The BRCA Tumor Suppressor Network in Chromosome Damage Repair by Homologous Recombination.

Mutations in the BRCA1 and BRCA2 genes predispose afflicted individuals to breast, ovarian, and other cancers. The BRCA-encoded products form complexes with other tumor suppressor proteins and with the recombinase enzyme RAD51 to mediate chromosome damage repair by homologous recombination and also to protect stressed DNA replication forks against spurious nucleolytic attrition. Understanding how the BRCA tumor suppressor network executes its biological functions would provide the foundation for developing targeted cancer therapeutics, but progress in this area has been greatly hampered by the challenge of obtaining purified BRCA complexes for mechanistic studies. In this article, we review how recent effort begins to overcome this technical challenge, leading to functional and structural insights into the biochemical attributes of these complexes and the multifaceted roles that they fulfill in genome maintenance. We also highlight the major mechanistic questions that remain.

Inadequate DNA Damage Repair Promotes Mammary Transdifferentiation, Leading to BRCA1 Breast Cancer.

Loss of BRCA1 p220 function often results in basal-like breast cancer (BLBC), but the underlying disease mechanism is largely opaque. In mammary epithelial cells (MECs), BRCA1 interacts with multiple proteins, including NUMB and HES1, to form complexes that participate in interstrand crosslink (ICL) DNA repair and MEC differentiation control. Unrepaired ICL damage results in aberrant transdifferentiation to a mesenchymal state of cultured, human basal-like MECs and to a basal/mesenchymal state in primary mouse luminal MECs. Loss of BRCA1, NUMB, or HES1 or chemically induced ICL damage in primary murine luminal MECs results in persistent DNA damage that triggers luminal to basal/mesenchymal transdifferentiation. In vivo single-cell analysis revealed a time-dependent evolution from normal luminal MECs to luminal progenitor-like tumor cells with basal/mesenchymal transdifferentiation during murine BRCA1 BLBC development. Growing DNA damage accompanied this malignant transformation.

The MRE11-RAD50-NBS1 Complex Conducts the Orchestration of Damage Signaling and Outcomes to Stress in DNA Replication and Repair.

Genomic instability in disease and its fidelity in health depend on the DNA damage response (DDR), regulated in part from the complex of meiotic recombination 11 homolog 1 (MRE11), ATP-binding cassette-ATPase (RAD50), and phosphopeptide-binding Nijmegen breakage syndrome protein 1 (NBS1). The MRE11-RAD50-NBS1 (MRN) complex forms a multifunctional DDR machine. Within its network assemblies, MRN is the core conductor for the initial and sustained responses to DNA double-strand breaks, stalled replication forks, dysfunctional telomeres, and viral DNA infection. MRN can interfere with cancer therapy and is an attractive target for precision medicine. Its conformations change the paradigm whereby kinases initiate damage sensing. Delineated results reveal kinase activation, posttranslational targeting, functional scaffolding, conformations storing binding energy and enabling access, interactions with hub proteins such as replication protein A (RPA), and distinct networks at DNA breaks and forks. MRN biochemistry provides prototypic insights into how it initiates, implements, and regulates multifunctional responses to genomic stress.

DNA Repair Network Analysis Reveals Shieldin as a Key Regulator of NHEJ and PARP Inhibitor Sensitivity.

Repair of damaged DNA is essential for maintaining genome integrity and for preventing genome-instability-associated diseases, such as cancer. By combining proximity labeling with quantitative mass spectrometry, we generated high-resolution interaction neighborhood maps of the endogenously expressed DNA repair factors 53BP1, BRCA1, and MDC1. Our spatially resolved interaction maps reveal rich network intricacies, identify shared and bait-specific interaction modules, and implicate previously concealed regulators in this process. We identified a novel vertebrate-specific protein complex, shieldin, comprising REV7 plus three previously uncharacterized proteins, RINN1 (CTC-534A2.2), RINN2 (FAM35A), and RINN3 (C20ORF196). Recruitment of shieldin to DSBs, via the ATM-RNF8-RNF168-53BP1-RIF1 axis, promotes NHEJ-dependent repair of intrachromosomal breaks, immunoglobulin class-switch recombination (CSR), and fusion of unprotected telomeres. Shieldin functions as a downstream effector of 53BP1-RIF1 in restraining DNA end resection and in sensitizing BRCA1-deficient cells to PARP inhibitors. These findings have implications for understanding cancer-associated PARPi resistance and the evolution of antibody CSR in higher vertebrates.

