SMARCAD1 Phosphorylation and Ubiquitination Are Required for Resection during DNA Double-Strand Break Repair

Chakraborty, Sharmistha; Pandita, Raj K.; Hambarde, Shashank; Mattoo, Abid R.; Charaka, Vijaya; Ahmed, Kazi Mokim; Iyer, Swaminathan P.; Hunt, Clayton R.; Pandita, Tej K.

doi:10.1016/j.isci.2018.03.016

Cited by 47 publications

(74 citation statements)

References 47 publications

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“…One possibility is that ATM directly stimulates the resection machinery and/or down-regulates inhibitors of resection. A non-exclusive possibility is that ATM recruits chromatin remodeling activities to DSBs (Chakraborty et al 2018).…”

Section: Discussionmentioning

confidence: 99%

Molecular structures and mechanisms of DNA break processing in mouse meiosis

Yamada

Hinch

Kamido

et al. 2019

Preprint

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Exonucleolytic resection, critical to repair double-strand breaks (DSBs) by recombination, is not well understood, particularly in mammalian meiosis. Here, we define structures of resected DSBs in mouse spermatocytes genome-wide at nucleotide resolution. Resection tracts averaged 1100 nucleotides, but with substantial fine-scale heterogeneity at individual hotspots. Surprisingly, EXO1 is not the major 5′→3′ exonuclease, but the DSB-responsive kinase ATM proved a key regulator of both initiation and extension of resection. In wild type, apparent intermolecular recombination intermediates clustered near to but offset from DSB positions, consistent with joint molecules with incompletely invaded 3′ ends. Finally, we provide evidence for PRDM9-dependent chromatin remodeling leading to increased accessibility at recombination sites. Our findings give insight into the mechanisms of DSB processing and repair in meiotic chromatin.

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Section: Discussionmentioning

confidence: 99%

Molecular structures and mechanisms of DNA break processing in mouse meiosis

Yamada

Hinch

Kamido

et al. 2019

Preprint

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“…To answer these questions, we employed our previously characterized 293T-based stable cell lines that harbor CRISPR-Cas9-knocked-in homing endonuclease I-SceI target sites into two euchromatic and two heterochromatic regions, in this case, on chromosome 1. As demonstrated in previously, we verified, for each of these four sites, that they a) retained relevant chromatin conformation-specific epigenetic marks, b) that they can each be efficiently cleaved by a transfected cDNA encoding the I-SceI mega-nuclease, and c) chromatin context does not affect repair efficiency (34,35). We used this system first, to evaluate the relative levels of ZEB1 and 53BP1 before and after induction of DSBs.…”

Section: Zeb1 Levels Increase 4-fold At Dsbs In Euchromatin Independmentioning

confidence: 57%

“…Chromosomal aberrations Chromosomal aberrations at metaphase were analyzed as previously described (34,69) EJ-DR-based DSB Repair Pathway Choice Assay I-SceI-mediated DSBs were induced in the EJ-DR cell line as described (40,41). Briefly, at the appropriate time after administration of RNAi and rescue constructs, cells were rinsed 1X in 37˚ PBS, and replenished with complete media (with 10% Tet-minus FCS) containing 1µM Sheild1 (Aobious) and 0.1 µM triamcinolone acteonide (Cayman).…”

Section: Survival Assaysmentioning

confidence: 99%

“…(35) DNA End-Resection Assays Isolation of genomic DNA, o/n digestion with indicated restriction enzymes, and subsequent qPCR analysis was performed as described (X). The extent of resection at an AsiSI-induced DSB was quantitated in asynchronous or synchronized DIvA cells as previously described (34). Synchronization was achieved using double thymidine block (Cohen ref), with G1 and G2 cells collected 15hr and 7hr post-release from the second block, respectively.…”

Section: I-scei Dsbsmentioning

confidence: 99%

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ZEB1 is Required for NHEJ-Mediated DSB Repair in Euchromatin

