Assembly and Dynamics of Molecular Machines in Genome Maintenance

摘要 DNA修复是一把双刃剑。准确修复这种有害的DNA损伤，如双- 链断裂、链间交联和受损的复制叉促进基因组稳定性。它还 使癌细胞获得更具侵略性的特征，并对辐射和DNA产生抵抗力。 有害的化疗药物此外，DNA修复的不及时部署和/或失调 机器可能会进一步破坏基因组的稳定性（这可能导致癌症），或者可能导致 有毒的修复中间体（可导致细胞死亡）。我们对以下问题的理解仍然存在重大差距： 分子事件，漏斗的中间，否则准确修复到“流氓”，基因组- 破坏稳定的机制 这项研究计划强调同源重组的分子机制，它是如何 整合到DNA复制，修复和重组（基因组稳定性的3R）中，以及它是如何 在分子途径中被挪用， 通过高度致突变的基因组不稳定机制。 我们的中心假设是，RAD 51重组酶，ssDNA结合蛋白RPA， 重组介体BRCA 2（人体）和Rad 52（酵母），以及DNA修复解旋酶被精细调节 通过多种因素，包括翻译后修饰，相互作用的伴侣蛋白，特异性DNA 结构和DNA损伤。这些因素影响蛋白质构象动力学和关键蛋白质- 蛋白质界面了解蛋白质可塑性和大分子组装动力学 DNA修复机器将向我们展示选择性操纵RAD 51活性的新方法， DNA复制和修复中的多功能DNA解旋酶。 我们正在利用和建立单分子生物化学，生物物理学和化学生物学的工具。 我们对核蛋白复合物的形成，活动和调节的独特观点 精心策划重组的根源在于我们能够利用其对单个人类DNA修复蛋白进行排序 天然的翻译后修饰，并探测和分离与不同的表面- 在单分子水平上的束缚蛋白和核蛋白复合物。我们的目标是提供一个完全 关于细胞如何平衡分子机器的组装和活动的新观点， 而且使基因组不稳定，并且能够用新的抗癌化疗剂改变这种平衡。

ABSTRACT Efficient DNA repair is a double-edged sword. Accurate repair of such deleterious DNA lesions as double- stranded breaks, inter-strand crosslinks, and damaged replication forks promotes genome stability. It also allows cancer cells to acquire a more aggressive character and develop resistance to radiation and DNA damaging chemotherapeutics. Additionally, untimely deployment and/or misregulation of the DNA repair machines may further destabilize the genome (which can lead to cancer) or may result in the accumulation of toxic repair intermediates (which can lead to cell death). Significant gaps remain in our understanding of the molecular events that funnel the intermediates of otherwise accurate repair into “rogue”, genome- destabilizing mechanisms. This research program emphasizes the molecular machinery of homologous recombination, how it is integrated into DNA replication, repair and recombination (the 3Rs of genome stability), and how it is misappropriated in the molecular pathways that process stalled DNA replication events and DNA breaks through highly mutagenic, genome destabilizing mechanisms. Our central hypothesis is that the activities of the RAD51 recombinase, the ssDNA-binding protein RPA, recombination mediators BRCA2 (in human) and Rad52 (in yeast), and DNA repair helicases are finely tuned by a variety of factors, which include posttranslational modifications, interacting partner proteins, specific DNA structures and DNA lesions. These factors affect the protein conformational dynamics and critical protein- protein interfaces. Understanding how the protein plasticity and kinetics of assembly of the macromolecular machines of DNA repair will show us new ways to selectively manipulate the activities of RAD51 and multifunctional DNA helicases in DNA replication and repair. We are leveraging and building the tools of single-molecule biochemistry, biophysics and chemical biology. Our unique perspective on the formation, activities and regulation of the nucleoprotein complexes orchestrating recombination is rooted in our ability to sort individual human DNA repair proteins with their native posttranslational modifications, and to probe and separate activities associated with different surface- tethered proteins and nucleoprotein complexes at the single-molecule level. Our goal is to provide an entirely new outlook on how the cell balances the assembly and activities of the molecular machines that can repair, but also destabilize, the genome, and to be able to alter this balance with new anticancer chemotherapeutics.