Mechanistic Analysis of a DNA End Processing Pathway Mediated by the Xenopus Werner Syndrome Protein

Toczylowski, Thomas; Yan, Hong

doi:10.1074/jbc.m605044200

Cited by 24 publications

(52 citation statements)

References 44 publications

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“…An initiating MRX-and Sae2-dependent endonucleolytic cleavage of 50-100-nt oligonucleotides from the 5 strand is followed by a highly processive resection step involving helicases and nucleases, including Sgs1 (RecQ homologue), Dna2, and Exo1. In vertebrates, several factors have been implicated in resection of DSBs, including DNA2 (Liao et al, 2008), Exo1 , and the two RecQ homologues BLM (Gravel et al, 2008; and WRN (Yan et al, 2005;Toczylowski and Yan, 2006). In our experiments, we show that both the BLM and WRN helicases are recruited independently of CtIP in S phase (Fig.…”

Section: Discussionsupporting

confidence: 48%

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Coordination of BRCA1/BARD1- and MRE11/RAD50/NBS1-Dependent DNA Transactions in Breast Tumor Suppression

Gautier¹

2011

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Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. REPORT DATE (DD-MM-YYYY)2. REPORT TYPE 3. DATES COVERED (From -To) 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER 5e. TASK NUMBER E-Mail:5f. WORK UNIT NUMBER The main objective of the proposal is to understand how the Mre11-Rad50-Nbs1 complex and the BRCA1-BARD1 complex interact on DNA and coordinate DNA transactions that are critical for the maintenance of genomic stability and to prevent breast tumor development. We have characterized the behavior of BRCA1/BARD1 on DNA, using a single molecule approach. We have developed new single molecule technologies to study the behavior of BRCA1/BARD1 and MRN complexes. Finally, we have established that MRN-BRAC1/BARD1 interactions are dispensable for MRN/CtIP dependent DNA end resection, the first step of homology-dependent repair of DNA double-strand breaks. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBER SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR'S ACRONYM(S) U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 SPONSOR/MONITOR'S REPORT NUMBER(S) DISTRIBUTION / AVAILABILITY STATEMENTNo subject terms provided. We have completed initial characterization of quantum dot(QD)-tagged Brac1/Bard1 interactions with dsDNA using our single molecule DNA curtain imaging technology. We have shown that the QD tagged protein complexes work in our single molecule assays, that the labeling is specific, and that the labeled proteins are well-behaved in our single molecule assays. We have also demonstrated that BRCA1/BARD1 binds double stranded DNA at apparently random locations, and that the protein appears to undergo one-dimensional (1D) diffusion along the DNA in a direction that is biased by buffer flow (see below) before finally stopping at a fixed location (unpublished). We do not yet know the significance of the 1D sliding behavior nor do we know what DNA features influence the binding distributions, but it seems reasonable to expect that these properties reflect mechanistic attributes of BRCA1/BARD1 as it searc...

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Section: Discussionsupporting

confidence: 48%

“…All three factors, which have been implicated in vertebrate resection (Yan et al, 2005;Toczylowski and Yan, 2006;Gravel et al, 2008;Liao et al, 2008;Zhu et al, 2008;Budd and Campbell, 2009), were enriched on damaged chromatin in mock-depleted S-phase extracts (Fig. 2 A).…”

Section: Characteristics Of Ctip-independent Resectionmentioning

confidence: 99%

Coordination of BRCA1/BARD1- and MRE11/RAD50/NBS1-Dependent DNA Transactions in Breast Tumor Suppression

