ABSTRACT
Knudson's "two-hit" paradigm posits that carcinogenesis requires inactivation of both copies of an autosomal tumor suppressor gene. Here, we report that the glycolytic metabolite methylglyoxal (MGO) transiently bypasses Knudson's paradigm by inactivating the breast cancer suppressor protein BRCA2 to elicit a cancer-associated, mutational single-base substitution (SBS) signature in nonmalignant mammary cells or patient-derived organoids. Germline monoallelic BRCA2 mutations predispose to these changes. An analogous SBS signature, again without biallelic BRCA2 inactivation, accompanies MGO accumulation and DNA damage in Kras-driven, Brca2-mutant murine pancreatic cancers and human breast cancers. MGO triggers BRCA2 proteolysis, temporarily disabling BRCA2's tumor suppressive functions in DNA repair and replication, causing functional haploinsufficiency. Intermittent MGO exposure incites episodic SBS mutations without permanent BRCA2 inactivation. Thus, a metabolic mechanism wherein MGO-induced BRCA2 haploinsufficiency transiently bypasses Knudson's two-hit requirement could link glycolysis activation by oncogenes, metabolic disorders, or dietary challenges to mutational signatures implicated in cancer evolution.
Subject(s)
BRCA2 Protein , Breast Neoplasms , Glycolysis , Pyruvaldehyde , Animals , BRCA2 Protein/metabolism , BRCA2 Protein/genetics , Mice , Humans , Female , Pyruvaldehyde/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Haploinsufficiency , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Mutation , DNA Damage , DNA Repair , Cell Line, TumorABSTRACT
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.
Subject(s)
BRCA1 Protein/genetics , BRCA2 Protein/genetics , Breast Neoplasms/genetics , Gene Regulatory Networks , Rad51 Recombinase/genetics , Recombinational DNA Repair , Tumor Suppressor Proteins/genetics , Ubiquitin-Protein Ligases/genetics , BRCA1 Protein/chemistry , BRCA1 Protein/metabolism , BRCA2 Protein/chemistry , BRCA2 Protein/metabolism , Binding Sites , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , DNA/chemistry , DNA/genetics , DNA/metabolism , DNA Breaks, Double-Stranded , DNA Replication , Female , Genome, Human , Genomic Instability , Humans , Models, Molecular , Protein Binding , Protein Structure, Secondary , Rad51 Recombinase/chemistry , Rad51 Recombinase/metabolism , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolismABSTRACT
While mutations affecting protein-coding regions have been examined across many cancers, structural variants at the genome-wide level are still poorly defined. Through integrative deep whole-genome and -transcriptome analysis of 101 castration-resistant prostate cancer metastases (109X tumor/38X normal coverage), we identified structural variants altering critical regulators of tumorigenesis and progression not detectable by exome approaches. Notably, we observed amplification of an intergenic enhancer region 624 kb upstream of the androgen receptor (AR) in 81% of patients, correlating with increased AR expression. Tandem duplication hotspots also occur near MYC, in lncRNAs associated with post-translational MYC regulation. Classes of structural variations were linked to distinct DNA repair deficiencies, suggesting their etiology, including associations of CDK12 mutation with tandem duplications, TP53 inactivation with inverted rearrangements and chromothripsis, and BRCA2 inactivation with deletions. Together, these observations provide a comprehensive view of how structural variations affect critical regulators in metastatic prostate cancer.
