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Enhancement of BLM-DNA2-Mediated Long-Range DNA End Resection by CtIP.

ABSTRACT: DNA double-strand break repair by homologous recombination entails the resection of DNA ends to reveal ssDNA tails, which are used to invade a homologous DNA template. CtIP and its yeast ortholog Sae2 regulate the nuclease activity of MRE11 in the initial stage of resection. Deletion of CtIP in the mouse or SAE2 in yeast engenders a more severe phenotype than MRE11 nuclease inactivation, indicative of a broader role of CtIP/Sae2. Here, we provide biochemical evidence that CtIP promotes long-range resection via the BLM-DNA2 pathway. Specifically, CtIP interacts with BLM and enhances its helicase activity, and it enhances DNA cleavage by DNA2. Thus, CtIP influences multiple aspects of end resection beyond MRE11 regulation.

SUBMITTER: Daley JM

PROVIDER: S-EPMC5689478 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Enhancement of BLM-DNA2-Mediated Long-Range DNA End Resection by CtIP.

Daley James M JM Jimenez-Sainz Judit J Wang Weibin W Miller Adam S AS Xue Xiaoyu X Nguyen Kevin A KA Jensen Ryan B RB Sung Patrick P

Cell reports 20171001 2

DNA double-strand break repair by homologous recombination entails the resection of DNA ends to reveal ssDNA tails, which are used to invade a homologous DNA template. CtIP and its yeast ortholog Sae2 regulate the nuclease activity of MRE11 in the initial stage of resection. Deletion of CtIP in the mouse or SAE2 in yeast engenders a more severe phenotype than MRE11 nuclease inactivation, indicative of a broader role of CtIP/Sae2. Here, we provide biochemical evidence that CtIP promotes long-rang ...[more]

PMID: 29020620

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CtIP promotes the motor activity of DNA2 to accelerate long-range DNA end resection.

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DNA end resection requires constitutive sumoylation of CtIP by CBX4.

Project description:DNA breaks are complex DNA lesions that can be repaired by two alternative mechanisms: non-homologous end-joining and homologous recombination. The decision between them depends on the activation of the DNA resection machinery, which blocks non-homologous end-joining and stimulates recombination. On the other hand, post-translational modifications play a critical role in DNA repair. We have found that the SUMO E3 ligase CBX4 controls resection through the key factor CtIP. Indeed, CBX4 depletion impairs CtIP constitutive sumoylation and DNA end processing. Importantly, mutating lysine 896 in CtIP recapitulates the CBX4-depletion phenotype, blocks homologous recombination and increases genomic instability. Artificial fusion of CtIP and SUMO suppresses the effects of both the non-sumoylatable CtIP mutant and CBX4 depletion. Mechanistically, CtIP sumoylation is essential for its recruitment to damaged DNA. In summary, sumoylation of CtIP at lysine 896 defines a subpopulation of the protein that is involved in DNA resection and recombination.The choice between non-homologous end-joining and homologous recombination to repair a DNA double-strand break depends on activation of the end resection machinery. Here the authors show that SUMO E3 ligase CBX4 sumoylates subpopulation of CtIP to regulate recruitment to breaks and resection.

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Project description: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 MRE11-RAD50-NBS1 (MRN). Subsequently, CtIP also stimulates the WRN/BLM-DNA2 pathway, coordinating thus both short and long-range resection. The structure of CtIP differs from its orthologues in yeast, as it contains a large internal unstructured region. Here, we conducted a domain analysis of CtIP to define the function of the internal region 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 CtIP region, further mapped to residues 550-600, may mediate a negative regulatory function to prevent over resection in vivo. The CtIP internal deletion mutant exhibited sensitivity to other DNA-damaging drugs, showing that upregulated resection may be instead toxic under different conditions. These experiments together identify a region within the central CtIP domain that negatively regulates DNA end resection.

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CtIP-mediated DNA resection is dispensable for IgH class switch recombination by alternative end-joining.

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Regulatory control of Sgs1 and Dna2 during eukaryotic DNA end resection.

Project description:In the repair of DNA double-strand breaks by homologous recombination, the DNA break ends must first be processed into 3' single-strand DNA overhangs. In budding yeast, end processing requires the helicase Sgs1 (BLM in humans), the nuclease/helicase Dna2, Top3-Rmi1, and replication protein A (RPA). Here, we use single-molecule imaging to visualize Sgs1-dependent end processing in real-time. We show that Sgs1 is recruited to DNA ends through Top3-Rmi1-dependent or -independent means, and in both cases Sgs1 is maintained in an immoble state at the DNA ends. Importantly, the addition of Dna2 triggers processive Sgs1 translocation, but DNA resection only occurs when RPA is also present. We also demonstrate that the Sgs1-Dna2-Top3-Rmi1-RPA ensemble can efficiently disrupt nucleosomes, and that Sgs1 itself possesses nucleosome remodeling activity. Together, these results shed light on the regulatory interplay among conserved protein factors that mediate the nucleolytic processing of DNA ends in preparation for homologous recombination-mediated chromosome damage repair.

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