19,818 research outputs found

    The probability of double-strand breaks in giant DNA decreases markedly as the DNA concentration increases

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    DNA double-strand breaks (DSBs) represent a serious source of damage for all living things and thus there have been many quantitative studies of DSBs both in vivo and in vitro. Despite this fact, the processes that lead to their production have not yet been clearly understood, and there is no established theory that can account for the statistics of their production, in particular, the number of DSBs per base pair per unit Gy, here denoted by P1, which is the most important parameter for evaluating the degree of risk posed by DSBs. Here, using the single-molecule observation method with giant DNA molecules (166 kbp), we evaluate the number of DSBs caused by gamma-ray irradiation. We find that P1 is nearly inversely proportional to the DNA concentration above a certain threshold DNA concentration. A simple model that accounts for the marked decrease of P1 shows that it is necessary to consider the characteristics of giant DNA molecules as semiflexible polymers to interpret the intrinsic mechanism of DSBs

    ATM and Artemis promote homologous recombination of radiation-induced DNA double-strand breaks in G2

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    Homologous recombination (HR) and non‐homologous end joining (NHEJ) represent distinct pathways for repairing DNA double‐strand breaks (DSBs). Previous work implicated Artemis and ATM in an NHEJ‐dependent process, which repairs a defined subset of radiation‐induced DSBs in G1‐phase. Here, we show that in G2, as in G1, NHEJ represents the major DSB‐repair pathway whereas HR is only essential for repair of ∼15% of X‐ or γ‐ray‐induced DSBs. In addition to requiring the known HR proteins, Brca2, Rad51 and Rad54, repair of radiation‐induced DSBs by HR in G2 also involves Artemis and ATM suggesting that they promote NHEJ during G1 but HR during G2. The dependency for ATM for repair is relieved by depleting KAP‐1, providing evidence that HR in G2 repairs heterochromatin‐associated DSBs. Although not core HR proteins, ATM and Artemis are required for efficient formation of single‐stranded DNA and Rad51 foci at radiation‐induced DSBs in G2 with Artemis function requiring its endonuclease activity. We suggest that Artemis endonuclease removes lesions or secondary structures, which inhibit end resection and preclude the completion of HR or NHEJ

    A Meiotic Checkpoint Alters Repair Partner Bias to Permit Inter-sister Repair of Persistent DSBs

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    Accurate meiotic chromosome segregation critically depends on the formation of inter-homolog crossovers initiated by double-strand breaks (DSBs). Inaccuracies in this process can drive aneuploidy and developmental defects, but how meiotic cells are protected from unscheduled DNA breaks remains unexplored. Here we define a checkpoint response to persistent meiotic DSBs in C. elegans that phosphorylates the synaptonemal complex (SC) to switch repair partner from the homolog to the sister chromatid. A key target of this response is the core SC component SYP-1, which is phosphorylated in response to ionizing radiation (IR) or unrepaired meiotic DSBs. Failure to phosphorylate (syp-16A) or dephosphorylate (syp-16D) SYP-1 in response to DNA damage results in chromosome non-dysjunction, hyper-sensitivity to IR-induced DSBs, and synthetic lethality with loss of brc-1BRCA1. Since BRC-1 is required for inter-sister repair, these observations reveal that checkpoint-dependent SYP-1 phosphorylation safeguards the germline against persistent meiotic DSBs by channelling repair to the sister chromatid.Cancer Research UK FC0010048UK Medical Research Council FC0010048Wellcome Trust FC0010048Ministerio de Economía y Competitividad BFU2016-75058-PEuropean Research Council ERC2014 AdG669898 TARLOO

    Risks from low dose/dose rate radiation: what an understanding of DNA damage response mechanisms can tell us

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    The DNA damage response (DDR) mechanisms represent a vital line of defense against exogenous and endogenous DNA damage to enhance two distinct outcomes, survival and the maintenance of genomic stability. The latter is critical for cancer avoidance. DDR processes encompass repair pathways and signal transduction mechanisms that activate cell cycle checkpoint arrest and apoptosis. DNA double strand breaks (DSBs) represent important radiation-induced lesions. The major DSB repair pathways are DNA non-homologous end-joining (NHEJ) and homologous recombination (HR) and ataxia telangiectasia mutated (ATM) activates the DSB signaling response. To evaluate the ability of these pathways to protect against low doses or dose rate radiation exposure, it is important to consider the fidelity of DSB repair and the sensitivity of checkpoint arrest and apoptosis. Radiation-induced DSBs are more complex than endogenously-induced DSBs, with the potential for multiple lesions to arise in close proximity. NHEJ, the major DSB repair pathway, cannot accurately reconstitute sequence information lost at DSBs. Both pathways have the potential to cause translocations by rejoining erroneous DNA ends. Thus, complete accuracy of repair cannot be guaranteed and the formation of translocations, which have the potential to initiate carcinogenesis, can arise. Additionally, the G2/M checkpoint has a defined sensitivity, allowing some chromosome breakage to occur. Thus, genomic rearrangements can potentially arise even if the G1/S checkpoint is efficient. The sensitivity of apoptosis is currently unclear but will likely differ between tissues. In summary, it is unlikely that the DDR mechanisms can fully protect cells from genomic rearrangements following exposure to low doses or dose rate radiation

