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.

Two redundant ubiquitin-dependent pathways of BRCA1 localization to DNA damage sites.

The tumor suppressor BRCA1 accumulates at sites of DNA damage in a ubiquitin-dependent manner. In this work, we revisit the role of RAP80 in promoting BRCA1 recruitment to damaged chromatin. We find that RAP80 acts redundantly with the BRCA1 RING domain to promote BRCA1 recruitment to DNA damage sites. We show that that RNF8 E3 ligase acts upstream of both the RAP80- and RING-dependent activities, whereas RNF168 acts uniquely upstream of the RING domain. BRCA1 RING mutations that do not impact BARD1 interaction, such as the E2 binding-deficient I26A mutation, render BRCA1 unable to accumulate at DNA damage sites in the absence of RAP80. Cells that combine BRCA1 I26A and mutations that disable the RAP80-BRCA1 interaction are hypersensitive to PARP inhibition and are unable to form RAD51 foci. Our results suggest that in the absence of RAP80, the BRCA1 E3 ligase activity is necessary for recognition of histone H2A Lys13/Lys15 ubiquitylation by BARD1, although we cannot rule out the possibility that the BRCA1 RING facilitates ubiquitylated nucleosome recognition in other ways.

ELOF1 is a transcription-coupled DNA repair factor that directs RNA polymerase II ubiquitylation.

Cells employ transcription-coupled repair (TCR) to eliminate transcription-blocking DNA lesions. DNA damage-induced binding of the TCR-specific repair factor CSB to RNA polymerase II (RNAPII) triggers RNAPII ubiquitylation of a single lysine (K1268) by the CRL4CSA ubiquitin ligase. How CRL4CSA is specifically directed towards K1268 is unknown. Here, we identify ELOF1 as the missing link that facilitates RNAPII ubiquitylation, a key signal for the assembly of downstream repair factors. This function requires its constitutive interaction with RNAPII close to K1268, revealing ELOF1 as a specificity factor that binds and positions CRL4CSA for optimal RNAPII ubiquitylation. Drug-genetic interaction screening also revealed a CSB-independent pathway in which ELOF1 prevents R-loops in active genes and protects cells against DNA replication stress. Our study offers key insights into the molecular mechanisms of TCR and provides a genetic framework of the interplay between transcriptional stress responses and DNA replication.

Untangling the crosstalk between BRCA1 and R-loops during DNA repair.

R-loops are RNA:DNA hybrids assembled during biological processes but are also linked to genetic instability when formed out of their natural context. Emerging evidence suggests that the repair of DNA double-strand breaks requires the formation of a transient R-loop, which eventually must be removed to guarantee a correct repair process. The multifaceted BRCA1 protein has been shown to be recruited at this specific break-induced R-loop, and it facilitates mechanisms in order to regulate R-loop removal. In this review, we discuss the different potential roles of BRCA1 in R-loop homeostasis during DNA repair and how these processes ensure faithful DSB repair.

The CIP2A-TOPBP1 axis safeguards chromosome stability and is a synthetic lethal target for BRCA-mutated cancer.

BRCA1/2-mutated cancer cells adapt to the genome instability caused by their deficiency in homologous recombination (HR). Identification of these adaptive mechanisms may provide therapeutic strategies to target tumors caused by the loss of these genes. In the present study, we report genome-scale CRISPR-Cas9 synthetic lethality screens in isogenic pairs of BRCA1- and BRCA2-deficient cells and identify CIP2A as an essential gene in BRCA1- and BRCA2-mutated cells. CIP2A is cytoplasmic in interphase but, in mitosis, accumulates at DNA lesions as part of a complex with TOPBP1, a multifunctional genome stability factor. Unlike PARP inhibition, CIP2A deficiency does not cause accumulation of replication-associated DNA lesions that require HR for their repair. In BRCA-deficient cells, the CIP2A-TOPBP1 complex prevents lethal mis-segregation of acentric chromosomes that arises from impaired DNA synthesis. Finally, physical disruption of the CIP2A-TOPBP1 complex is highly deleterious in BRCA-deficient tumors, indicating that CIP2A represents an attractive synthetic lethal therapeutic target for BRCA1- and BRCA2-mutated cancers.

PARP Inhibitor Resistance: A Tug-of-War in BRCA-Mutated Cells.

