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The internal region of CtIP functions as a regulator of DNA end resection

Citation

Howard, Sean et al. (2020), The internal region of CtIP functions as a regulator of DNA end resection, Dryad, Dataset, https://doi.org/10.5061/dryad.msbcc2fvd

Abstract

DNA double-strand breaks are repaired by end-joining or homologous recombination. A key-committing step of recombination is DNA end resection. In resection, phosphorylated CtIP first promotes the endonuclease of the MRE11-RAD50-NBS1 (MRN) complex. Subsequently, CtIP also stimulates the BLM-DNA2 helicase-nuclease, coordinating thus both short and long-range resection. The structure of CtIP differs from its orthologues in yeast, as it contains a large internal region. Here we conducted a domain analysis of the internal region of CtIP to define its function in DNA end resection. We found that residues 350-600 were entirely dispensable for resection in vitro. A mutant lacking these residues was unexpectedly more efficient than full-length CtIP in DNA end resection and homologous recombination in vivo, and consequently conferred resistance to lesions induced by the topoisomerase poison camptothecin, which require high MRN-CtIP-dependent resection activity for repair. This suggested that the internal region, further mapped to residues 550-600, may mediate a negative regulatory function to prevent over resection in vivo. Consequently, the CtIP internal deletion mutant exhibited sensitivity to other DNA-damaging drugs, showing that upregulated resection may be instead toxic under other conditions. These experiments together identify a region within the central CtIP domain that negatively regulates DNA end resection.

Methods

This dataset was collected by a variety of methods including cell-base flow cytometry reporters, Western blotting, RT-PCR, in vitro biochemical assays and clonogenic survival assays. 

Funding

Swiss National Science Foundation, Award: 31003A_175444

European Research Council, Award: 681630

European Molecular Biology Organization, Award: ALTF 1560-2015

Swiss National Science Foundation, Award: 31003A_175444