EXO1 protects BRCA1-deficient cells against toxic DNA lesions.

Inactivating mutations in the BRCA1 and BRCA2 genes impair DNA double-strand break (DSB) repair by homologous recombination (HR), leading to chromosomal instability and cancer. Importantly, BRCA1/2 deficiency also causes therapeutically targetable vulnerabilities. Here, we identify the dependency on the end resection factor EXO1 as a key vulnerability of BRCA1-deficient cells. EXO1 deficiency generates poly(ADP-ribose)-decorated DNA lesions during S phase that associate with unresolved DSBs and genomic instability in BRCA1-deficient but not in wild-type or BRCA2-deficient cells. Our data indicate that BRCA1/EXO1 double-deficient cells accumulate DSBs due to impaired repair by single-strand annealing (SSA) on top of their HR defect. In contrast, BRCA2-deficient cells retain SSA activity in the absence of EXO1 and hence tolerate EXO1 loss. Consistent with a dependency on EXO1-mediated SSA, we find that BRCA1-mutated tumors show elevated EXO1 expression and increased SSA-associated genomic scars compared with BRCA1-proficient tumors. Overall, our findings uncover EXO1 as a promising therapeutic target for BRCA1-deficient tumors.

BLM and BRCA1-BARD1 coordinate complementary mechanisms of joint DNA molecule resolution.

The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1, and RMI2 to form the BTR complex, which dissolves double Holliday junctions and DNA replication intermediates to promote sister chromatid disjunction before cell division. In its absence, structure-specific nucleases like the SMX complex (comprising SLX1-SLX4, MUS81-EME1, and XPF-ERCC1) can cleave joint DNA molecules instead, but cells deficient in both BTR and SMX are not viable. Here, we identify a negative genetic interaction between BLM loss and deficiency in the BRCA1-BARD1 tumor suppressor complex. We show that this is due to a previously overlooked role for BARD1 in recruiting SLX4 to resolve DNA intermediates left unprocessed by BLM in the preceding interphase. Consequently, cells with defective BLM and BRCA1-BARD1 accumulate catastrophic levels of chromosome breakage and micronucleation, leading to cell death. Thus, we reveal mechanistic insights into SLX4 recruitment to DNA lesions, with potential clinical implications for treating BRCA1-deficient tumors.

Mechanisms of RNF168 nucleosome recognition and ubiquitylation.

RNF168 plays a central role in the DNA damage response (DDR) by ubiquitylating histone H2A at K13 and K15. These modifications direct BRCA1-BARD1 and 53BP1 foci formation in chromatin, essential for cell-cycle-dependent DNA double-strand break (DSB) repair pathway selection. The mechanism by which RNF168 catalyzes the targeted accumulation of H2A ubiquitin conjugates to form repair foci around DSBs remains unclear. Here, using cryoelectron microscopy (cryo-EM), nuclear magnetic resonance (NMR) spectroscopy, and functional assays, we provide a molecular description of the reaction cycle and dynamics of RNF168 as it modifies the nucleosome and recognizes its ubiquitylation products. We demonstrate an interaction of a canonical ubiquitin-binding domain within full-length RNF168, which not only engages ubiquitin but also the nucleosome surface, clarifying how such site-specific ubiquitin recognition propels a signal amplification loop. Beyond offering mechanistic insights into a key DDR protein, our study aids in understanding site specificity in both generating and interpreting chromatin ubiquitylation.

Crucial roles of the BRCA1-BARD1 E3 ubiquitin ligase activity in homology-directed DNA repair.

The tumor-suppressor breast cancer 1 (BRCA1) in complex with BRCA1-associated really interesting new gene (RING) domain 1 (BARD1) is a RING-type ubiquitin E3 ligase that modifies nucleosomal histone and other substrates. The importance of BRCA1-BARD1 E3 activity in tumor suppression remains highly controversial, mainly stemming from studying mutant ligase-deficient BRCA1-BARD1 species that we show here still retain significant ligase activity. Using full-length BRCA1-BARD1, we establish robust BRCA1-BARD1-mediated ubiquitylation with specificity, uncover multiple modes of activity modulation, and construct a truly ligase-null variant and a variant specifically impaired in targeting nucleosomal histones. Cells expressing either of these BRCA1-BARD1 separation-of-function alleles are hypersensitive to DNA-damaging agents. Furthermore, we demonstrate that BRCA1-BARD1 ligase is not only required for DNA resection during homology-directed repair (HDR) but also contributes to later stages for HDR completion. Altogether, our findings reveal crucial, previously unrecognized roles of BRCA1-BARD1 ligase activity in genome repair via HDR, settle prior controversies regarding BRCA1-BARD1 ligase functions, and catalyze new efforts to uncover substrates related to tumor suppression.