Poly-(ADP)-ribose polymerase (PARP) inhibition is synthetic lethal with deficiency for homologous recombination (HR), a pathway essential for DNA double-strand break repair. PARP inhibitors (PARPi) therefore hold great promise for the treatment of tumors with disruptive mutations in BRCA1/2 or other HR factors. Unfortunately, PARPi resistance has proved to be a major problem in the clinic. Knowledge about PARPi resistance is expanding quickly, revealing four main mechanisms that alter drug availability, affect (de)PARylation enzymes, restore HR, or restore replication fork stability. We discuss how studies on resistance mechanisms have yielded important insights into the regulation of DNA double-strand break (DSB) repair and replication fork protection, and how these studies could pave the way for novel treatment options to target resistance mechanisms or acquired vulnerabilities.

The shieldin complex mediates 53BP1-dependent DNA repair.

53BP1 is a chromatin-binding protein that regulates the repair of DNA double-strand breaks by suppressing the nucleolytic resection of DNA termini1,2. This function of 53BP1 requires interactions with PTIP3 and RIF14-9, the latter of which recruits REV7 (also known as MAD2L2) to break sites10,11. How 53BP1-pathway proteins shield DNA ends is currently unknown, but there are two models that provide the best potential explanation of their action. In one model the 53BP1 complex strengthens the nucleosomal barrier to end-resection nucleases12,13, and in the other 53BP1 recruits effector proteins with end-protection activity. Here we identify a 53BP1 effector complex, shieldin, that includes C20orf196 (also known as SHLD1), FAM35A (SHLD2), CTC-534A2.2 (SHLD3) and REV7. Shieldin localizes to double-strand-break sites in a 53BP1- and RIF1-dependent manner, and its SHLD2 subunit binds to single-stranded DNA via OB-fold domains that are analogous to those of RPA1 and POT1. Loss of shieldin impairs non-homologous end-joining, leads to defective immunoglobulin class switching and causes hyper-resection. Mutations in genes that encode shieldin subunits also cause resistance to poly(ADP-ribose) polymerase inhibition in BRCA1-deficient cells and tumours, owing to restoration of homologous recombination. Finally, we show that binding of single-stranded DNA by SHLD2 is critical for shieldin function, consistent with a model in which shieldin protects DNA ends to mediate 53BP1-dependent DNA repair.

Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency.

Programmable nucleases, such as Cas9, are used for precise genome editing by homology-dependent repair (HDR). However, HDR efficiency is constrained by competition from other double-strand break (DSB) repair pathways, including non-homologous end-joining (NHEJ). We report the discovery of a genetically encoded inhibitor of 53BP1 that increases the efficiency of HDR-dependent genome editing in human and mouse cells. 53BP1 is a key regulator of DSB repair pathway choice in eukaryotic cells and functions to favor NHEJ over HDR by suppressing end resection, which is the rate-limiting step in the initiation of HDR. We screened an existing combinatorial library of engineered ubiquitin variants for inhibitors of 53BP1. Expression of one variant, named i53 (inhibitor of 53BP1), in human and mouse cells, blocked accumulation of 53BP1 at sites of DNA damage and improved gene targeting and chromosomal gene conversion with either double-stranded DNA or single-stranded oligonucleotide donors by up to 5.6-fold. Inhibition of 53BP1 is a robust method to increase efficiency of HDR-based precise genome editing.

ATM and CDK2 control chromatin remodeler CSB to inhibit RIF1 in DSB repair pathway choice.

CSB, a member of the SWI2/SNF2 superfamily, is implicated in DNA double-strand break (DSB) repair. However, how it regulates this repair process is poorly understood. Here we uncover that CSB interacts via its newly identified winged helix domain with RIF1, an effector of 53BP1, and that this interaction mediates CSB recruitment to DSBs in S phase. At DSBs, CSB remodels chromatin by evicting histones, which limits RIF1 and its effector MAD2L2 but promotes BRCA1 accumulation. The chromatin remodeling activity of CSB requires not only damage-induced phosphorylation on S10 by ATM but also cell cycle-dependent phosphorylation on S158 by cyclin A-CDK2. Both modifications modulate the interaction of the CSB N-terminal region with its ATPase domain, the activity of which has been previously reported to be autorepressed by the N-terminal region. These results suggest that ATM and CDK2 control the chromatin remodeling activity of CSB in the regulation of DSB repair pathway choice.

