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Genome-wide analysis of DNA-PK-bound MRN cleavage products supports a sequential model of DSB repair pathway choice

ABSTRACT: The Mre11-Rad50-Nbs1 (MRN) complex recognizes and processes DNA double-strand breaks for homologous recombination by performing short-range removal of 5ʹ strands. Endonucleolytic processing by MRN requires a stably bound protein at the break site—a role we postulate is played by DNA-dependent protein kinase (DNA-PK) in mammals. Here we interrogate the sites of MRN-dependent processing by isolating and sequencing DNA-PK-bound DNA fragments that are products of MRN cleavage. These intermediates are generated with highest efficiency when DNA-PK is catalytically blocked, yielding products within 200 bp of the break site, whereas DNA-PK products in the absence of kinase inhibition show much greater dispersal. Use of light-activated Cas9 to induce breaks facilitates temporal resolution of DNA-PK and Mre11 binding, showing that Mre11 and DNA-PK both bind to DNA ends before release of DNA-PK-bound products. These results support a sequential model of double-strand break repair involving collaborative interactions between homologous and non-homologous repair complexes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE218590 | GEO | 2023/09/18

REPOSITORIES: GEO

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Human U2OS DNApk GLASS-ChIP LC-MS/MS

Project description:The Mre11-Rad50-Nbs1 (MRN) complex recognizes and processes DNA double-strand breaks for homologous recombination by performing short-range removal of 5ʹ strands. Endonucleolytic processing by MRN requires a stably bound protein at the break site—a role we postulate is played by DNA-dependent protein kinase (DNA-PK) in mammals. Here we interrogate sites of MRN-dependent processing by identifying sites of CtIP association and by sequencing DNA- PK-bound DNA fragments that are products of MRN cleavage. These intermediates are generated most efficiently when DNA-PK is catalytically blocked, yielding products within 200 bp of the break site, whereas DNA-PK products in the absence of kinase inhibition show greater dispersal. Use of light-activated Cas9 to induce breaks facilitates temporal resolution of DNA- PK and Mre11 binding, showing that both complexes bind to DNA ends before release of DNA- PK-bound products. These results support a sequential model of double-strand break repair involving collaborative interactions between homologous and non-homologous repair complexes.

2023-10-24 | PXD045033 | Pride

DROSHA is recruited to DNA damage sites by the MRN complex to promote non-homologous end-joining

Project description:The DNA damage response (DDR) is the signaling cascade that recognizes DNA double-strand breaks (DSB) and promotes their resolution via the DNA repair pathways of Non-Homologous End Joining (NHEJ) or Homologous Recombination (HR). We and others have shown that DDR activation requires DROSHA. However, whether DROSHA exerts its functions by associating with damage sites, what controls its recruitment and how DROSHA influences DNA repair, remains poorly understood. Here we show that DROSHA associates to DSBs independently from transcription. Neither H2AX, nor ATM nor DNA-PK kinase activities are required for its recruitment to break site. Rather, DROSHA interacts with RAD50 and inhibition of MRN by Mirin treatment abolishes this interaction. MRN inactivation by RAD50 knockdown or mirin treatment prevents DROSHA recruitment to DSB and, as a consequence, also 53BP1 recruitment. During DNA repair, DROSHA inactivation reduces NHEJ and boosts HR frequency. Indeed, DROSHA knockdown also increase the association of downstream HR factors such as RAD51 to DNA ends. Overall, our results demonstrate that DROSHA is recruited at DSBs by the MRN complex and direct DNA repair toward NHEJ.

2021-03-15 | GSE166631 | GEO

DNA-PK Promotes DNA End Resection at DNA Double Strand Breaks in G0 cells [END-seq]

Project description:DNA double-strand break (DSB) repair by homologous recombination is confined to the S and G2 phases of the cell cycle partly due to 53BP1 antagonizing DNA end resection in G1 phase and non-cycling quiescent (G0) cells where DSBs must be repaired by non-homologous end joining (NHEJ). Unexpectedly, we uncovered extensive MRE11- and CtIP-dependent DNA end resection at DSBs in G0 mammalian cells. A whole genome CRISPR/Cas9 screen revealed the DNA-dependent kinase (DNA-PK) complex as a key factor in promoting DNA end resection in G0 cells. In agreement, depletion of FBXL12, which promotes ubiquitylation and removal of the KU70/KU80 subunits of DNA-PK from DSBs, promotes even more extensive resection in G0 cells. In contrast, a requirement for DNA-PK in promoting DNA end resection in cycling cells at the G1 or G2 phase cells was not observed. Our findings establish that DNA-PK uniquely promotes DNA end resection in G0, but not in G1 or G2 phase cells, and has important implications for DNA DSB repair in quiescent cells.