DNA-PKcs promotes fork reversal and chemoresistance.

The DNA-PKcs kinase mediates the repair of DNA double-strand breaks via classical non-homologous end joining (NHEJ). DNA-PKcs is also recruited to active replication forks, although a role for DNA-PKcs in the control of fork dynamics is unclear. Here, we identify a crucial role for DNA-PKcs in promoting fork reversal, a process that stabilizes stressed replication forks and protects genome integrity. DNA-PKcs promotes fork reversal and slowing in response to several replication stress-inducing agents in a manner independent of its role in NHEJ. Cells lacking DNA-PKcs activity show increased DNA damage during S-phase and cellular sensitivity to replication stress. Notably, prevention of fork slowing and reversal via DNA-PKcs inhibition efficiently restores chemotherapy sensitivity in BRCA2-deficient mammary tumors with acquired PARPi resistance. Together, our data uncover a new key regulator of fork reversal and show how DNA-PKcs signaling can be manipulated to alter fork dynamics and drug resistance in cancer.

RIF1 acts in DNA repair through phosphopeptide recognition of 53BP1.

The chromatin-binding protein 53BP1 promotes DNA repair by orchestrating the recruitment of downstream effectors including PTIP, RIF1, and shieldin to DNA double-strand break sites. While we know how PTIP recognizes 53BP1, the molecular details of RIF1 recruitment to DNA-damage sites remains undefined. Here, we report that RIF1 is a phosphopeptide-binding protein that directly interacts with three phosphorylated 53BP1 epitopes. The RIF1-binding sites on 53BP1 share an essential LxL motif followed by two closely apposed phosphorylated residues. Simultaneous mutation of these sites on 53BP1 abrogates RIF1 accumulation into ionizing-radiation-induced foci, but surprisingly, only fully compromises 53BP1-dependent DNA repair when an alternative mode of shieldin recruitment to DNA-damage sites is also disabled. Intriguingly, this alternative mode of recruitment still depends on RIF1 but does not require its interaction with 53BP1. RIF1 therefore employs phosphopeptide recognition to promote DNA repair but also modifies shieldin action independently of 53BP1 binding.

The dystonia gene THAP1 controls DNA double-strand break repair choice.

The Shieldin complex shields double-strand DNA breaks (DSBs) from nucleolytic resection. Curiously, the penultimate Shieldin component, SHLD1, is one of the least abundant mammalian proteins. Here, we report that the transcription factors THAP1, YY1, and HCF1 bind directly to the SHLD1 promoter, where they cooperatively maintain the low basal expression of SHLD1, thereby ensuring a proper balance between end protection and resection during DSB repair. The loss of THAP1-dependent SHLD1 expression confers cross-resistance to poly (ADP-ribose) polymerase (PARP) inhibitor and cisplatin in BRCA1-deficient cells and shorter progression-free survival in ovarian cancer patients. Moreover, the embryonic lethality and PARPi sensitivity of BRCA1-deficient mice is rescued by ablation of SHLD1. Our study uncovers a transcriptional network that directly controls DSB repair choice and suggests a potential link between DNA damage and pathogenic THAP1 mutations, found in patients with the neurodevelopmental movement disorder adult-onset torsion dystonia type 6.

Structural insight into BRCA1-BARD1 complex recruitment to damaged chromatin.

The BRCA1-BARD1 complex directs the DNA double-strand break (DSB) repair pathway choice to error-free homologous recombination (HR) during the S-G2 stages. Targeting BRCA1-BARD1 to DSB-proximal sites requires BARD1-mediated nucleosome interaction and histone mark recognition. Here, we report the cryo-EM structure of BARD1 bound to a ubiquitinated nucleosome core particle (NCPUb) at 3.1 Å resolution and illustrate how BARD1 simultaneously recognizes the DNA damage-induced mark H2AK15ub and DNA replication-associated mark H4K20me0 on the nucleosome. In vitro and in vivo analyses reveal that the BARD1-NCPUb complex is stabilized by BARD1-nucleosome interaction, BARD1-ubiquitin interaction, and BARD1 ARD domain-BARD1 BRCT domain interaction, and abrogating these interactions is detrimental to HR activity. We further identify multiple disease-causing BARD1 mutations that disrupt BARD1-NCPUb interactions and hence impair HR. Together, this study elucidates the mechanism of BRCA1-BARD1 complex recruitment and retention by DSB-flanking nucleosomes and sheds important light on cancer therapeutic avenues.