The structural basis of modified nucleosome recognition by 53BP1.

DNA double-strand breaks (DSBs) elicit a histone modification cascade that controls DNA repair. This pathway involves the sequential ubiquitination of histones H1 and H2A by the E3 ubiquitin ligases RNF8 and RNF168, respectively. RNF168 ubiquitinates H2A on lysine 13 and lysine 15 (refs 7, 8) (yielding H2AK13ub and H2AK15ub, respectively), an event that triggers the recruitment of 53BP1 (also known as TP53BP1) to chromatin flanking DSBs. 53BP1 binds specifically to H2AK15ub-containing nucleosomes through a peptide segment termed the ubiquitination-dependent recruitment motif (UDR), which requires the simultaneous engagement of histone H4 lysine 20 dimethylation (H4K20me2) by its tandem Tudor domain. How 53BP1 interacts with these two histone marks in the nucleosomal context, how it recognizes ubiquitin, and how it discriminates between H2AK13ub and H2AK15ub is unknown. Here we present the electron cryomicroscopy (cryo-EM) structure of a dimerized human 53BP1 fragment bound to a H4K20me2-containing and H2AK15ub-containing nucleosome core particle (NCP-ubme) at 4.5 Å resolution. The structure reveals that H4K20me2 and H2AK15ub recognition involves intimate contacts with multiple nucleosomal elements including the acidic patch. Ubiquitin recognition by 53BP1 is unusual and involves the sandwiching of the UDR segment between ubiquitin and the NCP surface. The selectivity for H2AK15ub is imparted by two arginine fingers in the H2A amino-terminal tail, which straddle the nucleosomal DNA and serve to position ubiquitin over the NCP-bound UDR segment. The structure of the complex between NCP-ubme and 53BP1 reveals the basis of 53BP1 recruitment to DSB sites and illuminates how combinations of histone marks and nucleosomal elements cooperate to produce highly specific chromatin responses, such as those elicited following chromosome breaks.

A mechanism for the suppression of homologous recombination in G1 cells.

DNA repair by homologous recombination is highly suppressed in G1 cells to ensure that mitotic recombination occurs solely between sister chromatids. Although many homologous recombination factors are cell-cycle regulated, the identity of the events that are both necessary and sufficient to suppress recombination in G1 cells is unknown. Here we report that the cell cycle controls the interaction of BRCA1 with PALB2-BRCA2 to constrain BRCA2 function to the S/G2 phases in human cells. We found that the BRCA1-interaction site on PALB2 is targeted by an E3 ubiquitin ligase composed of KEAP1, a PALB2-interacting protein, in complex with cullin-3 (CUL3)-RBX1 (ref. 6). PALB2 ubiquitylation suppresses its interaction with BRCA1 and is counteracted by the deubiquitylase USP11, which is itself under cell cycle control. Restoration of the BRCA1-PALB2 interaction combined with the activation of DNA-end resection is sufficient to induce homologous recombination in G1, as measured by RAD51 recruitment, unscheduled DNA synthesis and a CRISPR-Cas9-based gene-targeting assay. We conclude that the mechanism prohibiting homologous recombination in G1 minimally consists of the suppression of DNA-end resection coupled with a multi-step block of the recruitment of BRCA2 to DNA damage sites that involves the inhibition of BRCA1-PALB2-BRCA2 complex assembly. We speculate that the ability to induce homologous recombination in G1 cells with defined factors could spur the development of gene-targeting applications in non-dividing cells.

Mitosis inhibits DNA double-strand break repair to guard against telomere fusions.

Mitotic cells inactivate DNA double-strand break (DSB) repair, but the rationale behind this suppression remains unknown. Here, we unravel how mitosis blocks DSB repair and determine the consequences of repair reactivation. Mitotic kinases phosphorylate the E3 ubiquitin ligase RNF8 and the nonhomologous end joining factor 53BP1 to inhibit their recruitment to DSB-flanking chromatin. Restoration of RNF8 and 53BP1 accumulation at mitotic DSB sites activates DNA repair but is, paradoxically, deleterious. Aberrantly controlled mitotic DSB repair leads to Aurora B kinase-dependent sister telomere fusions that produce dicentric chromosomes and aneuploidy, especially in the presence of exogenous genotoxic stress. We conclude that the capacity of mitotic DSB repair to destabilize the genome explains the necessity for its suppression during mitosis, principally due to the fusogenic potential of mitotic telomeres.

