DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions. The swift recognition and faithful repair of such damage is crucial for the maintenance of genomic stability, as well as for cell and ...organismal fitness. Signalling by ubiquitin, SUMO and other ubiquitin-like modifiers (UBLs) orchestrates and regulates cellular responses to DSBs at multiple levels, often involving extensive crosstalk between these modifications. Recent findings have revealed compelling insights into the complex mechanisms by which ubiquitin and UBLs regulate protein interactions with DSB sites to promote accurate lesion repair and protection of genome integrity in mammalian cells. These advances offer new therapeutic opportunities for diseases linked to genetic instability.
DNA double-strand breaks (DSBs) are among the most cytotoxic types of DNA damage, which if left unrepaired can lead to mutations or gross chromosomal aberrations, and promote the onset of diseases ...associated with genomic instability such as cancer. One of the most discernible hallmarks of the cellular response to DSBs is the accumulation and local concentration of a plethora of DNA damage signaling and repair proteins in the vicinity of the lesion, initiated by ATM-mediated phosphorylation of H2AX (γ-H2AX) and culminating in the generation of distinct nuclear compartments, so-called Ionizing Radiation-Induced Foci (IRIF). The assembly of proteins at the DSB-flanking chromatin occurs in a highly ordered and strictly hierarchical fashion. To a large extent, this is achieved by regulation of protein–protein interactions triggered by a variety of post-translational modifications including phosphorylation, ubiquitylation, SUMOylation, and acetylation. Over the last decade, insight into the identity of proteins residing in IRIF and the molecular underpinnings of their retention at these structures has been vastly expanded. Despite such advances, however, our understanding of the biological relevance of such DNA repair foci still remains limited. In this review, we focus on recent discoveries on the mechanisms that govern the formation of IRIF, and discuss the implications of such findings in light of our understanding of the physiological importance of these structures.
Proliferating cell nuclear antigen (PCNA) has a central role in promoting faithful DNA replication, providing a molecular platform that facilitates the myriad protein-protein and protein-DNA ...interactions that occur at the replication fork. Numerous PCNA-associated proteins compete for binding to a common surface on PCNA; hence these interactions need to be tightly regulated and coordinated to ensure proper chromosome replication and integrity. Control of PCNA-protein interactions is multilayered and involves post-translational modifications, in particular ubiquitylation, accessory factors and regulated degradation of PCNA-associated proteins. This regulatory framework allows cells to maintain a fine-tuned balance between replication fidelity and processivity in response to DNA damage.
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.
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•Endogenous networks of BRCA1, 53BP1, and MDC1 are characterized by proximity proteomics•Shieldin is a 53BP1 effector complex in DNA double-strand break repair•Vertebrate-specific shieldin is required for antibody class-switch recombination•Deletion of shieldin confers resistance to PARP inhibitors in BRCA1-deficient cells
Application of proximity-based quantitative proteomics allows the characterization of endogenous protein networks among major DNA damage repair factors and reveals the role of the protein complex shieldin in regulating NHEJ, antibody class switching, and sensitivity to PARP inhibitors.
► DNA double-strand breaks (DSBs) trigger protein recruitment to DSB-flanking chromatin. ► Non-proteolytic ubiquitylation plays a key regulatory role in promoting this response. ► The RNF8/RNF168 E3 ...ligase cascade catalyzes DSB-associated chromatin ubiquitylation. ► Ubiquitin and SUMO co-regulate the DSB response by poorly understood mechanisms. ► Recent findings on how ubiquitin and SUMO orchestrate responses to DSBs are discussed.
DNA double-strand breaks (DSBs) represent the most destructive type of chromosomal lesion and trigger rapid chromatin restructuring accompanied by accumulation of proteins in the vicinity of the DSB. Non-proteolytic ubiquitylation of chromatin surrounding DSBs, mediated by the RNF8/RNF168 ubiquitin ligase cascade, has emerged as a key mechanism for restoration of genome integrity by licensing the DSB-modified chromatin to concentrate genome caretaker proteins such as 53BP1 and BRCA1 near the lesions. In parallel, SUMOylation of upstream DSB regulators is also required for execution of this ubiquitin-dependent chromatin response, but its molecular basis is currently unclear. Here, we discuss recent insights into how ubiquitin- and SUMO-dependent signaling processes cooperate to orchestrate protein interactions with sites of DNA damage to facilitate DSB repair.
The faithful segregation of eukaryotic chromosomes in mitosis requires that the genome be duplicated completely prior to anaphase. However, cells with large genomes sometimes fail to complete ...replication during interphase and instead enter mitosis with regions of incompletely replicated DNA. These regions are processed in early mitosis via a process known as mitotic DNA repair synthesis (MiDAS), but little is known about how cells switch from conventional DNA replication to MiDAS. Using the early embryo of the nematode
as a model system, we show that the TRAIP ubiquitin ligase drives replisome disassembly in response to incomplete DNA replication, thereby providing access to replication forks for other factors. Moreover, TRAIP is essential for MiDAS in human cells, and is important in both systems to prevent mitotic segregation errors. Our data indicate that TRAIP is a master regulator of the processing of incomplete DNA replication during mitosis in metazoa.