FANCM regulates repair pathway choice at stalled replication forks.

Repair pathway "choice" at stalled mammalian replication forks is an important determinant of genome stability; however, the underlying mechanisms are poorly understood. FANCM encodes a multi-domain scaffolding and motor protein that interacts with several distinct repair protein complexes at stalled forks. Here, we use defined mutations engineered within endogenous Fancm in mouse embryonic stem cells to study how Fancm regulates stalled fork repair. We find that distinct FANCM repair functions are enacted by molecularly separable scaffolding domains. These findings define FANCM as a key mediator of repair pathway choice at stalled replication forks and reveal its molecular mechanism. Notably, mutations that inactivate FANCM ATPase function disable all its repair functions and "trap" FANCM at stalled forks. We find that Brca1 hypomorphic mutants are synthetic lethal with Fancm null or Fancm ATPase-defective mutants. The ATPase function of FANCM may therefore represent a promising "druggable" target for therapy of BRCA1-linked cancer.

Temporally distinct post-replicative repair mechanisms fill PRIMPOL-dependent ssDNA gaps in human cells.

PRIMPOL repriming allows DNA replication to skip DNA lesions, leading to ssDNA gaps. These gaps must be filled to preserve genome stability. Using a DNA fiber approach to directly monitor gap filling, we studied the post-replicative mechanisms that fill the ssDNA gaps generated in cisplatin-treated cells upon increased PRIMPOL expression or when replication fork reversal is defective because of SMARCAL1 inactivation or PARP inhibition. We found that a mechanism dependent on the E3 ubiquitin ligase RAD18, PCNA monoubiquitination, and the REV1 and POLζ translesion synthesis polymerases promotes gap filling in G2. The E2-conjugating enzyme UBC13, the RAD51 recombinase, and REV1-POLζ are instead responsible for gap filling in S, suggesting that temporally distinct pathways of gap filling operate throughout the cell cycle. Furthermore, we found that BRCA1 and BRCA2 promote gap filling by limiting MRE11 activity and that simultaneously targeting fork reversal and gap filling enhances chemosensitivity in BRCA-deficient cells.

Loss of nuclear DNA ligase III reverts PARP inhibitor resistance in BRCA1/53BP1 double-deficient cells by exposing ssDNA gaps.

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1 identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 loss promotes formation of MRE11-mediated post-replicative ssDNA gaps in BRCA1-deficient and BRCA1/53BP1 double-deficient cells exposed to PARPi, leading to an accumulation of chromosomal abnormalities. LIG3 depletion also enhances efficacy of PARPi against BRCA1-deficient mammary tumors in mice, suggesting LIG3 as a potential therapeutic target.

REV1-Polζ maintains the viability of homologous recombination-deficient cancer cells through mutagenic repair of PRIMPOL-dependent ssDNA gaps.

BRCA1/2 mutant tumor cells display an elevated mutation burden, the etiology of which remains unclear. Here, we report that these cells accumulate ssDNA gaps and spontaneous mutations during unperturbed DNA replication due to repriming by the DNA primase-polymerase PRIMPOL. Gap accumulation requires the DNA glycosylase SMUG1 and is exacerbated by depletion of the translesion synthesis (TLS) factor RAD18 or inhibition of the error-prone TLS polymerase complex REV1-Polζ by the small molecule JH-RE-06. JH-RE-06 treatment of BRCA1/2-deficient cells results in reduced mutation rates and PRIMPOL- and SMUG1-dependent loss of viability. Through cellular and animal studies, we demonstrate that JH-RE-06 is preferentially toxic toward HR-deficient cancer cells. Furthermore, JH-RE-06 remains effective toward PARP inhibitor (PARPi)-resistant BRCA1 mutant cells and displays additive toxicity with crosslinking agents or PARPi. Collectively, these studies identify a protective and mutagenic role for REV1-Polζ in BRCA1/2 mutant cells and provide the rationale for using REV1-Polζ inhibitors to treat BRCA1/2 mutant tumors.

Proteome dynamics at broken replication forks reveal a distinct ATM-directed repair response suppressing DNA double-strand break ubiquitination.

