Meiotic recombination is initiated by SPO11-induced double-strand breaks (DSBs). In most mammals, the methyltransferase PRDM9 guides SPO11 targeting, and the ATM kinase controls meiotic DSB numbers. ...Following MRE11 nuclease removal of SPO11, the DSB is resected and loaded with DMC1 filaments for homolog invasion. Here, we demonstrate the direct detection of meiotic DSBs and resection using END-seq on mouse spermatocytes with low sample input. We find that DMC1 limits both minimum and maximum resection lengths, whereas 53BP1, BRCA1 and EXO1 play surprisingly minimal roles. Through enzymatic modifications to END-seq, we identify a SPO11-bound meiotic recombination intermediate (SPO11-RI) present at all hotspots. We propose that SPO11-RI forms because chromatin-bound PRDM9 asymmetrically blocks MRE11 from releasing SPO11. In Atm
spermatocytes, trapped SPO11 cleavage complexes accumulate due to defective MRE11 initiation of resection. Thus, in addition to governing SPO11 breakage, ATM and PRDM9 are critical local regulators of mammalian SPO11 processing.
DNA double-strand breaks (DSBs) arise during physiological transcription, DNA replication, and antigen receptor diversification. Mistargeting or misprocessing of DSBs can result in pathological ...structural variation and mutation. Here we describe a sensitive method (END-seq) to monitor DNA end resection and DSBs genome-wide at base-pair resolution in vivo. We utilized END-seq to determine the frequency and spectrum of restriction-enzyme-, zinc-finger-nuclease-, and RAG-induced DSBs. Beyond sequence preference, chromatin features dictate the repertoire of these genome-modifying enzymes. END-seq can detect at least one DSB per cell among 10,000 cells not harboring DSBs, and we estimate that up to one out of 60 cells contains off-target RAG cleavage. In addition to site-specific cleavage, we detect DSBs distributed over extended regions during immunoglobulin class-switch recombination. Thus, END-seq provides a snapshot of DNA ends genome-wide, which can be utilized for understanding genome-editing specificities and the influence of chromatin on DSB pathway choice.
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•END-seq provides a high-resolution view of DNA breaks and end resection•END-seq detects at least one DSB among 10,000 cells not harboring DSBs•END-seq maps DSBs during antigen receptor rearrangements and genome editing•END-seq can be used to study DSB formation/repair in various tissues and organisms
Canela et al. develop a sensitive and quantitative method that provides a landscape of DNA double-strand breaks and end resection in vivo prior to DNA repair. This opens up the possibility for better understanding the causes and consequences of genome instability.
Immunoglobulin class switch recombination (CSR) is initiated by activation‐induced cytidine deaminase (AID), an enzyme that deaminates cytidine residues in single‐stranded DNA. U:G mismatches created ...by AID are processed to produce lesions that recruit and activate DNA damage response proteins including Ataxia‐telangiectasia mutated (ATM), histone H2AX, Nijmegen breakage syndrome 1 (Nbs1), and p53 binding protein 1 (53BP1). Among these proteins, absence of 53BP1 produces the most severe impairment of class switching. Here, we demonstrate that AID is targeted normally to switch region DNA and that intra‐switch region recombination is enhanced in 53BP1–/– B cells. In addition, Sµ‐Sγ1 switch region junctions cloned from 53BP1–/– B cells show unusual insertions suggestive of failed class switching. Our data are consistent with a role for 53BP1 in stabilizing the synapsis of switch regions during CSR.
