Single Molecule Studies of DNA Error Recognition by Mismatch Repair Enzymes

项目概述：细胞分子错配修复（MMR）机器负责扫描和修复DNA，因为它不断受到各种压力的轰击。人类错配识别蛋白的突变与一半以上的遗传性非息肉病性结直肠癌和-15%的人类内胚层和卵巢癌有关。尽管迫切需要了解MMR蛋白如何识别受损DNA，但目前对错配损伤识别的分子机制知之甚少。本提案概述了一系列将单分子生物物理学方法与id扩散分析相结合的实验，以解决DNA修复领域长期存在的争论：错配修复蛋白如何定位和响应错配碱基？为了实现这一目标，将采用全内反射荧光显微镜（TIRFM）来监测单个荧光标记的MMR蛋白，这些蛋白与拴在微流控样品室钝化表面的DNA分子阵列结合。TIRFM技术将促进大肠杆菌蛋白MutS和MutL的直接可视化，它们共同促进了DNA错配识别的第一步。mutt和MutL同系物几乎存在于包括人类在内的所有生物体中。初步研究将观察atp依赖的mts - dna校对机制，随后将关注mts - mutl复合物在触发下游修复事件中的作用。MMR蛋白水解ATP在结合、校对和随后定位错配位点中的作用将通过位点导向蛋白诱变和不可水解ATP类似物的研究相结合来探索。相关性：许多常见的人类癌症发生时，错误的错配的双链DNA片段不被分子错配机制修复。本研究旨在揭示允许受损DNA被识别并最终被错配修复蛋白修复的复杂分子事件序列。

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.