Assembly and Dynamics of Molecular Machines in Genome Maintenance

基因组维护中分子机器的组装和动力学

• 批准号: 10593161
• 负责人: Maria Spies
• 金额: $ 38.43万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10593161
• 关键词: Affect; Aging; BRCA2 gene; Binding Proteins; Biochemistry; Biology; Biophysics; Cancer Etiology; Cell Death; Cells; Chemicals; Chromosomal Rearrangement; Complex; DNA; DNA Damage; DNA Repair; DNA Repair Gene; DNA Structure; DNA biosynthesis; DNA lesion; DNA replication fork; Defect; Disease; Equilibrium; Event; Fragile X Syndrome; Genetic; Genetic Recombination; Genome; Genome Stability; Goals; Human; Individual; Kinetics; Maintenance; Malignant Neoplasms; Mediator; Molecular; Molecular Machines; Myotonic Dystrophy; Nucleoproteins; Pathway interactions; Post-Translational Protein Processing; Process; Protein Conformation; Proteins; RAD52 gene; Rad51 recombinase; Radiation induced damage; Regulation; Research; Resistance development; Single-Stranded DNA; Sorting; Surface; Therapeutic; Yeasts; anti-cancer; anti-cancer therapeutic; cancer cell; crosslink; genome integrity; genotoxicity; helicase; homologous recombination; human DNA; macromolecular assembly; molecular dynamics; novel; programs; repaired; single molecule; tool

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.

抽象的 有效的DNA修复是一把双刃剑。精确修复此类已删除的DNA病变，例如双重 滞留的断裂，晶体间交联和损坏的复制叉可促进基因组稳定性。也是如此 允许癌细胞获得更具侵略性的特征和对辐射和DNA的发育抗性 有害的化学治疗药。另外，DNA修复不合时宜地部署和/或 机器可能会进一步破坏基因组（可能导致癌症），或者可能导致积累 有毒修复中间体（可能导致细胞死亡）。我们对 将其他精确修复的中间体有趣的分子事件变成“流氓”，基因组 - 破坏稳定的机制。 该研究计划强调了同源重组的分子机制 集成到DNA复制，修复和重组（基因组稳定性的3R），以及如何 在分子途径中被误解，该途径停滞了DNA复制事件和DNA断裂 通过高度诱变的基因组破坏了机制。 我们的中心假设是Rad51重组酶的活性，SSDNA结合蛋白RPA， 重组介质BRCA2（在人类）和RAD52（在酵母中），DNA修复解酶进行了细调 通过多种因素，包括翻译后修饰，相互作用的伴侣蛋白，特定的DNA 结构和DNA病变。这些因素会影响蛋白质构象动力学和关键蛋白质 - 蛋白质界面。了解大分子的蛋白质可塑性和动力学 DNA维修的机器将向我们展示新的方法，以选择性操纵Rad51和 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，活性和调节的独特观点 编排的重组植根于我们与他们的单个人类DNA修复蛋白分类的能力 天然翻译后修饰，并探测和分离与不同的表面相关的活动 单分子水平的束缚蛋白和核蛋白复合物。我们的目标是完全提供 细胞如何平衡可以修复的分子机器的组装和活动的新前景， 但也能使基因组稳定，并能够通过新的抗癌化学治疗剂改变这种平衡。