Characterizing resection at random and unique chromosome double-strand breaks and telomere ends.
Characterizing resection at random and unique chromosome double-strand breaks and telomere ends.
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DOI：
10.1007/978-1-61779-129-1_2
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发表时间：
2011
期刊：
影响因子：
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通讯作者：
Resnick MA
中科院分区：
文献类型：
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作者：
Ma W;Westmoreland J;Nakai W;Malkova A;Resnick MA
DNA双链断裂（DSB）末端的切除导致3 '单链尾巴，是DSB修复的早期事件，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。目前检查切除的技术仅限于具有确定底物（即ho -内切酶靶标）的体内系统模型。我们在这里提出了一种强大的分析方法，不仅可以分析通常具有“干净”双链末端的位点特异性dsb的切除，还可以分析随机的“脏端”dsb，如电离辐射和化疗药物产生的dsb。该实验是基于我们的发现，在脉冲场凝胶电泳（PFGE）中，具有单链DNA尾部的酵母染色体比没有尾部的酵母染色体流动性更低。结合使用环状染色体和单链DNA的酶切，可以很容易地检测和分析随机dsb的切除。这种移动转移试验提供了一个独特的机会来检查切除机制，DSB修复的早期事件，以及参与途径调节的因素。
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 “dirtyended” 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.