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

  • Wenjian Ma
  • Jim Westmoreland
  • Wataru Nakai
  • Anna Malkova
  • Michael A. ResnickEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 745)

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-Scemung bean nuclease telomere 

Notes

Acknowledgments

This work was supported by the Intramural Research Program of the NIEHS (NIH, DHHS) under project 1 Z01 ES065073 (MAR).

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Wenjian Ma
    • 1
  • Jim Westmoreland
    • 1
  • Wataru Nakai
    • 1
  • Anna Malkova
    • 2
  • Michael A. Resnick
    • 1
    Email author
  1. 1.Chromosome Stability SectionNational Institute of Environmental Health Sciences (NIEHS), NIHResearch Triangle ParkUSA
  2. 2.Biology DepartmentIndiana University Purdue UniversityIndianapolisUSA

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