Subject(s)
Genomic Structural Variation/genetics , Prostatic Neoplasms/genetics , Aged , Aged, 80 and over , BRCA2 Protein/metabolism , Cyclin-Dependent Kinases/metabolism , DNA Copy Number Variations , Exome , Gene Expression Profiling/methods , Genomics/methods , Humans , Male , Middle Aged , Mutation , Neoplasm Metastasis/genetics , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Tandem Repeat Sequences/genetics , Tumor Suppressor Protein p53/metabolism , Whole Genome Sequencing/methodsABSTRACT
Mutations truncating a single copy of the tumor suppressor, BRCA2, cause cancer susceptibility. In cells bearing such heterozygous mutations, we find that a cellular metabolite and ubiquitous environmental toxin, formaldehyde, stalls and destabilizes DNA replication forks, engendering structural chromosomal aberrations. Formaldehyde selectively depletes BRCA2 via proteasomal degradation, a mechanism of toxicity that affects very few additional cellular proteins. Heterozygous BRCA2 truncations, by lowering pre-existing BRCA2 expression, sensitize to BRCA2 haploinsufficiency induced by transient exposure to natural concentrations of formaldehyde. Acetaldehyde, an alcohol catabolite detoxified by ALDH2, precipitates similar effects. Ribonuclease H1 ameliorates replication fork instability and chromosomal aberrations provoked by aldehyde-induced BRCA2 haploinsufficiency, suggesting that BRCA2 inactivation triggers spontaneous mutagenesis during DNA replication via aberrant RNA-DNA hybrids (R-loops). These findings suggest a model wherein carcinogenesis in BRCA2 mutation carriers can be incited by compounds found pervasively in the environment and generated endogenously in certain tissues with implications for public health.
Subject(s)
BRCA2 Protein/genetics , Chromosome Aberrations/drug effects , Formaldehyde/toxicity , Genomic Instability/drug effects , Toxins, Biological/toxicity , DNA Damage , DNA Replication/drug effects , DNA-Binding Proteins/metabolism , Haploinsufficiency , HeLa Cells , Humans , MRE11 Homologue Protein , Proteome , Ribonuclease H/metabolismABSTRACT
Tumor suppressor BRCA2 functions in homology-directed repair (HDR), the protection of stalled replication forks, and the suppression of replicative gaps, but their relative contributions to genome integrity and chemotherapy response are under scrutiny. Here, we report that mouse and human cells require a RAD51 filament stabilization motif in BRCA2 for fork protection and gap suppression but not HDR. In mice, the loss of fork protection/gap suppression does not compromise genome stability or shorten tumor latency. By contrast, HDR deficiency increases spontaneous and replication stress-induced chromosome aberrations and tumor predisposition. Unlike with HDR, fork protection/gap suppression defects are also observed in Brca2 heterozygous cells, likely due to reduced RAD51 stabilization at stalled forks/gaps. Gaps arise from PRIMPOL activity, which is associated with 5-hydroxymethyl-2'-deoxyuridine sensitivity due to the formation of SMUG1-generated abasic sites and is exacerbated by poly(ADP-ribose) polymerase (PARP) inhibition. However, HDR proficiency has the major role in mitigating sensitivity to chemotherapeutics, including PARP inhibitors.
Subject(s)
BRCA2 Protein , DNA Replication , Rad51 Recombinase , Animals , Humans , Mice , BRCA2 Protein/metabolism , DNA Repair , Genomic Instability , Genomics , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Recombinational DNA RepairABSTRACT
Abasic sites are DNA lesions repaired by base excision repair. Cleavage of unrepaired abasic sites in single-stranded DNA (ssDNA) can lead to chromosomal breakage during DNA replication. How rupture of abasic DNA is prevented remains poorly understood. Here, using cryoelectron microscopy (cryo-EM), Xenopus laevis egg extracts, and human cells, we show that RAD51 nucleofilaments specifically recognize and protect abasic sites, which increase RAD51 association rate to DNA. In the absence of BRCA2 or RAD51, abasic sites accumulate as a result of DNA base methylation, oxidation, and deamination, inducing abasic ssDNA gaps that make replicating DNA fibers sensitive to APE1. RAD51 assembled on abasic DNA prevents abasic site cleavage by the MRE11-RAD50 complex, suppressing replication fork breakage triggered by an excess of abasic sites or POLθ polymerase inhibition. Our study highlights the critical role of BRCA2 and RAD51 in safeguarding against unrepaired abasic sites in DNA templates stemming from base alterations, ensuring genomic stability.
