title: Characterizing resection at random and unique chromosome double-strand breaks and telomere ends.
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.
authors.affiliation: Chromosome Stability Section, National Institute of Environmental Health Sciences (NIEHS), NIH, Research Triangle Park, NC 27709, USA. ma2@niehs.nih.gov
authors.forename: Wenjian
authors.initials: W
authors.lastname: Ma
authors.forename: Jim
authors.initials: J
authors.lastname: Westmoreland
authors.forename: Wataru
authors.initials: W
authors.lastname: Nakai
authors.forename: Anna
authors.initials: A
authors.lastname: Malkova
authors.forename: Michael A
authors.initials: MA
authors.lastname: Resnick
mesh_terms: D015139:Blotting, Southern; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000235:genetics; D016521:Electrophoresis, Gel, Pulsed-Field; D010940:Plant Proteins / Q000378:metabolism; D011839:Radiation, Ionizing; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D016615:Telomere / Q000235:genetics*
chemical_list: C526101:Mung Bean Nuclease; D010940:Plant Proteins; D015719:Single-Strand Specific DNA and RNA Endonucleases