Cells have evolved an elaborate DNA repair network to ensure complete and accurate DNA replication. Defects in these repair machineries can fuel genome instability and drive carcinogenesis while creating vulnerabilities that may be exploited in therapy. Here, we use nascent chromatin capture (NCC) proteomics to characterize the repair of replication-associated DNA double-strand breaks (DSBs) triggered by topoisomerase 1 (TOP1) inhibitors. We reveal profound changes in the fork proteome, including the chromatin environment and nuclear membrane interactions, and identify three classes of repair factors according to their enrichment at broken and/or stalled forks. ATM inhibition dramatically rewired the broken fork proteome, revealing that ataxia telangiectasia mutated (ATM) signalling stimulates DNA end resection, recruits PLK1, and concomitantly suppresses the canonical DSB ubiquitination response by preventing accumulation of RNF168 and BRCA1-A. This work and collection of replication fork proteomes provide a new framework to understand how cells orchestrate homologous recombination repair of replication-associated DSBs.

Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency.

Mutations in BRCA1 or BRCA2 (BRCA) is synthetic lethal with poly(ADP-ribose) polymerase inhibitors (PARPi). Lethality is thought to derive from DNA double-stranded breaks (DSBs) necessitating BRCA function in homologous recombination (HR) and/or fork protection (FP). Here, we report instead that toxicity derives from replication gaps. BRCA1- or FANCJ-deficient cells, with common repair defects but distinct PARPi responses, reveal gaps as a distinguishing factor. We further uncouple HR, FP, and fork speed from PARPi response. Instead, gaps characterize BRCA-deficient cells, are diminished upon resistance, restored upon resensitization, and, when exposed, augment PARPi toxicity. Unchallenged BRCA1-deficient cells have elevated poly(ADP-ribose) and chromatin-associated PARP1, but aberrantly low XRCC1 consistent with defects in backup Okazaki fragment processing (OFP). 53BP1 loss resuscitates OFP by restoring XRCC1-LIG3 that suppresses the sensitivity of BRCA1-deficient cells to drugs targeting OFP or generating gaps. We highlight gaps as a determinant of PARPi toxicity changing the paradigm for synthetic lethal interactions.

53BP1 Enforces Distinct Pre- and Post-resection Blocks on Homologous Recombination.

53BP1 activity drives genome instability and lethality in BRCA1-deficient mice by inhibiting homologous recombination (HR). The anti-recombinogenic functions of 53BP1 require phosphorylation-dependent interactions with PTIP and RIF1/shieldin effector complexes. While RIF1/shieldin blocks 5'-3' nucleolytic processing of DNA ends, it remains unclear how PTIP antagonizes HR. Here, we show that mutation of the PTIP interaction site in 53BP1 (S25A) allows sufficient DNA2-dependent end resection to rescue the lethality of BRCA1Δ11 mice, despite increasing RIF1 "end-blocking" at DNA damage sites. However, double-mutant cells fail to complete HR, as excessive shieldin activity also inhibits RNF168-mediated loading of PALB2/RAD51. As a result, BRCA1Δ1153BP1S25A mice exhibit hallmark features of HR insufficiency, including premature aging and hypersensitivity to PARPi. Disruption of shieldin or forced targeting of PALB2 to ssDNA in BRCA1D1153BP1S25A cells restores RNF168 recruitment, RAD51 nucleofilament formation, and PARPi resistance. Our study therefore reveals a critical function of shieldin post-resection that limits the loading of RAD51.

PCAF-Mediated Histone Acetylation Promotes Replication Fork Degradation by MRE11 and EXO1 in BRCA-Deficient Cells.

Stabilization of stalled replication forks is a prominent mechanism of PARP (Poly(ADP-ribose) Polymerase) inhibitor (PARPi) resistance in BRCA-deficient tumors. Epigenetic mechanisms of replication fork stability are emerging but remain poorly understood. Here, we report the histone acetyltransferase PCAF (p300/CBP-associated) as a fork-associated protein that promotes fork degradation in BRCA-deficient cells by acetylating H4K8 at stalled replication forks, which recruits MRE11 and EXO1. A H4K8ac binding domain within MRE11/EXO1 is required for their recruitment to stalled forks. Low PCAF levels, which we identify in a subset of BRCA2-deficient tumors, stabilize stalled forks, resulting in PARPi resistance in BRCA-deficient cells. Furthermore, PCAF activity is tightly regulated by ATR (ataxia telangiectasia and Rad3-related), which phosphorylates PCAF on serine 264 (S264) to limit its association and activity at stalled forks. Our results reveal PCAF and histone acetylation as critical regulators of fork stability and PARPi responses in BRCA-deficient cells, which provides key insights into targeting BRCA-deficient tumors and identifying epigenetic modulators of chemotherapeutic responses.