Correct End Use during End Joining of Multiple Chromosomal Double Strand Breaks Is Influenced by Repair Protein RAD50, DNA-dependent Protein Kinase DNA-PKcs, and Transcription Context

Gunn, Amanda; Bennardo, Nicole; Cheng, Anita; Stark, Jeremy M.

doi:10.1074/jbc.m111.309252

Cited by 95 publications

(111 citation statements)

References 70 publications

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“…4C. A similar result was also using a second inducible DSB system in a U2OS cell line carrying the EJ5-GFP reporter (23), where CTCF is also recruited to most sites flanking the I-Sce I induced break point (Fig. 4D).…”

Section: Ctcf Prevents Genomic Instabilitysupporting

confidence: 62%

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CTCF prevents genomic instability by promoting homologous recombination-directed DNA double-strand break repair

Lang

Zheng

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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CTCF is an essential epigenetic regulator mediating chromatin insulation, long-range regulatory interactions, and the organization of large topological domains in the nucleus. Phenotypes of CTCF haploinsufficient mutations in humans, knockout in mice, and depletion in cells are often consistent with impaired genome stability, but a role of CTCF in genome maintenance has not been fully investigated. Here, we report that CTCF maintains genome stability, is recruited to sites of DNA damage, and promotes homologous recombination repair of DNA double-strand breaks (DSBs). CTCF depletion increased chromosomal instability, marked by chromosome breakage and end fusions, elevated genotoxic stress-induced genomic DNA fragmentation, and activated the ataxia telangiectasia mutated (ATM) kinase. We show that CTCF could be recruited to drug-induced 53BP1 foci and known fragile sites, as well as to I-SceI endonuclease-induced DSBs. Laser irradiation analysis revealed that this recruitment depends on ATM, Nijmegen breakage syndrome (NBS), and the zinc finger DNA-binding domain of CTCF. We demonstrate that CTCF knockdown impaired homologous recombination (HR) repair of DSBs. Consistent with this, CTCF knockdown reduced the formation of γ-radiation-induced Rad51 foci, as well as the recruitment of Rad51 to laser-irradiated sites of DNA lesions and to I-SceI-induced DSBs. We further show that CTCF is associated with DNA HR repair factors MDC1 and AGO2, and directly interacts with Rad51 via its C terminus. These analyses establish a direct, functional role of CTCF in DNA repair and provide a potential link between genome organization and genome stability.CTCF | chromatin | genome stability | DNA repair | homologous recombination

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Section: Ctcf Prevents Genomic Instabilitysupporting

confidence: 62%

“…To obtain more direct evidence supporting the interaction of CTCF at sites of DNA damage, we investigated whether CTCF interacts with the DNA near DSB ends by ChIP in a U2OS cell line carrying the DR-GFP reporter (23,24) (Fig. S3C).…”

Section: Ctcf Prevents Genomic Instabilitymentioning

confidence: 99%

CTCF prevents genomic instability by promoting homologous recombination-directed DNA double-strand break repair

Lang

Zheng

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…We used human U2OS cells (provided by J. Bartek, Institute of Cancer Biology and Centre for Genotoxic Stress Research), U2OS DR-GFP (provided by M. Jasin, Sloan-Kettering Institute) and U2OS SA-GFP cells (described in ref. 34). We cultured all cells lines in DMEM (Invitrogen) supplemented with 10% (vol/vol) FCS (Eurobio), 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen) in a humidified atmosphere with 5% CO 2 at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…We followed exactly the same protocol to estimate single-strand annealing using U2OS SA-GFP cells that were established previously. 34 Antibodies. Primary antibodies used during immunoblot (IB) or immunofluorescence (IF) experiments are listed below.…”

Section: Supplemental Materialsmentioning

confidence: 99%

Differential contribution of HP1 proteins to DNA end resection and homology-directed repair

Soria

Almouzni

2013

Cell Cycle

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“…To assess the functional relevance of the observed preferential recruitment of DROSHA to NHEJ-prone AsiSI sites, we exploited the U2OS EJ5 cellular system, a well-established GFP-based reporter cell system in which the reconstitution of a functional GFP gene occurs after DNA damage generation and repair by NHEJ repair (Gunn et al, 2011), (Gunn and Stark, 2012) (see cartoon in Figure 5A). Interestingly, we observed that the number not peer-reviewed) is the author/funder.…”

Section: Drosha Controls Dna Repair By Nhejmentioning

confidence: 99%

DROSHA associates to DNA damage sites and is required for DNA repair

Cabrini

Roncador

Galbiati

et al. 2018

Preprint

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The DNA damage response (DDR) is the signaling cascade through which a cell recognizes DNA lesions, and promotes their resolution via the repair pathways of Non-Homologous End Joining (NHEJ), or Homologous Recombination (HR). We recently demonstrated that DROSHA boosts DDR signaling by processing damage-induced long non-coding RNAs into smaller DNA damage response RNAs (DDRNAs). However, the location at which DROSHA exerts its DDR functions, relative to sites of DNA damage, remains unknown.To investigate DROSHA's localization during DDR activation, we used the DiVA cellular system, which allows the controlled induction of several DNA double strand breaks (DSBs) in the human genome. Indeed, by genome wide chromatin immunoprecipitation followed by next generation sequencing, we demonstrate that DROSHA associates with DSBs. In support of this, DSB-recruitment of DROSHA is detectable at the single-cell level by Proximity Ligation Assay between DROSHA and known DDR markers, and by DNA damage in situ ligation followed by Proximity Ligation Assay (DI-PLA), which demonstrates proximity of DROSHA to DNA ends. DROSHA recruitment occurs at both genic and inter-genic DSBs, suggesting that its recruitment is independent from ongoing transcription preceding damage generation. DROSHA's recruitment to DNA lesions occurs throughout the cell cycle, and with a preference for NHEJ-prone DSBs. Consistently, inhibition of the HR pathway increases DROSHA recruitment, and DROSHA knock down strongly impairs NHEJ efficiency in a GFP-reporter cellular system for monitoring NHEJ DNA repair. Overall, these results demonstrate that DROSHA acts locally at sites of DNA damage to promote NHEJ DNA repair.

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Correct End Use during End Joining of Multiple Chromosomal Double Strand Breaks Is Influenced by Repair Protein RAD50, DNA-dependent Protein Kinase DNA-PKcs, and Transcription Context

Cited by 95 publications

References 70 publications

CTCF prevents genomic instability by promoting homologous recombination-directed DNA double-strand break repair

CTCF prevents genomic instability by promoting homologous recombination-directed DNA double-strand break repair

Differential contribution of HP1 proteins to DNA end resection and homology-directed repair

DROSHA associates to DNA damage sites and is required for DNA repair

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