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 的潜在机制。这一过程对健康的影响延伸到乳腺癌和卵巢癌，因为这些癌症与该途径的缺陷有关。提高对同源重组和 DNA 修复的了解将有助于更好地了解人类癌症的发展。该提案的重点是确定 DNA 修复蛋白 Rad51 和 Rad54 如何与 DNA 以及同源重组的其他蛋白质成分相互作用，以及 DNA 分子在同源重组过程中如何排列。该提议假设由 Rad51、Rad54 和 ssDNA 形成的突触前复合物使用 ATP 水解来搜索 dsDNA 的同源区域以驱动易位。此外，推测 ATP 水解是从 dsDNA 中去除核小体和其他 DNA 结合蛋白的载体，否则这些蛋白会阻碍进入 dsDNA 的同源区域。该项目的具体目标是 (1) 研究突触前复合体用于探测 dsDNA 的同源序列的生化机制，以及 (2) 详细说明突触前复合体与结合到 dsDNA 的核小体的相互作用。为了检验这一假设，将使用全内反射荧光显微镜观察涉及荧光标记 DNA 分子、Rad51 和 Rad54 的反应。该技术能够直接可视化单个蛋白质与单个 DNA 分子相互作用的情况。 DNA 修复是维持细胞健康绝对重要的过程。拟议的研究与癌症生物学直接相关，成功完成本研究中包含的实验将极大地有助于了解各种类型的癌症。

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.