    Regional gene repression by DNA double-strand breaks in G1 phase cells

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    DNA damage responses (DDR) to double-strand breaks (DSBs) alter cellular transcription programs at the genome-wide level. Through processes that are less well understood, DSBs also alter transcriptional responses locally, which may be important for efficient DSB repair. Here, we developed an approach to elucidate th

    Studying DNA Double-Strand Break Repair: An Ever-Growing Toolbox

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    To ward off against the catastrophic consequences of persistent DNA double-strand breaks (DSBs), eukaryotic cells have developed a set of complex signaling networks that detect these DNA lesions, orchestrate cell cycle checkpoints and ultimately lead to their repair. Collectively, these signaling networks comprise the DNA damage response (DDR). The current knowledge of the molecular determinants and mechanistic details of the DDR owes greatly to the continuous development of ground-breaking experimental tools that couple the controlled induction of DSBs at distinct genomic positions with assays and reporters to investigate DNA repair pathways, their impact on other DNA-templated processes and the specific contribution of the chromatin environment. In this review, we present these tools, discuss their pros and cons and illustrate their contribution to our current understanding of the DDR.European Research Council (ERC-2014-CoG 647344

    Endogenous topoisomerase II-mediated DNA breaks drive thymic cancer predisposition linked to ATM deficiency

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    The ATM kinase is a master regulator of the DNA damage response to double-strand breaks (DSBs) and a well-established tumour suppressor whose loss is the cause of the neurodegenerative and cancer-prone syndrome Ataxia-Telangiectasia (A-T). A-T patients and Atm−/− mouse models are particularly predisposed to develop lymphoid cancers derived from deficient repair of RAG-induced DSBs during V(D)J recombination. Here, we unexpectedly find that specifically disturbing the repair of DSBs produced by DNA topoisomerase II (TOP2) by genetically removing the highly specialised repair enzyme TDP2 increases the incidence of thymic tumours in Atm−/− mice. Furthermore, we find that TOP2 strongly colocalizes with RAG, both genome-wide and at V(D)J recombination sites, resulting in an increased endogenous chromosomal fragility of these regions. Thus, our findings demonstrate a strong causal relationship between endogenous TOP2-induced DSBs and cancer development, confirming these lesions as major drivers of ATM-deficient lymphoid malignancies, and potentially other conditions and cancer types.Junta de Andalucía SAF2010-21017, SAF2013-47343-P, SAF2014-55532-R, SAF2017-89619-R, CVI-7948European Research Council ERC-CoG-2014-64735

    A surge of late-occurring meiotic double-strand breaks rescues synapsis abnormalities in spermatocytes of mice with hypomorphic expression of SPO11

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    Meiosis is the biological process that, after a cycle of DNA replication, halves the cellular chromosome complement, leading to the formation of haploid gametes. Haploidization is achieved via two successive rounds of chromosome segregation, meiosis I and II. In mammals, during prophase of meiosis I, homologous chromosomes align and synapse through a recombination-mediated mechanism initiated by the introduction of DNA double-strand breaks (DSBs) by the SPO11 protein. In male mice, if SPO11 expression and DSB number are reduced below heterozygosity levels, chromosome synapsis is delayed, chromosome tangles form at pachynema, and defective cells are eliminated by apoptosis at epithelial stage IV at a spermatogenesis-specific endpoint. Whether DSB levels produced in Spo11 +/− spermatocytes represent, or approximate, the threshold level required to guarantee successful homologous chromosome pairing is unknown. Using a mouse model that expresses Spo11 from a bacterial artificial chromosome, within a Spo11 −/− background, we demonstrate that when SPO11 expression is reduced and DSBs at zygonema are decreased (approximately 40 % below wild-type level), meiotic chromosome pairing is normal. Conversely, DMC1 foci number is increased at pachynema, suggesting that under these experimental conditions, DSBs are likely made with delayed kinetics at zygonema. In addition, we provide evidences that when zygotene-like cells receive enough DSBs before chromosome tangles develop, chromosome synapsis can be completed in most cells, preventing their apoptotic elimination

    Requirement for PBAF in transcriptional repression and repair at DNA breaks in actively transcribed regions of chromatin

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    Actively transcribed regions of the genome are vulnerable to genomic instability. Recently, it was discovered that transcription is repressed in response to neighboring DNA double-strand breaks (DSBs). It is not known whether a failure to silence transcription flanking DSBs has any impact on DNA repair efficiency or whether chromatin remodelers contribute to the process. Here, we show that the PBAF remodeling complex is important for DSB-induced transcriptional silencing and promotes repair of a subset of DNA DSBs at early time points, which can be rescued by inhibiting transcription globally. An ATM phosphorylation site on BAF180, a PBAF subunit, is required for both processes. Furthermore, we find that subunits of the PRC1 and PRC2 polycomb group complexes are similarly required for DSB-induced silencing and promoting repair. Cancer-associated BAF180 mutants are unable to restore these functions, suggesting PBAF's role in repressing transcription near DSBs may contribute to its tumor suppressor activity
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