Quantitative genomic analysis of RecA protein binding during DNA double-strand break repair reveals RecBCD action in vivo
Quantitative genomic analysis of RecA protein binding during DNA double-strand break repair reveals RecBCD action in vivo
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DOI：
10.1073/pnas.1424269112
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发表时间：
2015-08-25
影响因子：
11.1
通讯作者：
Leach, David R. F.
中科院分区：
文献类型：
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作者：
Cockram, Charlotte A.;Filatenkova, Milana;Leach, David R. F.
在活细胞的背景下，了解分子机制，需要在体内生化分析，以补充建立在体外生物化学的新方法的发展。一个至关重要的分子机制是遗传重组，这是有益的遗传信息重组和DNA双链断裂修复（DSBR）所必需的。重组的核心是RecA（Rad 51）蛋白，它在DNA上组装成螺旋丝并介导遗传交换。在这里，我们开发了一种将染色质免疫沉淀与下一代测序（ChIP-Seq）和数学建模相结合的方法，以量化大肠杆菌染色体中单个DSB主动修复期间RecA蛋白结合。我们已经使用了定量基因组分析来推断在重组热点，被称为Chi的解旋酶/核酸酶RecBCD的RecA负载的关键体内分子参数。我们的基因组分析还显示，在lacZ位点的DSBR导致第二个RecBCD介导的DSBR事件发生在染色体的末端区域，超过1 Mb。
Understanding molecular mechanisms in the context of living cells requires the development of new methods of in vivo biochemical analysis to complement established in vitro biochemistry. A critically important molecular mechanism is genetic recombination, required for the beneficial reassortment of genetic information and for DNA double-strand break repair (DSBR). Central to recombination is the RecA (Rad51) protein that assembles into a spiral filament on DNA and mediates genetic exchange. Here we have developed a method that combines chromatin immunoprecipitation with next-generation sequencing (ChIP-Seq) and mathematical modeling to quantify RecA protein binding during the active repair of a single DSB in the chromosome of Escherichia coli. We have used quantitative genomic analysis to infer the key in vivo molecular parameters governing RecA loading by the helicase/nuclease RecBCD at recombination hot-spots, known as Chi. Our genomic analysis has also revealed that DSBR at the lacZ locus causes a second RecBCD-mediated DSBR event to occur in the terminus region of the chromosome, over 1 Mb away.