Subject(s)
BRCA2 Protein , DNA Damage , DNA Repair , DNA Replication , DNA, Single-Stranded , Rad51 Recombinase , Xenopus laevis , Humans , Rad51 Recombinase/metabolism , Rad51 Recombinase/genetics , BRCA2 Protein/genetics , BRCA2 Protein/metabolism , Animals , DNA, Single-Stranded/metabolism , DNA, Single-Stranded/genetics , Cryoelectron Microscopy , DNA Polymerase theta , DNA Methylation , DNA-Directed DNA Polymerase/metabolism , DNA-Directed DNA Polymerase/genetics , MRE11 Homologue Protein/metabolism , MRE11 Homologue Protein/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/geneticsABSTRACT
The repair of DNA by homologous recombination is an essential, efficient, and high-fidelity process that mends DNA lesions formed during cellular metabolism; these lesions include double-stranded DNA breaks, daughter-strand gaps, and DNA cross-links. Genetic defects in the homologous recombination pathway undermine genomic integrity and cause the accumulation of gross chromosomal abnormalities-including rearrangements, deletions, and aneuploidy-that contribute to cancer formation. Recombination proceeds through the formation of joint DNA molecules-homologously paired but metastable DNA intermediates that are processed by several alternative subpathways-making recombination a versatile and robust mechanism to repair damaged chromosomes. Modern biophysical methods make it possible to visualize, probe, and manipulate the individual molecules participating in the intermediate steps of recombination, revealing new details about the mechanics of genetic recombination. We review and discuss the individual stages of homologous recombination, focusing on common pathways in bacteria, yeast, and humans, and place particular emphasis on the molecular mechanisms illuminated by single-molecule methods.
Subject(s)
DNA/genetics , Escherichia coli/genetics , Recombination, Genetic , Recombinational DNA Repair , Saccharomyces cerevisiae/genetics , Chromosome Aberrations , DNA/metabolism , DNA Damage , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/metabolism , Escherichia coli/metabolism , Exodeoxyribonuclease V/genetics , Exodeoxyribonuclease V/metabolism , Exodeoxyribonucleases/genetics , Exodeoxyribonucleases/metabolism , Gene Expression Regulation , Genomic Instability , Humans , Rad52 DNA Repair and Recombination Protein/genetics , Rad52 DNA Repair and Recombination Protein/metabolism , RecQ Helicases/genetics , RecQ Helicases/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Single Molecule ImagingABSTRACT
Homologous recombination (HR) is essential for error-free repair of DNA double-strand breaks, perturbed replication forks (RFs), and post-replicative single-stranded DNA (ssDNA) gaps. To initiate HR, the recombination mediator and tumor suppressor protein BRCA2 facilitates nucleation of RAD51 on ssDNA prior to stimulation of RAD51 filament growth by RAD51 paralogs. Although ssDNA binding by BRCA2 has been implicated in RAD51 nucleation, the function of double-stranded DNA (dsDNA) binding by BRCA2 remains unclear. Here, we exploit single-molecule (SM) imaging to visualize BRCA2-mediated RAD51 nucleation in real time using purified proteins. We report that BRCA2 nucleates and stabilizes RAD51 on ssDNA either directly or through an unappreciated diffusion-assisted delivery mechanism involving binding to and sliding along dsDNA, which requires the cooperative action of multiple dsDNA-binding modules in BRCA2. Collectively, our work reveals two distinct mechanisms of BRCA2-dependent RAD51 loading onto ssDNA, which we propose are critical for its diverse functions in maintaining genome stability and cancer suppression.
Subject(s)
BRCA2 Protein , Rad51 Recombinase , Humans , BRCA2 Protein/genetics , BRCA2 Protein/metabolism , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , DNA-Binding Proteins/metabolism , DNA, Single-Stranded/genetics , DNA/metabolism , DNA Repair , Protein BindingABSTRACT
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.
Subject(s)
DNA Breaks, Double-Stranded , Drug Resistance, Neoplasm , Drug Resistance, Neoplasm/genetics , DNA Damage , DNA End-Joining Repair , DNA/genetics , DNA Replication , DNA RepairABSTRACT
Aberrant replication causes cells lacking BRCA2 to enter mitosis with under-replicated DNA, which activates a repair mechanism known as mitotic DNA synthesis (MiDAS). Here, we identify genome-wide the sites where MiDAS reactions occur when BRCA2 is abrogated. High-resolution profiling revealed that these sites are different from MiDAS at aphidicolin-induced common fragile sites in that they map to genomic regions replicating in the early S-phase, which are close to early-firing replication origins, are highly transcribed, and display R-loop-forming potential. Both transcription inhibition in early S-phase and RNaseH1 overexpression reduced MiDAS in BRCA2-deficient cells, indicating that transcription-replication conflicts (TRCs) and R-loops are the source of MiDAS. Importantly, the MiDAS sites identified in BRCA2-deficient cells also represent hotspots for genomic rearrangements in BRCA2-mutated breast tumors. Thus, our work provides a mechanism for how tumor-predisposing BRCA2 inactivation links transcription-induced DNA damage with mitotic DNA repair to fuel the genomic instability characteristic of cancer cells.
