability for ssDNA-dependent strand exchange. We further show that the RAD51D-XRCC2 paralog complex remodels RAD51-X3C into a pentameric RAD51-X3CDX2 assembly by engaging the exposed RAD51C surface and disrupting contacts that stabilize the octamer. This remodeling exposes the RAD51 DNA-binding interface, enhances RAD51-ssDNA filament assembly, and promotes strand exchange on RPA-coated ssDNA, and yields a filament-compatible paralog assembly that integrates into ssDNA-bound RAD51 filaments. Together, these findings establish paralog exchange as a mechanism that converts an autoinhibited RAD51-X3C octamer into an activated RAD51-X3CDX2 pentamer to regulate RAD51 filament formation during HR and replication fork preservation.

Homologous recombination (HR) removes DNA double-strand breaks (DSBs) and preserves stressed DNA replication forks. Successful HR execution requires the tumor suppressor BRCA2, which harbors distinct DNA-binding domains (DBDs): one that possesses three oligonucleotide/oligosaccharide-binding (OB) folds (OB-DBD) and another residing in the C-terminal recombinase binding domain (CTRB-DBD). Here, we employ multi-faceted approaches to delineate the contributions of these domains toward HR and replication fork maintenance. We show that OB-DBD and CTRB-DBD confer single-strand DNA (ssDNA)- and dsDNA-binding capabilities, respectively, and that BRCA2 variants mutated in either domain are impaired in their ability to load the recombinase RAD51 onto ssDNA pre-occupied by RPA. While the CTRB-DBD mutant is modestly affected by DNA break repair, it exhibits a strong defect in the protection of stressed replication forks. In contrast, the OB-DBD is indispensable for both BRCA2 functions. Our study thus defines the unique contributions of the two BRCA2 DBDs in genome maintenance.

Antagonistic activities of the 53BP1 axis and the tumor suppressor BRCA1-BARD1 determine whether DNA double-strand breaks (DSBs) are repaired by end joining or homologous recombination. We show that the CTC1-STN1-TEN1 (CST) complex, a central 53BP1 axis component, suppresses DNA end resection by EXO1 and the BLM-DNA2 helicase-nuclease complex but acts by distinct mechanisms in restricting these entities. Whereas BRCA1-BARD1 alleviates the CST-imposed EXO1 blockade, it has little effect on BLM-DNA2 restriction. CST mutants impaired for DNA binding or BLM-EXO1 interaction exhibit a hyper-resection phenotype and render BRCA1-deficient cells resistant to poly(ADP-ribose) polymerase (PARP) inhibitors. Our findings mechanistically define the crucial role of CST in DNA DSB repair pathway choice and have implications for understanding cancer therapy resistance stemming from dysfunction of the 53BP1 axis.

Abstract Senataxin (SETX), a putative RNA-DNA helicase, is recruited to transcription pause sites via the tumor suppressor BRCA1. Here, we define the mechanism by which SETX-BRCA1 resolves transcription-associated R-loops to prevent deleterious outcomes. Specifically, we show that SETX unwinds R-loops, and that the complex of BRCA1 and its obligatory partner BARD1 binds R-loops and stimulates R-loop unwinding by SETX. Importantly, BRCA1-BARD1 alleviates the inhibitory effect of RAD52 on SETX-mediated R-loop unwinding. We also demonstrate that phosphorylation of Ser642 in SETX promotes its interaction with BRCA1 via the tandem BRCT domain of the latter. Accordingly, mutations in the catalytic domain or Ser642 in SETX lead to R-loop accumulation, transcription-replication conflicts, replication fork stalling, and DNA double strand breaks in human cells. Our results thus establish the molecular basis for functional synergy between SETX and BRCA1-BARD1 in R-loop resolution and the mitigation of transcription-replication conflicts to preserve genome integrity.

The key functions of the tumor suppressor BRCA2 include repairing DNA damages, such as DNA double-strand breaks (DSBs) or inter-strand crosslinks, and protecting stalled replication forks upon degradation. Thus, deleterious mutations in BRCA2 entail various cancers and Fanconi anemia. The BRCA2 protein possesses a composite of DNA-binding domains consisting of three Oligonucleotide Binding (OB) folds and a C-terminal region known as the C-terminal Recombinase Binding region (CTRB). The latter is encoded by the last gene exon (exon 27 in humans), also harboring the RAD51 recombinase binding region. The importance of these dual interactions in the functionality of the BRCA2 protein was examined in this study using biochemical and cell biological analyses. Firstly, we showed that the aberrations in both DNA and RAD51 binding in the CTRB lead to defects in DNA double-strand break (DSB) repair and replication fork preservation. Furthermore, the significance of DNA binding via OB folds and CTRB was explored through combinatory mutations that separately impair the DNA binding of OB folds and CTRB. The results reveal that the DNA binding of OB folds guides the recognition of ssDNA, while the CTRB facilitates interaction with dsDNA. We demonstrate that DNA binding through OB-folds is essential for RAD51-mediated homologous recombination, while the CTRB mainly plays a role in protecting DNA from nucleolytic degradation at the replication fork. These results highlight the distinctive roles of BRCA2 DNA-binding modules that contribute to the multifaceted actions of BRCA2 in DNA damage repair and replication fork protection. This study was supported by NIH grants, R50 CA265315 (Y.K.), R01 ES007061 (P.S.), and R35 CA241801 (P.S.)

The Bloom syndrome (BLM) helicase is critical for alternative lengthening of telomeres (ALT), a homology-directed repair (HDR)-mediated telomere maintenance mechanism that is prevalent in cancers of mesenchymal origin. The DNA substrates that BLM engages to direct telomere recombination during ALT remain unknown. Here, we determine that BLM helicase acts on lagging strand telomere intermediates that occur specifically in ALT-positive cells to assemble a replication-associated DNA damage response. Loss of ATRX was permissive for BLM localization to ALT telomeres in S and G2, commensurate with the appearance of telomere C-strand-specific single-stranded DNA (ssDNA). DNA2 nuclease deficiency increased 5′-flap formation in a BLM-dependent manner, while telomere C-strand, but not G-strand, nicks promoted ALT. These findings define the seminal events in the ALT DNA damage response, linking aberrant telomeric lagging strand DNA replication with a BLM-directed HDR mechanism that sustains telomere length in a subset of human cancers.

The licensing step of DNA double-strand break repair by homologous recombination entails resection of DNA ends to generate a single-stranded DNA template for assembly of the repair machinery consisting of the RAD51 recombinase and ancillary factors1. DNA end resection is mechanistically intricate and reliant on the tumour suppressor complex BRCA1-BARD1 (ref. 2). Specifically, three distinct nuclease entities-the 5'-3' exonuclease EXO1 and heterodimeric complexes of the DNA endonuclease DNA2, with either the BLM or WRN helicase-act in synergy to execute the end resection process3. A major question concerns whether BRCA1-BARD1 directly regulates end resection. Here, using highly purified protein factors, we provide evidence that BRCA1-BARD1 physically interacts with EXO1, BLM and WRN. Importantly, with reconstituted biochemical systems and a single-molecule analytical tool, we show that BRCA1-BARD1 upregulates the activity of all three resection pathways. We also demonstrate that BRCA1 and BARD1 harbour stand-alone modules that contribute to the overall functionality of BRCA1-BARD1. Moreover, analysis of a BARD1 mutant impaired in DNA binding shows the importance of this BARD1 attribute in end resection, both in vitro and in cells. Thus, BRCA1-BARD1 enhances the efficiency of all three long-range DNA end resection pathways during homologous recombination in human cells.