通过单分子解链力分析检测MSH2-MSH6的高亲和力和滑动钳模式。

Detection of high-affinity and sliding clamp modes for MSH2-MSH6 by single-molecule unzipping force analysis.

作者信息

Jiang Jingjing, Bai Lu, Surtees Jennifer A, Gemici Zekeriyya, Wang Michelle D, Alani Eric

机构信息

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.

出版信息

Mol Cell. 2005 Dec 9;20(5):771-81. doi: 10.1016/j.molcel.2005.10.014.

DOI:10.1016/j.molcel.2005.10.014

PMID:16337600

Abstract

Mismatch repair (MMR) is initiated by MutS family proteins (MSH) that recognize DNA mismatches and recruit downstream repair factors. We used a single-molecule DNA-unzipping assay to probe interactions between S. cerevisiae MSH2-MSH6 and a variety of DNA mismatch substrates. This work revealed a high-specificity binding state of MSH proteins for mismatch DNA that was not observed in bulk assays and allowed us to measure the affinity of MSH2-MSH6 for mismatch DNA as well as its footprint on DNA surrounding the mismatch site. Unzipping analysis with mismatch substrates containing an end blocked by lac repressor allowed us to identify MSH proteins present on DNA between the mismatch and the block, presumably in an ATP-dependent sliding clamp mode. These studies provide a high-resolution approach to study MSH interactions with DNA mismatches and supply evidence to support and refute different models proposed for initiation steps in MMR.

摘要

错配修复（MMR）由MutS家族蛋白（MSH）启动，这些蛋白识别DNA错配并招募下游修复因子。我们使用单分子DNA解链分析来探测酿酒酵母MSH2-MSH6与多种DNA错配底物之间的相互作用。这项工作揭示了MSH蛋白对错配DNA的高特异性结合状态，这在大量分析中未观察到，并且使我们能够测量MSH2-MSH6对错配DNA的亲和力及其在错配位点周围DNA上的足迹。对含有被乳糖阻遏物封闭末端的错配底物进行解链分析，使我们能够鉴定出存在于错配与封闭末端之间DNA上的MSH蛋白，推测其处于ATP依赖的滑动夹模式。这些研究提供了一种高分辨率方法来研究MSH与DNA错配的相互作用，并为支持和反驳针对MMR起始步骤提出的不同模型提供了证据。