DNA double-strand breaks (DSBs) are highly toxic lesions that can drive genetic instability. To preserve genome integrity, organisms have evolved several DSB repair mechanisms, of which nonhomologous ...end-joining (NHEJ) and homologous recombination (HR) represent the two most prominent. It has recently become apparent that multiple layers of regulation exist to ensure these repair pathways are accurate and restricted to the appropriate cellular contexts. Such regulation is crucial, as failure to properly execute DSB repair is known to accelerate tumorigenesis and is associated with several human genetic syndromes. Here, we review recent insights into the mechanisms that influence the choice between competing DSB repair pathways, how this is regulated during the cell cycle, and how imbalances in this equilibrium result in genome instability.
Covalent DNA-protein crosslinks (DPCs, also known as protein adducts) of topoisomerases and other proteins with DNA are highly toxic DNA lesions. Of note, chemical agents that induce DPCs include ...widely used classes of chemotherapeutics. Their bulkiness blocks virtually every chromatin-based process and makes them intractable for repair by canonical repair pathways. Distinct DPC repair pathways employ unique points of attack and are crucial for the maintenance of genome stability. Tyrosyl-DNA phosphodiesterases (TDPs) directly hydrolyse the covalent linkage between protein and DNA. The MRE11-RAD50-NBS1 (MRN) nuclease complex targets the DNA component of DPCs, excising the fragment affected by the lesion, whereas proteases of the spartan (SPRTN)/weak suppressor of SMT3 protein 1 (Wss1) family target the protein component. Loss of these pathways renders cells sensitive to DPC-inducing chemotherapeutics, and DPC repair pathways are thus attractive targets for combination cancer therapy.
DNA is subject to constant chemical modification and damage, which eventually results in variable mutation rates throughout the genome. Although detailed molecular mechanisms of DNA damage and repair ...are well understood, damage impact and execution of repair across a genome remain poorly defined.
To bridge the gap between our understanding of DNA repair and mutation distributions, we developed a novel method, AP-seq, capable of mapping apurinic sites and 8-oxo-7,8-dihydroguanine bases at approximately 250-bp resolution on a genome-wide scale. We directly demonstrate that the accumulation rate of apurinic sites varies widely across the genome, with hot spots acquiring many times more damage than cold spots. Unlike single nucleotide variants (SNVs) in cancers, damage burden correlates with marks for open chromatin notably H3K9ac and H3K4me2. Apurinic sites and oxidative damage are also highly enriched in transposable elements and other repetitive sequences. In contrast, we observe a reduction at chromatin loop anchors with increased damage load towards inactive compartments. Less damage is found at promoters, exons, and termination sites, but not introns, in a seemingly transcription-independent but GC content-dependent manner. Leveraging cancer genomic data, we also find locally reduced SNV rates in promoters, coding sequence, and other functional elements.
Our study reveals that oxidative DNA damage accumulation and repair differ strongly across the genome, but culminate in a previously unappreciated mechanism that safeguards the regulatory and coding regions of genes from mutations.
The appropriate execution of DNA double-strand break (DSB) repair is critical for genome stability and tumor avoidance. 53BP1 and BRCA1 directly influence DSB repair pathway choice by regulating 5′ ...end resection, but how this is achieved remains uncertain. Here we report that Rif1−/− mice are severely compromised for 53BP1-dependent class switch recombination (CSR) and fusion of dysfunctional telomeres. The inappropriate accumulation of RIF1 at DSBs in S phase is antagonized by BRCA1, and deletion of Rif1 suppresses toxic nonhomologous end joining (NHEJ) induced by PARP inhibition in Brca1-deficient cells. Mechanistically, RIF1 is recruited to DSBs via the N-terminal phospho-SQ/TQ domain of 53BP1, and DSBs generated by ionizing radiation or during CSR are hyperresected in the absence of RIF1. Thus, RIF1 and 53BP1 cooperate to block DSB resection to promote NHEJ in G1, which is antagonized by BRCA1 in S phase to ensure a switch of DSB repair mode to homologous recombination.
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► RIF1 is essential for 53BP1-dependent CSR and fusion of dysfunctional telomeres ► BRCA1 antagonizes RIF1 in S phase to prevent error-prone repair by toxic NHEJ ► N-terminal phospho-SQ/TQ domain of 53BP1 interacts with and recruits RIF1 to DSBs ► RIF1 and 53BP1 promote NHEJ in G1 by blocking 5′ end resection of DSBs
T loops and telomeric G-quadruplex (G4) DNA structures pose a potential threat to genome stability and must be dismantled to permit efficient telomere replication. Here we implicate the helicase ...RTEL1 in the removal of telomeric DNA secondary structures, which is essential for preventing telomere fragility and loss. In the absence of RTEL1, T loops are inappropriately resolved by the SLX4 nuclease complex, resulting in loss of the telomere as a circle. Depleting SLX4 or blocking DNA replication abolished telomere circles (TCs) and rescued telomere loss in RTEL1−/− cells but failed to suppress telomere fragility. Conversely, stabilization of telomeric G4-DNA or loss of BLM dramatically enhanced telomere fragility in RTEL1-deficient cells but had no impact on TC formation or telomere loss. We propose that RTEL1 performs two distinct functions at telomeres: it disassembles T loops and also counteracts telomeric G4-DNA structures, which together ensure the dynamics and stability of the telomere.
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► RTEL1 disassembles T loops to maintain telomere integrity ► In RTEL1−/− cells, T loop resolution by SLX4 removes the telomere as a circle ► RTEL1 and BLM operate in separate pathways to prevent telomere fragility ► G-quadruplex DNA structures are a major source of telomere fragility
The DNA helicase RTEL1 acts during DNA replication to unfold telomeric T loops and G4-DNA structures; in its absence, T loops are cleaved and resolved as circular telomere fragments, resulting in ectopic recombination and telomere-length instability.