Genetta

Abbas

Pandita

et al. 2020

Preprint

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Ionizing radiation-induced DSBs are repaired primarily by the Non-Homologous End Joining (NHEJ) pathway, but the details of how this is regulated in different chromatin contexts are far from understood. We have discovered a novel response to DSBs that promotes NHEJ selectively in euchromatin, based on a novel interaction between the EMT-inducing transcriptional repressor ZEB1, and the wellstudied NHEJ-promoting DNA repair factor 53BP1. Using a number of approaches, we have discovered that the ZEB1-53BP1 association is amplified following exposure of cells to IR and that they co-localize at IR-induced foci (IRIF). Depletion of ZEB1 enhances radio-sensitivity and increases IR-induced chromosomal aberrations in an ATM-independent manner. The very rapid recruitment-within 2 seconds-of ZEB1 to euchromatic DSBs is like-wise ATM-independent, but DNA-PK-dependent and is required for subsequent recruitment of 53BP1. ZEB1 promotes NHEJ and inhibits HR through its homeodomain by inducing 53BP1permissive, pro-NHEJ/anti-HR chromatin modifications. Lastly, depletion of ZEB1 increases hyper-resection at DSBs and inhibits physiological DSB repair. These results support the argument that ZEB1 plays an essential role in DSB repair in euchromatin by establishing a 53BP1-permissive/pro-NHEJ chromatin environment.

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“…2E), two well-established defects observed in ATR-deficient cells. (19) Chromatin immunoprecipitation real-time quantitative PCR analysis found that BRUCE is recruited to DNA damage sites induced by the endonuclease I-SecI in a human fibroblast DR95 cell line, which expresses a pDR-GFP plasmid containing I-SceI cleavage sequence (21) (Fig. 2F).…”

Section: Bruce Accumulates At Dna Damage Sites and Protects Against Rmentioning

confidence: 99%

The BRUCE‐ATR Signaling Axis Is Required for Accurate DNA Replication and Suppression of Liver Cancer Development

et al. 2019

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Replication fork stability during DNA replication is vital for maintenance of genomic stability and suppression of cancer development in mammals. ATR (ataxia‐telangiectasia mutated [ATM] and RAD3‐related) is a master regulatory kinase that activates the replication stress response to overcome replication barriers. Although many downstream effectors of ATR have been established, the upstream regulators of ATR and the effect of such regulation on liver cancer remain unclear. The ubiquitin conjugase BRUCE (BIR Repeat containing Ubiquitin‐Conjugating Enzyme) is a guardian of chromosome integrity and activator of ATM signaling, which promotes DNA double‐strand break repair through homologous recombination. Here we demonstrate the functions for BRUCE in ATR activation in vitro and liver tumor suppression in vivo. BRUCE is recruited to induced DNA damage sites. Depletion of BRUCE inhibited multiple ATR‐dependent signaling events during replication stress, including activation of ATR itself, phosphorylation of its downstream targets CHK1 and RPA, and the mono‐ubiquitination of FANCD2. Consequently, BRUCE deficiency resulted in stalled DNA replication forks and increased firing of new replication origins. The in vivo impact of BRUCE loss on liver tumorigenesis was determined using the hepatocellular carcinoma model induced by genotoxin diethylnitrosamine. Liver‐specific knockout of murine Bruce impaired ATR activation and exacerbated inflammation, fibrosis and hepatocellular carcinoma, which exhibited a trabecular architecture, closely resembling human hepatocellular carcinoma (HCC). In humans, the clinical relevance of BRUCE down‐regulation in liver disease was found in hepatitis, cirrhosis, and HCC specimens, and deleterious somatic mutations of the Bruce gene was found in human hepatocellular carcinoma in the Cancer Genome Atlas database. Conclusion: These findings establish a BRUCE‐ATR signaling axis in accurate DNA replication and suppression of liver cancer in mice and humans and provides a clinically relevant HCC mouse model.

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SMARCAD1 Phosphorylation and Ubiquitination Are Required for Resection during DNA Double-Strand Break Repair

Cited by 47 publications

References 47 publications

Molecular structures and mechanisms of DNA break processing in mouse meiosis

Molecular structures and mechanisms of DNA break processing in mouse meiosis

ZEB1 is Required for NHEJ-Mediated DSB Repair in Euchromatin

The BRUCE‐ATR Signaling Axis Is Required for Accurate DNA Replication and Suppression of Liver Cancer Development

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