ATR, activated by replication stress, protects replication forks locally and suppresses origin firing globally. Here, we show that these functions of ATR are mechanistically coupled. Although ...initially stable, stalled forks in ATR-deficient cells undergo nucleus-wide breakage after unscheduled origin firing generates an excess of single-stranded DNA that exhausts the nuclear pool of RPA. Partial reduction of RPA accelerated fork breakage, and forced elevation of RPA was sufficient to delay such “replication catastrophe” even in the absence of ATR activity. Conversely, unscheduled origin firing induced breakage of stalled forks even in cells with active ATR. Thus, ATR-mediated suppression of dormant origins shields active forks against irreversible breakage via preventing exhaustion of nuclear RPA. This study elucidates how replicating genomes avoid destabilizing DNA damage. Because cancer cells commonly feature intrinsically high replication stress, this study also provides a molecular rationale for their hypersensitivity to ATR inhibitors.
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•RPA is rate limiting for shielding replication forks against breakage•ATR signaling prevents RPA exhaustion by restraining origin firing•Exhaustion of RPA leads to pan-nuclear “replication catastrophe”•RPA exhaustion explains the hypersensitivity of cancer cells to ATR inhibitors
ATR-mediated suppression of dormant origins limits the generation of single-stranded DNA and prevents the exhaustion of the nuclear RPA reservoir, therefore protecting cells against “replication catastrophe.”
Activation of the ATR kinase following perturbations to DNA replication relies on a complex mechanism involving ATR recruitment to RPA-coated single-stranded DNA via its binding partner ATRIP and ...stimulation of ATR kinase activity by TopBP1. Here, we discovered an independent ATR activation pathway in vertebrates, mediated by the uncharacterized protein ETAA1 (Ewing's tumour-associated antigen 1). Human ETAA1 accumulates at DNA damage sites via dual RPA-binding motifs and promotes replication fork progression and integrity, ATR signalling and cell survival after genotoxic insults. Mechanistically, this requires a conserved domain in ETAA1 that potently and directly stimulates ATR kinase activity independently of TopBP1. Simultaneous loss of ETAA1 and TopBP1 gives rise to synthetic lethality characterized by massive genome instability and abrogation of ATR-dependent signalling. Our findings demonstrate that parallel TopBP1- and ETAA1-mediated pathways underlie ATR activation and that their combined action is essential for coping with replication stress.
Protein recruitment to DNA double-strand breaks (DSBs) relies on ubiquitylation of the surrounding chromatin by the RING finger ubiquitin ligases RNF8 and RNF168. Flux through this pathway is opposed ...by several deubiquitylating enzymes (DUBs), including OTUB1 and USP3. By analyzing the effect of individually overexpressing the majority of human DUBs on RNF8/RNF168-mediated 53BP1 retention at DSB sites, we found that USP44 and USP29 powerfully inhibited this response at the level of RNF168 accrual. Both USP44 and USP29 promoted efficient deubiquitylation of histone H2A, but unlike USP44, USP29 displayed nonspecific reactivity toward ubiquitylated substrates. Moreover, USP44 but not other H2A DUBs was recruited to RNF168-generated ubiquitylation products at DSB sites. Individual depletion of these DUBs only mildly enhanced accumulation of ubiquitin conjugates and 53BP1 at DSBs, suggesting considerable functional redundancy among cellular DUBs that restrict ubiquitin-dependent protein assembly at DSBs. Our findings implicate USP44 in negative regulation of the RNF8/RNF168 pathway and illustrate the usefulness of DUB overexpression screens for identification of antagonizers of ubiquitin-dependent cellular responses.
Background: The RNF8/RNF168 E3 ligase cascade promotes ubiquitin-dependent protein assembly at DNA double-strand breaks (DSBs).
Results: An overexpression screen identified new deubiquitylating enzymes (DUBs) opposing the RNF8/RNF168 pathway in human cells.
Conclusion: USP44 counteracts RNF168-dependent ubiquitylation of proteins at DSB sites, including histone H2A.
Significance: Identification of antagonizers of the RNF8/RNF168 pathway is crucial for understanding how cells regulate DSB repair.
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.
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•Comprehensive proteomics of replication forks damaged by TOP1 inhibition•Broken and stalled forks show distinct repairomes and chromatin environments•Rewiring of the broken fork proteome by ATM inhibition toward DSB ubiquitination•PLK1, NDRG3, and UBAP2 are promoting repair of broken forks by HR
By systematic proteomics profiling of replication forks challenged by the topoisomerase I inhibitor camptothecin, Nakamura et al. identify dedicated repair factors for broken replication forks, characterize their chromatin environment, and reveal that ATM and PLK1 promote homologous recombination by suppressing the canonical DNA double-strand break ubiquitination response at broken forks.
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