53BP1 is a reader of the DNA-damage-induced H2A Lys 15 ubiquitin mark.

53BP1 (also called TP53BP1) is a chromatin-associated factor that promotes immunoglobulin class switching and DNA double-strand-break (DSB) repair by non-homologous end joining. To accomplish its function in DNA repair, 53BP1 accumulates at DSB sites downstream of the RNF168 ubiquitin ligase. How ubiquitin recruits 53BP1 to break sites remains unknown as its relocalization involves recognition of histone H4 Lys 20 (H4K20) methylation by its Tudor domain. Here we elucidate how vertebrate 53BP1 is recruited to the chromatin that flanks DSB sites. We show that 53BP1 recognizes mononucleosomes containing dimethylated H4K20 (H4K20me2) and H2A ubiquitinated on Lys 15 (H2AK15ub), the latter being a product of RNF168 action on chromatin. 53BP1 binds to nucleosomes minimally as a dimer using its previously characterized methyl-lysine-binding Tudor domain and a carboxy-terminal extension, termed the ubiquitination-dependent recruitment (UDR) motif, which interacts with the epitope formed by H2AK15ub and its surrounding residues on the H2A tail. 53BP1 is therefore a bivalent histone modification reader that recognizes a histone 'code' produced by DSB signalling.

Expression of the BRCA1 complex member BRE predicts disease free survival in breast cancer.

Breast cancer is one of the leading causes of cancer mortality in women. Recent advances in gene expression profiling have indicated that breast cancer is a heterogeneous disease and the current prognostication using clinico-pathological features is not sufficient to fully predict therapy response and disease outcome. In this retrospective study, we show that expression levels of BRE, which encodes a member of the BRCA1 DNA damage repair complex, predicted disease-free survival (DFS) in non-familial breast cancer patients. The predictive value of BRE expression depended on whether patients received radiotherapy as a part of their primary treatment. In radiotherapy-treated patients, high BRE expression predicted a favorable DFS (hazard ratio (HR) = 0.47, 95 % confidence interval (CI) = 0.28-0.78, p = 0.004), while in non-treated patients, high BRE expression predicted an adverse prognosis (HR = 2.59, 95 % CI = 1.00-6.75, p = 0.05). Among radiotherapy-treated patients, the prognostic impact of BRE expression was confined to patients with smaller tumors (HR = 0.23, 95 % CI = 0.068-0.75, p = 0.015) and it remained an independent factor after correction for the other prognostic factors age, tumor size, lymph node involvement, and histological grade (HR = 0.50, CI = 0.27-0.90, p = 0.021). In addition, high BRE expression predicted a favorable relapse-free survival in a publicly available dataset of 2,324 breast cancer patients (HR = 0.59, CI = 0.51-0.68, p < 0.001). These data indicate that BRE is an interesting candidate for future functional studies aimed at developing targeted therapies.

High BRE expression predicts favorable outcome in adult acute myeloid leukemia, in particular among MLL-AF9-positive patients.

Aberrations in protein ubiquitination have recently been identified in the pathogenesis of acute myeloid leukemia (AML). We studied whether expression changes of more than 1600 ubiquitination related genes correlated with clinical outcome in 525 adult AML patients. High expression of one of these genes, BRE, was observed in 3% of the cases and predicted favorable prognosis independently of known prognostic factors (5-year overall survival: 57%). Remarkably, unsupervised expression profiling showed that 86% of high BRE-expressing patients were confined to a previously unrecognized cluster. High BRE expression was mutually exclusive with FLT3 ITD, CEBPA, IDH1, and IDH2 mutations, EVI1 overexpression, and favorable karyotypes. In contrast, high BRE expression co-occurred strongly with FAB M5 morphology and MLL-AF9 fusions. Within the group of MLL-AF9-positive patients, high BRE expression predicted superior survival, while normal BRE expression predicted extremely poor survival (5-year overall survival of 80% vs 0%, respectively, P = .0002). Both the co-occurrence of high BRE expression with MLL-AF9 and its prognostic impact were confirmed in an independent cohort of 436 AML patients. Thus, high BRE expression defines a novel subtype of adult AML characterized by a favorable prognosis. This work contributes to improved risk stratification in AML, especially among MLL-AF9-positive patients.