Mechanism of homology search in homologous recombination by Rad51 and Rad54.

描述（由申请人提供）：本研究的目的是阐明DNA修复蛋白如何定位和修复受损DNA的机制。这一过程对健康的影响延伸到乳腺癌和卵巢癌，因为这些癌症与这一途径的缺陷有关。对同源重组和DNA修复的进一步了解将有助于更好地了解人类癌症的发展。本研究的重点是确定DNA修复蛋白Rad51和Rad54如何与DNA和其他同源重组蛋白组分相互作用，以及在同源重组过程中DNA分子如何排列。该研究假设Rad51、Rad54和ssDNA形成的突触前复合物通过ATP水解来寻找dsDNA的同源区域，从而驱动易位。此外，假设ATP水解是一种载体，可以从dsDNA中去除核小体和其他DNA结合蛋白，否则会阻碍进入dsDNA的同源区域。该项目的具体目标是：(1)研究突触前复合物用于探测dsDNA同源序列的生化机制；(2)详细描述突触前复合物与dsDNA结合的核小体的相互作用。为了验证这一假设，将使用全内反射荧光显微镜观察涉及荧光标记的DNA分子Rad51和Rad54的反应。这项技术能够直接可视化单个蛋白质与单个DNA分子的相互作用。

DESCRIPTION (provided by applicant): The goal of this research is to elucidate the mechanisms underlying how DNA repair proteins locate and repair damaged DNA. The health implications of this process extend to breast and ovarian cancers, as these cancers are associated with defects in this pathway. Improved understanding of homologous recombination and DNA repair will in turn allow better understanding of the development of human cancers. The focus of this proposal is to determine how the DNA repair proteins Rad51 and Rad54 interact with DNA and with other protein components of homologous recombination, and how DNA molecules are aligned during homologous recombination. This proposal hypothesizes that the presynaptic complex formed by Rad51, Rad54, and ssDNA searches dsDNA for regions of homology using ATP hydrolysis to drive translocation. In addition, it is hypothesized that ATP hydrolysis is a vehicle for the removal of nucleosomes and other DNA binding proteins from the dsDNA that would otherwise hinder access to homologous regions of dsDNA. The specific aims of this project are to (1) investigate the biochemical mechanism used by the presynaptic complex to probe dsDNA for homologous sequence and to (2) detail the interaction of the presynaptic complex with nucleosomes bound to the dsDNA. To test this hypothesis, reactions involving fluorescently labeled DNA molecules, Rad51, and Rad54 will be observed using total internal reflection fluorescent microscopy. This technique is able to directly visualize individual proteins interacting with individual molecules of DNA. DNA repair is an absolutely essential process to maintain cellular health. The proposed research is directly relevant to cancer biology, and the successful completion of the experiments contained in this research will contribute immensely to understanding of various types of cancers.