Recombination/Repair Complex in Human Cells

描述（由申请人提供）：人细胞中的增殖修复复合物广泛的基因组不稳定性是人肿瘤的标志。基因组不稳定性最常见的表现是大规模的染色体重排。这种染色体重排可导致杂合性丢失（洛）或新的基因融合，所述基因融合包括已知含有致癌基因和/或肿瘤抑制基因的染色体区域。部分同源序列之间的重排修复（同源重组或单链退火）是许多这些基因组重排事件的常见DMA特征，通常被错配修复机制抑制。这项研究的长期目标是了解在人类肿瘤发生过程中导致染色体畸变的生物物理和分子过程及其对治疗药物耐药性的贡献。在上一个授权期间，我们详细介绍了基础重组蛋白hRAD 51的独特生化功能，hXRCC 2和hRAD 51 D以及hMSH 2-hMSH 6和hMSH 4-hXRCC 2和hRAD 51 D的生物化学作用的局限性和令人惊讶的地方。此外，我们详细描述了这些DNA修复蛋白与许多其他DNA修复组分的单独物理相互作用，这些DNA修复组分构成了一个非常大的重组修复蛋白质组（Proteome）虽然我们的生物物理学研究的意义已经显而易见，但复杂蛋白质相互作用的生理相关性仍然是个谜。我们的生物物理学研究已经产生了控制蛋白质相互作用和/或功能的ATP结合蛋白（A蛋白）分子开关的概念。当与充分研究的原型细菌RecA蛋白相比时，关于人RecA同源物（hRAD 51、hXRCC 2、hRAD 51 D）的分子开关功能出现了许多重要的问题。在这一更新补助金申请中，我们建议通过以下方式增强我们对人类细胞中重组修复的理解：I.生物物理学表征的RAD 51-parabolic分子开关，II.）用组蛋白-DNA底物对hRAD 51-parasitic活性的生物物理分析，III.）建立阻止由RR和IV启动的同源异型（HOER）和/或单链退火（SSA）重组事件的MMR机制。）体内和体外人RR蛋白质组的检查。这些研究很可能将大大有助于理解维持人类染色体稳定性的过程，并为设计更有效的治疗方法以及旨在控制人类肿瘤中发现的染色体不稳定性的预防性治疗方式提供框架

DESCRIPTION (provided by applicant): Recombination Repair Complex in Human Cells Widespread genomic instability is a hallmark of human tumors. The most frequent manifestations of genomic instability are large-scale chromosomal rearrangements. Such chromosomal rearrangements can lead to Loss-of-Heterozygosity (LOH) or novel gene fusions encompassing chromosome regions known to contain oncogenes and/or tumor suppressor genes. Recombination repair between partially homologous sequences (homeologous recombination or single-stranded annealing) is a common DMA signature of many of these genomic rearrangement events and is normally suppressed by the mismatch repair machinery. The long-term goal of this research is to understand the biophysical and molecular processes that lead to chromosomal aberrations during human tumorigenesis and their contribution to therapeutic drug resistance. In the last granting period we detailed unique biochemical functions of the fundamental recombination proteins hRAD51, hXRCC2 and hRAD51D as well as both the limitations and surprises to the biochemical role(s) of hMSH2-hMSH6 and hMSH4-hMSH5 in recombination In addition we detailed the individual physical interaction of these DMA repair proteins with numerous other DMA repair components that make up a very large recombination repair complex (Proteome). While the significance of our biophysical studies has been readily apparent, the physiological relevance of complex protein interaction has remained enigmatic. Our biophysical studies have engendered the concept of an ATP-binding protein (A-protein) molecular switch that controls protein interaction(s) and/or function(s). A number of important questions have arisen regarding molecular switch function(s) of the human RecA homplogs (hRAD51, hXRCC2, hRAD51D) when compared with the well-studied prototypical bacterial RecA protein. In this renewal grant application we propose to enhance our understanding of recombination repair in human cells via: I.) Biophysical characterization of the RAD51-paralog molecular switch, II.) Biophysical analysis of hRAD51-paralog activities with histone-DNA substrates, III.) Establish the MMR mechanism(s) that deters homeologous (HOER) and/or single-stranded annealing (SSA) recombination events initiated by RR, and IV.) Examination of the human RR Proteome in vivo and in vitro. It is likely that these studies will significantly contribute to understanding the processes involved in maintaining human chromosome stability and should provide a framework for the design of more efficacious therapeutic methodologies as well as preventive therapeutic modalities designed to control the chromosome instability found in human tumors