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Viral Chromosome Conformation Capture (V3C) Assays for Identifying Trans-interaction Sites between Lytic Viruses and the Cellular Genome

机译:病毒染色体构象捕获(V3C)测定以识别裂解病毒和细胞基因组之间的反式相互作用位点

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摘要

The folding mechanisms of the mammalian genome package our genetic material into the nucleus, and in doing so, dictate its appropriate replication and expression. Chromosome conformation capture technology has enabled the dissection of the folding principles of the cellular genome. This has led to a better understanding of the role played by architectural proteins in forming and dissolving 3D-chromatin-structure. These assays are based on the principle of crosslinking distant cellular sites that are proximal to each other in 3D space using formaldehyde followed by digestion of formed hybrid DNA junctions. Invading viruses, such as the lytic parvovirus Minute Virus of Mice (MVM), establish distinct replication centers within the nuclear environment at cellular sites that preferentially undergo DNA damage, but do not integrate into the cellular DNA. We have adapted chromosome conformation capture technology to study the trans-interaction between MVM and the cellular genome, which we have dubbed V3C, which can be extended to a whole-genome analysis we term V3C-seq. This protocol describes the procedure for performing, as well as analyzing V3C-seq assays, and can be adapted for mapping the cellular interaction sites of any non-integrating DNA virus.
机译:哺乳动物基因组的折叠机制将我们的遗传物质包装到细胞核中,并决定了其适当的复制和表达。染色体构象捕获技术已使解剖细胞基因组的折叠原理成为可能。这导致人们对建筑蛋白在形成和溶解3D染色质结构中所起的作用有了更好的了解。这些测定法基于以下原理:使用甲醛交联在3D空间中彼此邻近的远处细胞位置,然后消化形成的杂合DNA连接。诸如裂解性细小病毒小鼠微小病毒(MVM)之类的入侵病毒会在细胞核内的环境中建立不同的复制中心,这些复制中心优先遭受DNA破坏,但不会整合到细胞DNA中。我们已经采用了染色体构象捕获技术来研究MVM和细胞基因组之间的反式相互作用,我们将其称为V3C,它可以扩展到我们称为V3C-seq的全基因组分析。该协议描述了执行和分析V3C-seq分析的程序,并且可以适用于绘制任何非整合型DNA病毒的细胞相互作用位点。

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