Subject(s)
DNA Replication , Mitosis , Aphidicolin/pharmacology , BRCA2 Protein/genetics , Chromosome Fragile Sites/genetics , DNA/genetics , DNA Damage , Genomic Instability , Humans , Mitosis/geneticsABSTRACT
RAD51 and the breast cancer suppressor BRCA2 have critical functions in DNA double-strand (dsDNA) break repair by homologous recombination and the protection of newly replicated DNA from nucleolytic degradation. The recombination function of RAD51 requires its binding to single-stranded DNA (ssDNA), whereas binding to dsDNA is inhibitory. Using reconstituted MRE11-, EXO1-, and DNA2-dependent nuclease reactions, we show that the protective function of RAD51 unexpectedly depends on its binding to dsDNA. The BRC4 repeat of BRCA2 abrogates RAD51 binding to dsDNA and accordingly impairs the function of RAD51 in protection. The BRCA2 C-terminal RAD51-binding segment (TR2) acts in a dominant manner to overcome the effect of BRC4. Mechanistically, TR2 stabilizes RAD51 binding to dsDNA, even in the presence of BRC4, promoting DNA protection. Our data suggest that RAD51's dsDNA-binding capacity may have evolved together with its function in replication fork protection and provide a mechanistic basis for the DNA-protection function of BRCA2.
Subject(s)
DNA, Single-Stranded , Rad51 Recombinase , BRCA2 Protein/genetics , BRCA2 Protein/metabolism , DNA/genetics , DNA Breaks, Double-Stranded , DNA Repair , DNA Replication , DNA, Single-Stranded/genetics , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolismABSTRACT
POLθ promotes repair of DNA double-strand breaks (DSBs) resulting from collapsed forks in homologous recombination (HR) defective tumors. Inactivation of POLθ results in synthetic lethality with the loss of HR genes BRCA1/2, which induces under-replicated DNA accumulation. However, it is unclear whether POLθ-dependent DNA replication prevents HR-deficiency-associated lethality. Here, we isolated Xenopus laevis POLθ and showed that it processes stalled Okazaki fragments, directly visualized by electron microscopy, thereby suppressing ssDNA gaps accumulating on lagging strands in the absence of RAD51 and preventing fork reversal. Inhibition of POLθ DNA polymerase activity leaves fork gaps unprotected, enabling their cleavage by the MRE11-NBS1-CtIP endonuclease, which produces broken forks with asymmetric single-ended DSBs, hampering BRCA2-defective cell survival. These results reveal a POLθ-dependent genome protection function preventing stalled forks rupture and highlight possible resistance mechanisms to POLθ inhibitors.
Subject(s)
DNA Replication , DNA-Binding Proteins , MRE11 Homologue Protein/genetics , MRE11 Homologue Protein/metabolism , DNA-Binding Proteins/genetics , Homologous Recombination/genetics , DNAABSTRACT
RAD51 facilitates replication fork reversal and protects reversed forks from nuclease degradation. Although potentially a useful replication stress response mechanism, unregulated fork reversal can cause genome instability. Here we show that RADX, a single-strand DNA binding protein that binds to and destabilizes RAD51 nucleofilaments, can either inhibit or promote fork reversal depending on replication stress levels. RADX inhibits fork reversal at elongating forks, thereby preventing fork slowing and collapse. Paradoxically, in the presence of persistent replication stress, RADX localizes to stalled forks to generate reversed fork structures. Consequently, inactivating RADX prevents fork-reversal-dependent telomere dysfunction in the absence of RTEL1 and blocks nascent strand degradation when fork protection factors are inactivated. Addition of RADX increases SMARCAL1-dependent fork reversal in conditions in which pre-binding RAD51 to a model fork substrate is inhibitory. Thus, RADX directly interacts with RAD51 and single-strand DNA to confine fork reversal to persistently stalled forks.
