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DNA Recombination

Volume 745 of the series Methods in Molecular Biology pp 15-31

Date:

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

  • Wenjian MaAffiliated withChromosome Stability Section, National Institute of Environmental Health Sciences (NIEHS), NIH
  • , Jim WestmorelandAffiliated withChromosome Stability Section, National Institute of Environmental Health Sciences (NIEHS), NIH
  • , Wataru NakaiAffiliated withChromosome Stability Section, National Institute of Environmental Health Sciences (NIEHS), NIH
  • , Anna MalkovaAffiliated withBiology Department, Indiana University Purdue University
  • , Michael A. ResnickAffiliated withChromosome Stability Section, National Institute of Environmental Health Sciences (NIEHS), NIH Email author 

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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.

Key words

DNA double-strand break repair resection pulsed-field gel electrophoresis (PFGE) ionizing radiation HO endonuclease I-SceI mung bean nuclease telomere