Single Molecule Studies of DNA Error Recognition by Mismatch Repair Enzymes

描述（由申请人提供）：项目摘要：细胞分子错配修复（MMR）机制负责扫描和修复DNA，因为它是由各种应力连续轰击。人类错配识别蛋白中的突变与人类中超过一半的遗传性非息肉病性结肠直肠癌和约15%的内胚层癌和卵巢癌有关。尽管迫切需要了解MMR蛋白如何识别受损DNA，但目前关于错配损伤识别的分子机制几乎没有一致意见。该提案概述了一系列的实验，结合联合收割机单分子生物物理学方法与ID扩散测定，以解决一个长期存在的争论在DNA修复领域：错配修复蛋白如何定位和响应错配碱基？为了实现这一点，将采用全内反射荧光显微镜（TIRFM）来监测与DNA分子阵列结合的单个荧光标记的MMR蛋白，所述DNA分子阵列被拴系到微流体样品室的钝化表面。TIRFM技术将有助于直接观察E。大肠杆菌蛋白MutS和MutL，它们共同促进DNA错配识别的第一步。MutS和MutL同源物存在于几乎所有生物体中，包括人类。最初的研究将观察ATP依赖的MutS-DNA校对机制，随后将关注MutS-MutL复合物在触发下游修复事件中的作用。MMR蛋白在结合、校对和随后定位错配位点中的ATP水解作用将通过定点蛋白质诱变和对不可水解的ATP类似物的研究相结合来探测。相关性：许多常见的人类癌症发生在双链体DNA的错误错配片段没有被分子错配机制修复时。这项研究的目的是解开复杂的分子事件序列，使受损的DNA被识别并最终被错配修复蛋白修复。

DESCRIPTION (provided by applicant): Project Summary: Cellular molecular mismatch repair (MMR) machinery is charged with scanning and repairing DNA as it is continuously bombarded by a variety of stresses. Mutations in human mismatch recognition proteins have been implicated in over half of hereditary non-polyposis colorectal cancers and -15% of endodermal and ovarian cancers in humans. Despite the urgent need to understand how MMR proteins recognize damaged DNA, there is currently little agreement about the molecular mechanism of mismatch damage recognition. This proposal outlines a series of experiments that combine single-molecule biophysics methodologies with ID-diffusion assays to solve a long-standing debate in the DNA repair field: How do mismatch repair proteins locate and respond to mispaired bases? To accomplish this, total internal reflection fluorescence microscopy (TIRFM) will be employed to monitor individual fluorescently labeled MMR proteins bound to arrays of DNA molecules that are tethered to the passivated surface of a microfluidic sample chamber. The TIRFM technique will facilitate direct visualization of the E. coli proteins MutS and MutL, which together facilitate the first steps in DNA mismatch recognition. MutS and MutL homologues are found in nearly all organisms, including humans. Initial studies will observe the ATP-dependent MutS-DNA proofreading mechanism, and will subsequently focus to the role of MutS-MutL complexes in triggering downstream repair events. The role of ATP hydrolysis by MMR proteins in binding, proofreading, and subsequently locating a mismatch site will be probed by a combination of site-directed protein mutagenesis and studies on non-hydrolysable ATP analogs. I Relevance: Many common human cancers occur when an erroneously mispaired segment of duplex DNA is not repaired by the molecular mismatch machinery. This research aims to unravel the complicated sequence of molecular events that allow damaged DNA to be recognized and ultimately repaired by mismatch repair proteins.