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

描述(申请人提供)：这项研究的目标是阐明DNA修复蛋白如何定位和修复受损的DNA的潜在机制。这一过程的健康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修复的更好理解将反过来允许更好地理解人类癌症的发展。该提案的重点是确定DNA修复蛋白RAD51和Rad54如何与DNA和同源重组的其他蛋白质组分相互作用，以及DNA分子在同源重组过程中如何对齐。该建议假设由RAD51、Rad54和ssDNA形成的突触前复合体通过ATP水解驱动易位来搜索dsDNA的同源区域。此外，还假设ATP水解是从dsDNA中去除核小体和其他DNA结合蛋白的载体，否则会阻碍dsDNA同源区域的访问。本项目的具体目的是(1)研究突触前复合体用来探测dsDNA同源序列的生化机制，以及(2)详细说明突触前复合体与与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。这项技术能够直接显示单独的蛋白质与DNA的单独分子相互作用。 DNA修复是维持细胞健康的绝对必要的过程。这项拟议的研究与癌症生物学直接相关，这项研究中所包含的实验的成功完成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.