The neuronal genome is particularly sensitive to loss or attenuation of DNA repair, and many neurological diseases ensue when DNA repair is impaired. It is well-established that the neuronal genome ...is subjected to stochastic DNA damage, most likely because of extensive oxidative stress in the brain. However, recent studies have identified unexpected high levels of 'programmed' DNA breakage in neurons, which we propose arise during physiological DNA metabolic processes intrinsic to neuronal development, differentiation and maintenance. The role of programmed DNA breaks in normal neuronal physiology and disease remains relatively unexplored thus far. However, bulk and single-cell sequencing analyses of neurodegenerative diseases have revealed age-related somatic mutational signatures that are enriched in regulatory regions of the genome. Here, we explore a paradigm of DNA repair in neurons, in which the genome is safeguarded from erroneous impacts of programmed genome breakage intrinsic to normal neuronal function.
DNA double-strand breaks (DSBs) represent a threat to the genome because they can lead to the loss of genetic information and chromosome rearrangements. The DNA repair protein p53 binding protein 1 ...(53BP1) protects the genome by limiting nucleolytic processing of DSBs by a mechanism that requires its phosphorylation, but whether 53BP1 does so directly is not known. Here, we identify Rap1-interacting factor 1 (Rif1) as an ATM (ataxia-telangiectasia mutated) phosphorylation-dependent interactor of 53BP1 and show that absence of Rif1 results in 5′-3′ DNA-end resection in mice. Consistent with enhanced DNA resection, Rif1 deficiency impairs DNA repair in the G 1 and S phases of the cell cycle, interferes with class switch recombination in B lymphocytes, and leads to accumulation of chromosome DSBs.
The DNA damage response (DDR) protein 53BP1 protects DNA ends from excessive resection in G1, and thereby favors repair by nonhomologous end-joining (NHEJ) as opposed to homologous recombination ...(HR). During S phase, BRCA1 antagonizes 53BP1 to promote HR. The pro-NHEJ and antirecombinase functions of 53BP1 are mediated in part by RIF1, the only known factor that requires 53BP1 phosphorylation for its recruitment to double-strand breaks (DSBs). Here, we show that a 53BP1 phosphomutant, 53BP18A, comprising alanine substitutions of the eight most N-terminal S/TQ phosphorylation sites, mimics 53BP1 deficiency by restoring genome stability in BRCA1-deficient cells yet behaves like wild-type 53BP1 with respect to immunoglobulin class switch recombination (CSR). 53BP18A recruits RIF1 but fails to recruit the DDR protein PTIP to DSBs, and disruption of PTIP phenocopies 53BP18A. We conclude that 53BP1 promotes productive CSR and suppresses mutagenic DNA repair through distinct phosphodependent interactions with RIF1 and PTIP.
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•The mechanisms by which 53BP1 promotes NHEJ and inhibits HR are distinct•PTIP and RIF1 separate 53BP1 functions in productive and mutagenic DNA repair•PTIP promotes genome instability in BRCA1-deficient cells by inhibiting DSB resection•PTIP is required for NHEJ of dysfunctional telomeres
The DNA repair protein 53BP1 relies on two different cofactors to mediate its productive function in B cell class switching and its mutagenic function in BRCA1-deficient cells, raising the possibility of targeting one without disrupting the other.
Defective DNA repair by homologous recombination (HR) is thought to be a major contributor to tumorigenesis in individuals carrying
Brca1 mutations. Here, we show that DNA breaks in Brca1-deficient ...cells are aberrantly joined into complex chromosome rearrangements by a process dependent on the nonhomologous end-joining (NHEJ) factors 53BP1 and DNA ligase 4. Loss of
53BP1 alleviates hypersensitivity of
Brca1 mutant cells to PARP inhibition and restores error-free repair by HR. Mechanistically,
53BP1 deletion promotes ATM-dependent processing of broken DNA ends to produce recombinogenic single-stranded DNA competent for HR. In contrast,
Lig4 deficiency does not rescue the HR defect in
Brca1 mutant cells but prevents the joining of chromatid breaks into chromosome rearrangements. Our results illustrate that HR and NHEJ compete to process DNA breaks that arise during DNA replication and that shifting the balance between these pathways can be exploited to selectively protect or kill cells harboring
Brca1 mutations.