Subject(s)
DNA Replication/genetics , DNA-Binding Proteins/genetics , Genomic Instability/genetics , Replication Origin/genetics , Cell Line , Cell Line, Tumor , DNA Breaks, Double-Stranded , DNA Helicases/genetics , DNA Repair/genetics , DNA, Single-Stranded/genetics , HEK293 Cells , HeLa Cells , Humans , Protein Binding/genetics , Rad51 Recombinase/geneticsABSTRACT
Homologous recombination (HR) is an essential DNA double-strand break (DSB) repair mechanism, which is frequently inactivated in cancer. During HR, RAD51 forms nucleoprotein filaments on RPA-coated, resected DNA and catalyzes strand invasion into homologous duplex DNA. How RAD51 displaces RPA and assembles into long HR-proficient filaments remains uncertain. Here, we employed single-molecule imaging to investigate the mechanism of nematode RAD-51 filament growth in the presence of BRC-2 (BRCA2) and RAD-51 paralogs, RFS-1/RIP-1. BRC-2 nucleates RAD-51 on RPA-coated DNA, whereas RFS-1/RIP-1 acts as a "chaperone" to promote 3' to 5' filament growth via highly dynamic engagement with 5' filament ends. Inhibiting ATPase or mutation in the RFS-1 Walker box leads to RFS-1/RIP-1 retention on RAD-51 filaments and hinders growth. The rfs-1 Walker box mutants display sensitivity to DNA damage and accumulate RAD-51 complexes non-functional for HR in vivo. Our work reveals the mechanism of RAD-51 nucleation and filament growth in the presence of recombination mediators.
Subject(s)
Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/genetics , Carrier Proteins/genetics , DNA, Helminth/genetics , DNA-Binding Proteins/genetics , Rad51 Recombinase/genetics , Recombinational DNA Repair , Animals , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , Carrier Proteins/metabolism , DNA Breaks, Double-Stranded , DNA, Helminth/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Mutation , Protein Binding , Rad51 Recombinase/metabolism , Replication Protein A/genetics , Replication Protein A/metabolism , Signal Transduction , Single Molecule ImagingABSTRACT
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.
Subject(s)
DNA Breaks, Single-Stranded , DNA Primase/metabolism , DNA Repair , DNA Replication , DNA, Neoplasm/biosynthesis , DNA-Directed DNA Polymerase/metabolism , G2 Phase , Multifunctional Enzymes/metabolism , Neoplasms/metabolism , S Phase , Antineoplastic Agents/pharmacology , BRCA1 Protein/genetics , BRCA1 Protein/metabolism , BRCA2 Protein/metabolism , Cell Line, Tumor , DNA Helicases/genetics , DNA Helicases/metabolism , DNA Primase/genetics , DNA, Neoplasm/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/genetics , Genomic Instability , HEK293 Cells , Humans , MRE11 Homologue Protein/genetics , MRE11 Homologue Protein/metabolism , Multifunctional Enzymes/genetics , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/pathology , Nucleotidyltransferases/genetics , Nucleotidyltransferases/metabolism , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Time Factors , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , UbiquitinationABSTRACT
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.
Subject(s)
DNA Breaks, Single-Stranded , DNA Primase/metabolism , DNA Replication , DNA, Neoplasm/biosynthesis , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/metabolism , Multifunctional Enzymes/metabolism , Neoplasms/enzymology , Nucleotidyltransferases/metabolism , Recombinational DNA Repair , Animals , Antineoplastic Agents/pharmacology , BRCA1 Protein/genetics , BRCA1 Protein/metabolism , BRCA2 Protein/genetics , BRCA2 Protein/metabolism , Cell Line, Tumor , DNA Primase/genetics , DNA, Neoplasm/genetics , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , DNA-Directed DNA Polymerase/genetics , Female , HEK293 Cells , Humans , Mice, Nude , Multifunctional Enzymes/genetics , Mutation , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/pathology , Nucleic Acid Synthesis Inhibitors/pharmacology , Nucleotidyltransferases/antagonists & inhibitors , Nucleotidyltransferases/genetics , Uracil-DNA Glycosidase/genetics , Uracil-DNA Glycosidase/metabolism , Xenograft Model Antitumor AssaysABSTRACT
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.
