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首页> 外文期刊>Yeast >A new method to efficiently induce a site-specific double-strand break in the fission yeast Schizosaccharomyces pombe
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A new method to efficiently induce a site-specific double-strand break in the fission yeast Schizosaccharomyces pombe

机译：在裂殖酵母裂殖酵母中有效诱导位点特异性双链断裂的新方法

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Double-strand DNA breaks are a serious threat to cellular viability and yeast systems have proved invaluable in helping to understand how these potentially toxic lesions are sensed and repaired. An important method to study the processing of DNA breaks in the budding yeast Saccharomyces cerevisiae is to introduce a unique double-strand break into the genome by regulating the expression of the site-specific HO endonuclease with a galactose inducible promoter. Variations of the HO site-specific DSB assay have been adapted to many organisms, but the methodology has seen only limited use in the fission yeast Schizosaccharomyces pombe because of the lack of a promoter capable of inducing endonuclease expression on a relatively short time scale (similar to 1 h). We have overcome this limitation by developing a new assay in which expression of the homing endonuclease I-PpoI is tightly regulated with a tetracycline-inducible promoter. We show that induction of the I-PpoI endonuclease produces rapid cutting of a defined cleavage site (> 80% after 1 h), efficient cell cycle arrest and significant accumulation of the checkpoint protein Crb2 at break-adjacent regions in a manner that is analogous to published findings with DSBs produced by an acute exposure to ionizing irradiation. This assay provides an important new tool for the fission yeast community and, because many aspects of mammalian chromatin organization have been well-conserved in Sz. pombe but not in S. cerevisiae, also offers an attractive system to decipher the role of chromatin structure in modulating the repair of double-stranded DNA breaks. Copyright (C) 2012 John Wiley & Sons, Ltd.
机译：双链DNA断裂严重威胁着细胞的生存能力，酵母系统已被证明对帮助理解如何感测和修复这些潜在的毒性损伤具有不可估量的价值。研究芽孢酿酒酵母中DNA断裂的处理的一种重要方法是通过用半乳糖诱导型启动子调节位点特异性HO核酸内切酶的表达，将独特的双链断裂引入基因组。 HO位点特异性DSB测定的变化已适应许多生物，但由于缺乏能够在较短时间内诱导核酸内切酶表达的启动子，该方法仅在裂变酵母裂殖酵母中得到有限的应用（相似至1小时）。我们通过开发一种新的测定法克服了这一局限，在该测定法中，归巢核酸内切酶I-PpoI的表达受到四环素诱导型启动子的严格调控。我们显示，I-PpoI核酸内切酶的诱导产生快速切割一个确定的切割位点（1小时后> 80％），有效的细胞周期停滞以及在检查点附近区域的检查点蛋白Crb2的大量积累，其方式类似公开暴露于电离辐射下产生的DSB的发现。该测定法为裂变酵母群落提供了重要的新工具，并且因为哺乳动物染色质组织的许多方面在Sz中都得到了很好的保存。 pombe（但不在酿酒酵母中）也提供了一个有吸引力的系统，可用来解释染色质结构在调节双链DNA断裂修复中的作用。版权所有（C）2012 John Wiley＆Sons，Ltd.

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