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► 53BP1 and Brca1 regulate the choice between HR and NHEJ pathways for DNA repair ► 53BP1 deletion promotes HR by increasing ATM-dependent resection of DNA breaks ► 53BP1 mutation protects Brca1-deficient cells from death induced by PARP inhibition ► Lig4 joins unrepaired DNA breaks into radial chromosomes in Brca1 mutant cells
Mechanistic understanding of how ionizing radiation induces type I interferon signaling and how to amplify this signaling module should help to maximize the efficacy of radiotherapy. In the current ...study, we report that inhibitors of the DNA damage response kinase ATR can significantly potentiate ionizing radiation‐induced innate immune responses. Using a series of mammalian knockout cell lines, we demonstrate that, surprisingly, both the cGAS/STING‐dependent DNA‐sensing pathway and the MAVS‐dependent RNA‐sensing pathway are responsible for type I interferon signaling induced by ionizing radiation in the presence or absence of ATR inhibitors. The relative contributions of these two pathways in type I interferon signaling depend on cell type and/or genetic background. We propose that DNA damage‐elicited double‐strand DNA breaks releases DNA fragments, which may either activate the cGAS/STING‐dependent pathway or—especially in the case of AT‐rich DNA sequences—be transcribed and initiate MAVS‐dependent RNA sensing and signaling. Together, our results suggest the involvement of two distinct pathways in type I interferon signaling upon DNA damage. Moreover, radiation plus ATR inhibition may be a promising new combination therapy against cancer.
Synopsis
Type I interferon signaling plays key roles in cancer radiotherapy with ionizing radiation (IR). Here, exploration of combinatory effects of IR and DNA damage kinase inhibitors reveals surprising involvement of distinct cytosolic nucleic acid‐sensing pathways in interferon response induction.
Inhibition of DNA damage response kinase ATR (ATRi) significantly potentiates IR‐induced type I interferon response in multiple human and murine cancer cells.
MAVS‐dependent RNA sensing pathway is indispensable for interferon signaling induced by combined IR+ATRi in some human cells.
Both cGAS/STING‐dependent cytosolic DNA‐ and MAVS‐dependent cytosolic RNA‐sensing pathways contribute to IR+ATRi‐induced interferon signaling to varying extent in different cell lines.
DNA damage‐elicited AT‐rich DNA mediates type I interferon signaling in a subset of human cells.
Surprisingly, both cGAS/STING‐dependent DNA‐ and MAVS‐dependent RNA‐sensing pathways contribute to the effects of combined radiotherapy and blocked DNA damage signaling, depending on cellular context.
The integrity of the genome is frequently challenged by double-strand breaks in the DNA. Defects in the cellular response to double-strand breaks are a major cause of cancer and other age-related ...pathologies; therefore, much effort has been directed at understanding the enzymatic mechanisms involved in recognizing, signalling and repairing double-strand breaks. Recent work indicates that chromatin - the fibres into which DNA is packaged with a proteinaceous structural polymer - has an important role in initiating, propagating and terminating this cellular response to DNA damage.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Translocations arise when an end of one chromosome break is mistakenly joined to an end from a different chromosome break. Since translocations can lead to developmental disease and cancer, it is ...important to understand the mechanisms leading to these chromosome rearrangements. We review how characteristics of the sources and the cellular responses to chromosome breaks contribute to the accumulation of multiple chromosome breaks at the same moment in time. We also discuss the important role for chromosome break location; how translocation potential is impacted by the location of chromosome breaks both within chromatin and within the nucleus, as well as the effect of altered mobility of chromosome breaks. A common theme in work addressing both temporal and spatial contributions to translocation is that there is no shortage of examples of factors that promote translocation in one context, but have no impact or the opposite impact in another. Accordingly, a clear message for future work on translocation mechanism is that unlike normal DNA metabolic pathways, it isn't easily modeled as a simple, linear pathway that is uniformly followed regardless of differing cellular contexts.
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