Subject(s)
BRCA1 Protein/genetics , DNA Replication/drug effects , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Animals , Cell Line , Cisplatin/pharmacology , DNA/genetics , DNA/metabolism , DNA, Single-Stranded/genetics , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Fanconi Anemia Complementation Group Proteins/genetics , Homologous Recombination/drug effects , Humans , Mice, Inbred NOD , RNA Helicases/genetics , Rad51 Recombinase/genetics , Replication Protein A/genetics , Tumor Suppressor p53-Binding Protein 1/geneticsABSTRACT
Targeting poly(ADP-ribose) glycohydrolase (PARG) is currently explored as a therapeutic approach to treat various cancer types, but we have a poor understanding of the specific genetic vulnerabilities that would make cancer cells susceptible to such a tailored therapy. Moreover, the identification of such vulnerabilities is of interest for targeting BRCA2;p53-deficient tumors that have acquired resistance to poly(ADP-ribose) polymerase inhibitors (PARPi) through loss of PARG expression. Here, by performing whole-genome CRISPR/Cas9 drop-out screens, we identify various genes involved in DNA repair to be essential for the survival of PARG;BRCA2;p53-deficient cells. In particular, our findings reveal EXO1 and FEN1 as major synthetic lethal interactors of PARG loss. We provide evidence for compromised replication fork progression, DNA single-strand break repair, and Okazaki fragment processing in PARG;BRCA2;p53-deficient cells, alterations that exacerbate the effects of EXO1/FEN1 inhibition and become lethal in this context. Since this sensitivity is dependent on BRCA2 defects, we propose to target EXO1/FEN1 in PARPi-resistant tumors that have lost PARG activity. Moreover, EXO1/FEN1 targeting may be a useful strategy for enhancing the effect of PARG inhibitors in homologous recombination-deficient tumors.
Subject(s)
Neoplasms , Tumor Suppressor Protein p53 , Humans , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , DNA Repair , DNA Damage , Neoplasms/drug therapy , Neoplasms/genetics , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Glycoside Hydrolases/genetics , Glycoside Hydrolases/metabolism , Flap Endonucleases/genetics , Flap Endonucleases/metabolism , Flap Endonucleases/therapeutic use , Exodeoxyribonucleases/genetics , DNA Repair Enzymes/geneticsABSTRACT
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.
Subject(s)
BRCA1 Protein/deficiency , BRCA2 Protein/deficiency , DNA Repair Enzymes/metabolism , DNA Replication , Exodeoxyribonucleases/metabolism , Histones/metabolism , MRE11 Homologue Protein/metabolism , p300-CBP Transcription Factors/metabolism , Acetylation/drug effects , Amino Acid Sequence , Ataxia Telangiectasia Mutated Proteins/metabolism , BRCA1 Protein/metabolism , BRCA2 Protein/metabolism , Breast Neoplasms/genetics , Cell Line, Tumor , DNA Replication/drug effects , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lysine/metabolism , Models, Biological , Mutation/genetics , Phosphorylation/drug effects , Phosphoserine/metabolism , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Protein Binding/drug effects , p300-CBP Transcription Factors/chemistry , p300-CBP Transcription Factors/geneticsABSTRACT
The APEX2 gene encodes APE2, a nuclease related to APE1, the apurinic/apyrimidinic endonuclease acting in base excision repair. Loss of APE2 is lethal in cells with mutated BRCA1 or BRCA2, making APE2 a prime target for homologous recombination-defective cancers. However, because the function of APE2 in DNA repair is poorly understood, it is unclear why BRCA-deficient cells require APE2 for viability. Here we present the genetic interaction profiles of APE2, APE1, and TDP1 deficiency coupled to biochemical and structural dissection of APE2. We conclude that the main role of APE2 is to reverse blocked 3' DNA ends, problematic lesions that preclude DNA synthesis. Our work also suggests that TOP1 processing of genomic ribonucleotides is the main source of 3'-blocking lesions relevant to APEX2-BRCA1/2 synthetic lethality. The exquisite sensitivity of BRCA-deficient cells to 3' blocks indicates that they represent a tractable vulnerability in homologous recombination-deficient tumor cells.