Following the discovery in 1998 of γ-H2AX, the first histone modification induced by DNA damage, interest in the changes to chromatin induced by DNA damage has exploded, and a vast amount of ...information has been generated. However, there has been a discrepancy between our rapidly advancing knowledge of how chromatin responds to DNA damage and the understanding of why cells mobilize large segments of chromatin to protect the genome against destabilizing effects posed by tiny DNA lesions. Recent research has provided insights into these issues and suggests that chromatin responses induced by DNA damage are not simply the accumulation of 'nuclear foci' but are mechanisms required to guard genome integrity.
Proliferating cells rely on the so-called DNA replication checkpoint to ensure orderly completion of genome duplication, and its malfunction may lead to catastrophic genome disruption, including ...unscheduled firing of replication origins, stalling and collapse of replication forks, massive DNA breakage, and, ultimately, cell death. Despite many years of intensive research into the molecular underpinnings of the eukaryotic replication checkpoint, the mechanisms underlying the dismal consequences of its failure remain enigmatic. A recent development offers a unifying model in which the replication checkpoint guards against global exhaustion of rate-limiting replication regulators. Here we discuss how such a mechanism can prevent catastrophic genome disruption and suggest how to harness this knowledge to advance therapeutic strategies to eliminate cancer cells that inherently proliferate under increased DNA replication stress.
This Perspective provides an opinionated view on catastrophic DNA breakage caused by exhausting rate-limiting replication regulators. The authors highlight the unexpected notion that despite qualifying as irreversible damage to the genome, the concept of replication catastrophe reveals basic principles of physiological DNA replication, which opens new opportunities for cancer therapy.
Failure to complete DNA replication is a stochastic by-product of genome doubling in almost every cell cycle. During mitosis, under-replicated DNA (UR-DNA) is converted into DNA lesions, which are ...inherited by daughter cells and sequestered in 53BP1 nuclear bodies (53BP1-NBs). The fate of such cells remains unknown. Here, we show that the formation of 53BP1-NBs interrupts the chain of iterative damage intrinsically embedded in UR-DNA. Unlike clastogen-induced 53BP1 foci that are repaired throughout interphase, 53BP1-NBs restrain replication of the embedded genomic loci until late S phase, thus enabling the dedicated RAD52-mediated repair of UR-DNA lesions. The absence or malfunction of 53BP1-NBs causes premature replication of the affected loci, accompanied by genotoxic RAD51-mediated recombination. Thus, through adjusting replication timing and repair pathway choice at under-replicated loci, 53BP1-NBs enable the completion of genome duplication of inherited UR-DNA and prevent the conversion of stochastic under-replications into genome instability.
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.”
DNA replication requires coordination between replication fork progression and deoxynucleotide triphosphate (dNTP)–generating metabolic pathways. We find that perturbation of ribonucleotide reductase ...(RNR) in humans elevates reactive oxygen species (ROS) that are detected by peroxiredoxin 2 (PRDX2). In the oligomeric state, PRDX2 forms a replisome-associated ROS sensor, which binds the fork accelerator TIMELESS when exposed to low levels of ROS. Elevated ROS levels generated by RNR attenuation disrupt oligomerized PRDX2 to smaller subunits, whose dissociation from chromatin enforces the displacement of TIMELESS from the replisome. This process instantly slows replication fork progression, which mitigates pathological consequences of replication stress. Thus, redox signaling couples fluctuations of dNTP biogenesis with replisome activity to reduce stress during genome duplication. We propose that cancer cells exploit this pathway to increase their adaptability to adverse metabolic conditions.
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.
