Monitoring DNA recombination initiated by HO endonuclease.
Monitoring DNA recombination initiated by HO endonuclease.
复制标题
监测由 H2O 核酸内切酶引发的 DNA 重组。
DOI：
10.1007/978-1-61779-998-3_25
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Haber,JamesE
中科院分区：
文献类型：
--
作者：
Sugawara,Neal;Haber,JamesE
DNA双链断裂（DSBs）已被证明是酵母和其他生物中非常有效的重组起始物。单个位点特异性DSB启动同源DNA修复事件，如基因转换、断裂诱导复制和单链退火，以及非同源末端连接、微同源介导的末端连接和新的端粒添加。当修复被延迟或阻止时，单个DSB可以触发检查点介导的细胞周期阻滞。在出芽酵母中，在半乳糖诱导启动子的控制下表达HO内切酶，为研究这些过程提供了一种在体内独特位点同步诱导DSB的方法。我们描述了如何使用HO内切酶来研究交配型（MAT）转换中的重组事件。南方印迹通过检查DSB的形成、断裂时DNA的降解和产物的形成，提供了该过程的概述。变性凝胶和槽型斑点以及PCR为跟踪野生型和突变型细胞的切除进展提供了重要的工具。PCR在允许我们跟踪某些重组中间体的动力学方面也很重要，例如修复DNA合成的起始或在matswitching过程中去除非同源Y序列。最后，染色质免疫沉淀被用于跟踪关键蛋白向DSB的募集以及DSB修复的后续步骤。
DNA double-strand breaks (DSBs) have proven to be very potent initiators of recombination in yeast and other organisms. A single, site-specific DSB initiates homologous DNA repair events such as gene conversion, break-induced replication, and single-strand annealing, as well as nonhomologous end joining, microhomology-mediated end joining, and new telomere addition. When repair is either delayed or prevented, a single DSB can trigger checkpoint-mediated cell cycle arrest. In budding yeast, expressing the HO endonuclease under the control of a galactose-inducible promoter has been instrumental in the study of these processes by providing us a way to synchronously induce a DSB at a unique site in vivo. We describe how the HO endonuclease has been used to study the recombination events in mating-type (MAT) switching. Southern blots provide an overview of the process by allowing one to examine the formation of the DSB, DNA degradation at the break, and formation of the product. Denaturing gels and slot blots as well as PCR have provided important tools to follow the progression of resection in wild-type and mutant cells. PCR has also been important in allowing us to follow the kinetics of certain recombination intermediates such as the initiation of repair DNA synthesis or the removal of nonhomologous Y sequences duringMATswitching. Finally chromatin immunoprecipitation has been used to follow the recruitment of key proteins to the DSB and in subsequent steps in DSB repair.
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影响因子：
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作者：
G. Pfeifer;A. Riggs
通讯作者：
G. Pfeifer;A. Riggs
影响因子：
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作者：
Ira Herskowitz;Robert Jensen
通讯作者：
Ira Herskowitz;Robert Jensen
影响因子：
16
作者：
Wolner, B;van Komen, S;Peterson, CL
通讯作者：
Peterson, CL
影响因子：
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作者：
Sugawara, N;Wang, X;Haber, JE
通讯作者：
Haber, JE
DOI：
10.1016/0168-9525(92)90329-3
发表时间：
1992-12
期刊：
Trends in genetics : TIG
影响因子：
--
作者：
J. Haber
通讯作者：
J. Haber