2022-05-16 | GSE186085 | GEO

DNA-PK Promotes DNA End Resection at DNA Double Strand Breaks in G0 cells [ChIP-Seq]

2022-05-16 | GSE186086 | GEO

Integrative Structural Model of DNA-PK Synapsis

Project description:In non-homologous end joining repair of DNA double strand breaks, DNA dependent protein kinase catalytic subunit (DNA-PKcs) and Ku70/80 binds the free DNA ends forming the holoenzyme DNA-PK. DNA-PK synapses across the break to tether the broken ends in the initial long range synaptic complex. We generated an integrative structural model of DNA-PK synapsis at a precision of 13.5Å with crosslinking mass spectrometry (XL-MS) restraints. While our hydrogen deuterium exchange (HX) analysis revealed an allosteric axis in DNA-PK connecting DNA binding (including the plug domain) to the kinase domain. Our model presents a symmetrical synapsis primarily through head to head interactions and protection of the DNA by previously uncharacterized a plug domain. The offset of the DNA in our model allows access to downstream processing enzymes, while the combination of DNA binding and kinase loading creates a tensed state that could have roles in the re-arrangement/dissociation of DNA-PKcs as the repair process progresses.

2021-01-20 | PXD017931 | Pride

Mre11 suppresses replication stress and gene amplification proficiency

Project description:Gene amplification, copy-number increases of particular genes and surrounding genomic segments, promotes cancer progression and acquired therapy resistance. Thus, understanding genetic traits that confer gene amplification proficiency is important. The primary step for gene amplification is spontaneous DNA rearrangements initiated by DNA breaks. Here we show that mammalian cells became gene amplification-proficient when we knocked down Mre11/Rad50/Nbs1 (MRN) complex, a multifunctional complex that guards the genome from DNA breaks. Cells with reduced Mre11 experienced severe replication stress, with marked increases of single-stranded breaks followed by double-stranded breaks during DNA replication. Such breaks underlay for the increase in spontaneous gene amplification. Other traits associated with replication stress, such as impaired intra-S phase checkpoint and global transcriptional changes in DNA metabolism genes also contributed to gene amplification proficiency. Our results define Mre11 deficiency as a cause of replication stress and gene amplification proficiency and provide a candidate marker for aggressive cancer phenotypes. We sequenced four samples: 2 control, GFP-expressing samples and 2 Mre11 knockdown cells

2015-03-05 | E-GEOD-59487 | biostudies-arrayexpress

Physical interaction with Spo11 mediates the localisation of Mre11 to chromatin in meiosis and promotes its nuclease activity

Project description:Meiotic recombination is of central importance for the proper segregation of homologous chromosomes and is initiated by DNA double-strand breaks (DSB) that depend on Spo11 and Mre11. Calibrated chromatin immunoprecipitation (ChIP) for Mre11 shows that Spo11 promotes Mre11 recruitment to chromatin, independent of DSB formation. A C-terminal deletion mutant deficient in Spo11 interaction severely reduces the association of Mre11 with meiotic chromatin. Consistent with the reduction of Mre11 foci in this mutant, it strongly impedes DSB formation, leading to spore death.

2024-01-25 | GSE253302 | GEO

RecG Directs DNA Synthesis During Double-Strand Break Repair [ChIP-seq]

Project description:In the bacterium Escherichia coli, RecG directs DNA synthesis during the repair of DNA double-strand breaks by homologous recombination. Examination of RecA binding during double-strand break repair in Escherichia coli in the presence and absence of RecG protein

2016-05-19 | E-GEOD-77181 | biostudies-arrayexpress

Mre11 suppresses replication stress and gene amplification proficiency

2015-03-05 | GSE59487 | GEO

Tyrosine kinase c-Abl couples RNA polymerase II transcription to an RNA-dependent DNA damage response

Project description:Various modes of DNA repair counteract genotoxic DNA double-strand breaks (DSBs) to maintain genome stability. Recent findings suggest that the human DNA damage response (DDR) utilises damage-induced small RNA for efficient repair of DSBs. However, production and processing of RNA is poorly understood. Here we show that localised induction of DSBs triggers phosphorylation of RNA polymerase II (RNAPII) on carboxy-terminal domain (CTD) residue tyrosine-1 in an Mre11-Rad50-Nbs1 (MRN) complex-dependent manner. CTD Tyr1-phosphorylated RNAPII synthetises, strand-specific, damage-responsive transcripts (DARTs). DART synthesis occurs via formation of transient RNA-DNA hybrid (R-loop) intermediates. Impaired R-loop formation attenuates DART synthesis, impairs recruitment of repair factors and delays the DDR. Collectively, we provide mechanistic insight in RNA-dependent DSB repair.

2019-02-04 | GSE96825 | GEO

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