To safeguard genome integrity in response to DNA double-strand breaks (DSBs), mammalian cells mobilize the neighbouring chromatin to shield DNA ends against excessive resection that could undermine ...repair fidelity and cause damage to healthy chromosomes
. This form of genome surveillance is orchestrated by 53BP1, whose accumulation at DSBs triggers sequential recruitment of RIF1 and the shieldin-CST-POLα complex
. How this pathway reflects and influences the three-dimensional nuclear architecture is not known. Here we use super-resolution microscopy to show that 53BP1 and RIF1 form an autonomous functional module that stabilizes three-dimensional chromatin topology at sites of DNA breakage. This process is initiated by accumulation of 53BP1 at regions of compact chromatin that colocalize with topologically associating domain (TAD) sequences, followed by recruitment of RIF1 to the boundaries between such domains. The alternating distribution of 53BP1 and RIF1 stabilizes several neighbouring TAD-sized structures at a single DBS site into an ordered, circular arrangement. Depletion of 53BP1 or RIF1 (but not shieldin) disrupts this arrangement and leads to decompaction of DSB-flanking chromatin, reduction in interchromatin space, aberrant spreading of DNA repair proteins, and hyper-resection of DNA ends. Similar topological distortions are triggered by depletion of cohesin, which suggests that the maintenance of chromatin structure after DNA breakage involves basic mechanisms that shape three-dimensional nuclear organization. As topological stabilization of DSB-flanking chromatin is independent of DNA repair, we propose that, besides providing a structural scaffold to protect DNA ends against aberrant processing, 53BP1 and RIF1 safeguard epigenetic integrity at loci that are disrupted by DNA breakage.
Accumulation of repair proteins on damaged chromosomes is required to restore genomic integrity. However, the mechanisms of protein retention at the most destructive chromosomal lesions, the DNA ...double-strand breaks (DSBs), are poorly understood. We show that RNF8, a RING-finger ubiquitin ligase, rapidly assembles at DSBs via interaction of its FHA domain with the phosphorylated adaptor protein MDC1. This is accompanied by an increase in DSB-associated ubiquitylations and followed by accumulation of 53BP1 and BRCA1 repair proteins. Knockdown of RNF8 or disruption of its FHA or RING domains impaired DSB-associated ubiquitylation and inhibited retention of 53BP1 and BRCA1 at the DSB sites. In addition, we show that RNF8 can ubiquitylate histone H2A and H2AX, and that its depletion sensitizes cells to ionizing radiation. These data suggest that MDC1-mediated and RNF8-executed histone ubiquitylation protects genome integrity by licensing the DSB-flanking chromatin to concentrate repair factors near the DNA lesions.
DNA double-strand breaks (DSBs) not only interrupt the genetic information, but also disrupt the chromatin structure, and both impairments require repair mechanisms to ensure genome integrity. We ...showed previously that RNF8-mediated chromatin ubiquitylation protects genome integrity by promoting the accumulation of repair factors at DSBs. Here, we provide evidence that, while RNF8 is necessary to trigger the DSB-associated ubiquitylations, it is not sufficient to sustain conjugated ubiquitin in this compartment. We identified RNF168 as a novel chromatin-associated ubiquitin ligase with an ability to bind ubiquitin. We show that RNF168 interacts with ubiquitylated H2A, assembles at DSBs in an RNF8-dependent manner, and, by targeting H2A and H2AX, amplifies local concentration of lysine 63-linked ubiquitin conjugates to the threshold required for retention of 53BP1 and BRCA1. Thus, RNF168 defines a new pathway involving sequential ubiquitylations on damaged chromosomes and uncovers a functional cooperation between E3 ligases in genome maintenance.
Intrinsically disordered proteins can phase separate from the soluble intracellular space, and tend to aggregate under pathological conditions. The physiological functions and molecular triggers of ...liquid demixing by phase separation are not well understood. Here we show in vitro and in vivo that the nucleic acid-mimicking biopolymer poly(ADP-ribose) (PAR) nucleates intracellular liquid demixing. PAR levels are markedly induced at sites of DNA damage, and we provide evidence that PAR-seeded liquid demixing results in rapid, yet transient and fully reversible assembly of various intrinsically disordered proteins at DNA break sites. Demixing, which relies on electrostatic interactions between positively charged RGG repeats and negatively charged PAR, is amplified by aggregation-prone prion-like domains, and orchestrates the earliest cellular responses to DNA breakage. We propose that PAR-seeded liquid demixing is a general mechanism to dynamically reorganize the soluble nuclear space with implications for pathological protein aggregation caused by derailed phase separation.
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