Ma Wenjian, Westmoreland Jim, Nakai Wataru, Malkova Anna, Resnick Michael A
Chromosome Stability Section, National Institute of Environmental Health Sciences (NIEHS), NIH, Research Triangle Park, NC 27709, USA.
Methods Mol Biol. 2011;745:15-31. doi: 10.1007/978-1-61779-129-1_2.
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双链断裂（DSB）末端的切除会产生3′单链尾巴，这是DSB修复的早期事件，并且可能是修复途径选择和最终基因组稳定性的关键决定因素。目前用于检测切除的技术仅限于具有特定底物的体内模型系统（即HO内切酶靶点）。我们在此展示一种强大的检测方法，该方法不仅可以分析具有“干净”双链末端的位点特异性DSB的切除，还可以分析随机的“脏末端”DSB，例如由电离辐射和化疗药物产生的DSB。该检测方法基于我们的发现，即由切除产生的具有单链DNA尾巴的酵母染色体在脉冲场凝胶电泳（PFGE）过程中的移动性比没有尾巴的酵母染色体低。结合使用环状染色体和单链DNA的酶切修剪，可以轻松检测和分析随机DSB的切除。这种迁移率变化检测为研究切除机制、DSB修复的早期事件以及途径调节中涉及的因素提供了独特的机会。