Covalent DNA-protein crosslinks (DPCs) are toxic DNA lesions that interfere with essential chromatin transactions, such as replication and transcription. Little was known about DPC-specific repair ...mechanisms until the recent identification of a DPC-processing protease in yeast. The existence of a DPC protease in higher eukaryotes is inferred from data in Xenopus laevis egg extracts, but its identity remains elusive. Here we identify the metalloprotease SPRTN as the DPC protease acting in metazoans. Loss of SPRTN results in failure to repair DPCs and hypersensitivity to DPC-inducing agents. SPRTN accomplishes DPC processing through a unique DNA-induced protease activity, which is controlled by several sophisticated regulatory mechanisms. Cellular, biochemical, and structural studies define a DNA switch triggering its protease activity, a ubiquitin switch controlling SPRTN chromatin accessibility, and regulatory autocatalytic cleavage. Our data also provide a molecular explanation on how SPRTN deficiency causes the premature aging and cancer predisposition disorder Ruijs-Aalfs syndrome.
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•The SPRTN metalloprotease repairs DNA-protein crosslinks•A DNA switch controls SPRTN’s protease activity•A ubiquitin switch controls chromatin access of SPRTN•Structural insights reveal unique features of the SPRTN/Wss1 protease family
Stingele et al. discover the SPRTN metalloprotease to be crucial for DNA-protein crosslink repair in higher eukaryotes. In addition, several regulatory principles constraining SPRTN’s potentially toxic activity are described: a ubiquitin switch controlling chromatin access, a DNA switch triggering protease activity, and a negative feedback loop based on autocatalytic cleavage.
Inappropriate homologous recombination (HR) causes genomic instability and cancer. In yeast, the UvrD family helicase Srs2 is recruited to sites of DNA replication by SUMO-modified PCNA, where it ...acts to restrict HR by disassembling toxic RAD51 nucleofilaments. How human cells control recombination at replication forks is unknown. Here, we report that the protein PARI, containing a UvrD-like helicase domain, is a PCNA-interacting partner required for preservation of genome stability in human and DT40 chicken cells. Using cell-based and biochemical assays, we show that PARI restricts unscheduled recombination by interfering with the formation of RAD51-DNA HR structures. Finally, we show that PARI knockdown suppresses the genomic instability of Fanconi Anemia/BRCA pathway-deficient cells. Thus, we propose that PARI is a long sought-after factor that suppresses inappropriate recombination events at mammalian replication forks.
► PARI is a UvrD domain protein that interacts with PCNA and SUMO ► PARI is required for genomic stability in human and chicken cells ► PARI restricts inappropriate recombination by controlling RAD51 on DNA ► PARI downregulation improves recombination in Fanconi Anemia pathway-deficient cells
Following irradiation, numerous DNA-damage-responsive proteins rapidly redistribute into microscopically visible subnuclear aggregates, termed ionising-radiation-induced foci (IRIF). How the ...enrichment of proteins on damaged chromatin actually relates to DNA repair remains unclear. Here, we use super-resolution microscopy to examine the spatial distribution of BRCA1 and 53BP1 proteins within single IRIF at subdiffraction-limit resolution, yielding an unprecedented increase in detail that was not previously apparent by conventional microscopy. Consistent with a role for 53BP1 in promoting DNA double-strand break repair by non-homologous end joining, 53BP1 enrichment in IRIF is most prominent in the G0/G1 cell cycle phases, where it is enriched in dense globular structures. By contrast, as cells transition through S phase, the recruitment of BRCA1 into the core of IRIF is associated with an exclusion of 53BP1 to the focal periphery, leading to an overall reduction of 53BP1 occupancy at DNA damage sites. Our data suggest that the BRCA1-associated IRIF core corresponds to chromatin regions associated with repair by homologous recombination, and the enrichment of BRCA1 in IRIF represents a temporal switch in the DNA repair program. We propose that BRCA1 antagonises 53BP1-dependent DNA repair in S phase by inhibiting its interaction with chromatin proximal to damage sites. Furthermore, the genomic instability exhibited by BRCA1-deficient cells might result from a failure to efficiently exclude 53BP1 from such regions during S phase.
Telomere maintenance critically depends on the distinct activities of telomerase, which adds telomeric repeats to solve the end replication problem, and RTEL1, which dismantles DNA secondary ...structures at telomeres to facilitate replisome progression. Here, we establish that reversed replication forks are a pathological substrate for telomerase and the source of telomere catastrophe in Rtel1−/− cells. Inhibiting telomerase recruitment to telomeres, but not its activity, or blocking replication fork reversal through PARP1 inhibition or depleting UBC13 or ZRANB3 prevents the rapid accumulation of dysfunctional telomeres in RTEL1-deficient cells. In this context, we establish that telomerase binding to reversed replication forks inhibits telomere replication, which can be mimicked by preventing replication fork restart through depletion of RECQ1 or PARG. Our results lead us to propose that telomerase inappropriately binds to and inhibits restart of reversed replication forks within telomeres, which compromises replication and leads to critically short telomeres.
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•Blocking telomerase recruitment rescues telomere catastrophe in Rtel1−/− cells•Reversed replication forks within telomeres are aberrantly bound by telomerase•Blocking fork reversal rescues telomere catastrophe induced by telomerase•Telomerase prevents restart of reversed replication forks at telomeres
Telomerase can, paradoxically, contribute to telomere shortening by stabilizing stalled replication forks at chromosome ends.
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