Gautier¹

2011

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“…Dna2 antibody was raised in rabbits against a bacterially expressed His-tagged fusion protein containing the N-terminal 712 amino acids of Xenopus Dna2 protein. Antibodies against Xenopus Exo1, PCNA, RPA, CtIP, Mre11, WRN, NBS1 and Chk2 have been described before (26,31,52,63,87,88). For immunodepletion, 10 μl protein A agarose beads coupled with 50 μl of the protein antiserum or 50 μl each of two antisera for double-depletion were incubated with 50 μl NPE for 45 min at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…To dissect how resection is initiated at clean DSBs in the presence of all resection activities, we used the Xenopus nucleoplasmic extract (NPE) isolated from synthetic nuclei—a cell-free system that faithfully recapitulates the proper DNA damage response in S and G2 phases of the cell cycle (52,59,63,81–86). Our results indicate that like blocked DSBs, resection initiation of clean DSBs also occurs via endocleavage of the 5′ strand DNA; however, Dna2, but not CtIP–MRN, is the primary nuclease for this process.…”

Section: Introductionmentioning

confidence: 99%

Dna2 initiates resection at clean DNA double-strand breaks

Paudyal

Yan

et al. 2017

Nucleic Acids Research

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Nucleolytic resection of DNA double-strand breaks (DSBs) is essential for both checkpoint activation and homology-mediated repair; however, the precise mechanism of resection, especially the initiation step, remains incompletely understood. Resection of blocked ends with protein or chemical adducts is believed to be initiated by the MRN complex in conjunction with CtIP through internal cleavage of the 5′ strand DNA. However, it is not clear whether resection of clean DSBs with free ends is also initiated by the same mechanism. Using the Xenopus nuclear extract system, here we show that the Dna2 nuclease directly initiates the resection of clean DSBs by cleaving the 5′ strand DNA ∼10–20 nucleotides away from the ends. In the absence of Dna2, MRN together with CtIP mediate an alternative resection initiation pathway where the nuclease activity of MRN apparently directly cleaves the 5′ strand DNA at more distal sites. MRN also facilitates resection initiation by promoting the recruitment of Dna2 and CtIP to the DNA substrate. The ssDNA-binding protein RPA promotes both Dna2- and CtIP–MRN-dependent resection initiation, but a RPA mutant can distinguish between these pathways. Our results strongly suggest that resection of blocked and clean DSBs is initiated via distinct mechanisms.

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“…During DNA end resection, Dna2 works together with Sgs1-Top3-Rmi1 complex in yeast and Sgs1 ortholog BLM in cultured human cells [71,[85][86][87]114]. Studies in Xenopus egg extracts as well as human cells show that another RecQ family of helicase Werner syndrome RecQ-like helicase (WRN) also promotes resection by unwinding the DNA ends and making it accessible for Dna2 [115][116][117][118][119]. Although Dna2 functions as both a helicase and a nuclease, only the nuclease activity is essential for the extension of DNA end resection [87,120,121].…”

Section: Extension Of Resection By Exo1 and Dna2mentioning

confidence: 99%

Sharpening the ends for repair: mechanisms and regulation of DNA resection

Paudyal¹,

You²

2016

ABBS

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DNA end resection is a key process in the cellular response to DNA double-strand break damage that is essential for genome maintenance and cell survival. Resection involves selective processing of 5′ ends of broken DNA to generate ssDNA overhangs, which in turn control both DNA repair and checkpoint signaling. DNA resection is the first step in homologous recombination-mediated repair and a prerequisite for the activation of the ataxia telangiectasia mutated and Rad3-related (ATR)-dependent checkpoint that coordinates repair with cell cycle progression and other cellular processes. Resection occurs in a cell cycle-dependent manner and is regulated by multiple factors to ensure an optimal amount of ssDNA required for proper repair and genome stability. Here, we review the latest findings on the molecular mechanisms and regulation of the DNA end resection process and their implications for cancer formation and treatment.

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Mechanistic Analysis of a DNA End Processing Pathway Mediated by the Xenopus Werner Syndrome Protein

Cited by 24 publications

References 44 publications

Coordination of BRCA1/BARD1- and MRE11/RAD50/NBS1-Dependent DNA Transactions in Breast Tumor Suppression

Coordination of BRCA1/BARD1- and MRE11/RAD50/NBS1-Dependent DNA Transactions in Breast Tumor Suppression

Dna2 initiates resection at clean DNA double-strand breaks

Sharpening the ends for repair: mechanisms and regulation of DNA resection

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