Analysis of Chromatin Structure at Meiotic DSB Sites in Yeasts
Analysis of Chromatin Structure at Meiotic DSB Sites in Yeasts
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DOI：
10.1007/978-1-59745-527-5_16
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发表时间：
2009-01-01
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影响因子：
--
通讯作者：
Ohta, Kunihiro
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文献类型：
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作者：
Hirota, Kouji;Fukuda, Tomoyuki;Ohta, Kunihiro
减数分裂的主要特征之一是高频率的同源重组，这不仅赋予后代遗传多样性，而且确保染色体的适当分离。减数分裂重组由DNA双链断裂启动，需要许多蛋白质，包括催化核心Spo11。在这方面，像转录和修复等，重组被致密的染色质结构阻碍，因为反式作用因子不能容易地接近DNA。这种抑制作用必须在重组开始之前减轻。事实上，许多研究小组通过使用核酸酶作为探针来评估局部DNA可及性，表明重组热点周围的染色质凝聚程度较低。在这里，我们描述了一种方法来分析染色质结构的重组热点的酵母酿酒酵母和裂殖酵母粟酒裂殖酵母。这种方法，结合微球菌核酸酶（MNase）消化染色质DNA和随后的Southern印迹，预计将提供信息，染色质周围的热点。此外，由于MNase优先靶向接头DNA，也可以确定热点周围的几个核小体的位置。我们的协议是一个非常强大的方式来分析几个感兴趣的区域，并可以应用于其他目的。
One of the major features of meiosis is a high frequency of homologous recombination that not only confers genetic diversity to a successive generation but also ensures proper segregation of chromosomes. Meiotic recombination is initiated by DNA double-strand breaks that require many proteins including the catalytic core, Spo11. In this regard, like transcription and repair, etc., recombination is hindered by a compacted chromatin structure because trans-acting factors cannot easily access the DNA. Such inhibitory effects Must be alleviated prior to recombination initiation. Indeed, a number of groups showed that chromatin around recombination hotspots is less condensed, by using nucleases as a probe to assess local DNA accessibility. Here we describe a method to analyze chromatin structure of a recombination hotspot in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. This method, combining micrococcal nuclease (MNase) digestion of chromatin DNA and Subsequent Southern blotting, is expected to provide information as to chromatin context around a hotspot. Moreover, by virtue of MNase preferentially targeting linker DNA, positions of several nucleosomes surrounding a hotspot can also be determined. Our protocol is a very powerful way to analyze several regions of interest and can be applied to other purposes.