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Non redundant functions of ATM and DNAPKcs in response to clean DNA Double Strand Breaks.

ABSTRACT: DNA Double Strand Breaks (DSBs) are harmful lesions that require rapid detection and repair in order to avoid toxic genomic rearrangements. DSBs elicit the so called DNA Damage Response (DDR), largely relying on ataxia telangiectasia mutated (ATM) and DNA Protein Kinase (DNAPK), two members of the PI3K-like kinase family, whose respective functions during the sequential steps of the DDR remains controversial. Using the DIvA cell line, expressing the AsiSI restriction enzyme, we have investigated the role of ATM and DNAPK in several aspects of the DDR upon induction of multiple clean DSBs throughout the human genome. High resolution mapping revealed that they are activated and spread in cis on a confined region surrounding all DSBs, independently of the pathway used for repair. However, a thorough analysis of repair kinetics, H2AX domain establishment and H2AX foci structure and dynamics revealed non overlapping functions for the two kinases once recruited at DSBs. Our results suggest that ATM is not solely acting on chromatin marks but also on chromatin organisation in order to ensure repair accuracy and survival.

ORGANISM(S): Homo sapiens

SUBMITTER: Gaelle LEGUBE

PROVIDER: E-MTAB-2992 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Cohesin profiling at DSBs

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2012-01-01 | E-TABM-1164 | biostudies-arrayexpress

ChIP-chip profiling of human gammaH2AX spreading around DNA double strand breaks

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Chromatin compartmentalization regulates the response to DNA damage

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2021-01-29 | E-MTAB-8793 | biostudies-arrayexpress

Nuclear poly(A)-binding protein 1 is an ATM target and essential for DNA double-strand break rep

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2017-11-30 | PXD005913 | Pride

A POLD3/BLM dependent pathway handles DSBs in transcribed chromatin upon excessive RNA:DNA hybrids accumulation

Project description:Transcriptionally active loci are particularly prone to breakage and mounting evidence suggest that DNA Double-Strand Breaks arising in active genes are handled by a dedicated repair pathway, Transcription-Coupled DSB Repair (TC-DSBR), that entails R-loop accumulation and dissolution. Here, we uncovered a function for the Bloom RecQ DNA helicase (BLM) in TC-DSBR in human cells. BLM is recruited in a transcription dependent-manner at DSBs where it fosters resection, RAD51 binding and accurate Homologous Recombination repair. However, in an R-loop dissolution-deficient background, we found that BLM promotes cell death. We report that upon excessive R-loop accumulation, DNA synthesis is enhanced at DSBs, in a anner that depends on BLM and POLD3. Altogether our work unveils a role for BLM at DSBs in active chromatin, and highlights the toxic potential of RNA:DNA hybrids that accumulate at transcription-associated DSBs.

2022-04-13 | E-MTAB-11592 | biostudies-arrayexpress

Role of Polycomb group proteins in the DNA damage response – a reassessment

Project description:We have investigated whether components of Polycomb repressive complex 1 (PRC1) are recruited to double-strand breaks (DSBs) generated by inducible expression of the AsiSI restriction enzyme in normal human fibroblasts. Using chromatin immunoprecipitation and deep sequencing (ChIP-seq), which detects PRC1 proteins at common sites throughout the genome, we did not find evidence for recruitment of PRC1 components to AsiSI-induced DSBs. In contrast, the S2056 phosphorylated form of DNA-PKcs and other DNA repair proteins were detected at a subset of AsiSI sites that are predominantly at the 5’ ends of transcriptionally active genes. Our data question the idea that PcG protein recruitment provides a link between DSB repairs and transcriptional repression. Single chromatin preparations from treated and untreated cells (+/- OHT), immunoprecipitated with two antibodies (pDNA-PKcs and MEL18) were sequenced as technical replicates, along with the corresponding input DNAs

2014-07-06 | E-GEOD-55605 | biostudies-arrayexpress

Transcriptionally active chromatin channels DNA double-strand breaks to RAD51-dependent repair

Project description:Cells have developed effective mechanisms, namely homologous recombination (HR) and non-homologous end-joining (NHEJ), to repair DNA double-strand breaks (DSBs), which are considered to be the most deleterious type of damage that can challenge genome integrity. While these pathways coexist to repair DSBs, the mechanisms by which one of these pathways is chosen to repair a particular DSB remain unclear. Here, we show that the chromatin context in which a break occurs participates in this choice and that transcriptionnaly active chromatin channels repair to HR. By using a human cell line expressing a restriction enzyme fused to the ligand binding domain of the oestrogen receptor (AsiSI-ER)2,3, together with a genome wide chromatin immunoprecipitation-sequencing (ChIP-seq) approach, we establish that distinct DSBs induced across the genome are not necessarily repaired by the same pathway. Indeed, we identify an HR-prone subset of DSBs that recruit the HR protein RAD51, undergo resection, and rely on RAD51 for efficient repair. These DSBs are located in actively transcribed genes, and repair at such DSBs can be switched to RAD51-independent repair pathway upon transcriptional inhibition. Moreover, we show that HR is targeted to transcribed loci thanks to the elongation-associated H3K36me3 histone mark. Indeed depletion of HYPB, the main H3K36 tri methyltransferase severally impedes the use of HR at those DSBs. Our study, thereby demonstrates a clear role for chromatin in DSB repair pathway choice in human cells.

2014-03-23 | E-MTAB-1241 | biostudies-arrayexpress

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2022-09-06 | PXD035420 | Pride

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