Characterizing Resection at Random and Unique Chromosome Double-Strand Breaks and Telomere Ends

Wenjian Ma; Jim Westmoreland; Wataru Nakai; Anna Malkova; Michael A Resnick

doi:10.1007/978-1-61779-129-1_2

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Book chapter

Characterizing Resection at Random and Unique Chromosome Double-Strand Breaks and Telomere Ends

Wenjian Ma, Jim Westmoreland, Wataru Nakai, Anna Malkova and Michael A Resnick

DNA Recombination, pp.15-31

Methods in Molecular Biology, Humana Press

04/28/2011

DOI: 10.1007/978-1-61779-129-1_2

PMCID: PMC4857595

PMID: 21660686

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https://www.ncbi.nlm.nih.gov/pmc/articles/4857595View

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Abstract

Resection of DNA double-strand break (DSB) ends, which results in 3′ single-stranded tails, is an early event of DSB repair and can be a critical determinant in choice of repair pathways and eventual genome stability. Current techniques for examining resection are restricted to model in vivo systems with defined substrates (i.e., HO-endonuclease targets). We present here a robust assay that can analyze not only the resection of site-specific DSBs which typically have “clean” double-strand ends but also random “dirty-ended” DSBs such as those generated by ionizing radiation and chemotherapeutic agents. The assay is based on our finding that yeast chromosomes with single-stranded DNA tails caused by resection are less mobile during pulsed-field gel electrophoresis (PFGE) than those without a tail. In combination with the use of a circular chromosome and enzymatic trimming of single-stranded DNA, resection of random DSBs can be easily detected and analyzed. This mobility-shift assay provides a unique opportunity to examine the mechanisms of resection, early events in DSB repair, as well as factors involved in pathway regulation.

DNA

double-strand break repair

HO endonuclease

ionizing radiation

mung bean nuclease

pulsed-field gel electrophoresis (PFGE)

resection

telomere

Details

Title: Subtitle: Characterizing Resection at Random and Unique Chromosome Double-Strand Breaks and Telomere Ends
Creators: Wenjian Ma - Chromosome Stability Section, National Institute of Environmental Health Sciences (NIEHS), NIH, Research Triangle Park, USA
Jim Westmoreland - Chromosome Stability Section, National Institute of Environmental Health Sciences (NIEHS), NIH, Research Triangle Park, USA
Wataru Nakai - Chromosome Stability Section, National Institute of Environmental Health Sciences (NIEHS), NIH, Research Triangle Park, USA
Anna Malkova - Biology Department, Indiana University Purdue University, Indianapolis, USA
Michael A Resnick - Chromosome Stability Section, National Institute of Environmental Health Sciences (NIEHS), NIH, Research Triangle Park, USA
Resource Type: Book chapter
Publication Details: DNA Recombination, pp.15-31
Series: Methods in Molecular Biology
DOI: 10.1007/978-1-61779-129-1_2
PMID: 21660686
PMCID: PMC4857595
NLM abbreviation: Methods Mol Biol
eISSN: 1940-6029
ISSN: 1064-3745
Publisher: Humana Press; Totowa, NJ
Language: English
Date published: 04/28/2011
Academic Unit: Biology
Record Identifier: 9984217533702771

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