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.

摘要 高效的DNA修复是一把双刃剑。精确修复双核等有害DNA损伤 链状断裂、链间交联和受损的复制叉会促进基因组的稳定性。它还 使癌细胞获得更具侵袭性的特征，并对辐射和DNA产生抵抗力 破坏性的化疗药物。此外，不合时宜地部署和/或错误地调节DNA修复 机器可能会进一步破坏基因组的稳定(这可能会导致癌症)，或者可能导致堆积 有毒的修复中间体(这可能导致细胞死亡)。在我们对……的理解上仍然存在重大差距 分子事件将原本精确修复的中间产物输送到“无赖”的基因组中- 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3Rs)中，以及它是如何 在处理停滞的DNA复制事件和DNA断裂的分子途径中被挪用 通过高度诱变的基因组破坏稳定的机制。 我们的中心假设是RAD51重组酶，单链DNA结合蛋白RPA， 重组介体BRCA2(在人中)和Rad52(在酵母中)和DNA修复解旋酶被微调 受多种因素的影响，包括翻译后修饰、相互作用的伴侣蛋白、特定的DNA 结构和DNA损伤。这些因素影响蛋白质的构象动力学和关键蛋白质。 蛋白质界面。了解蛋白质的可塑性和大分子组装的动力学 DNA修复机器将向我们展示选择性地操纵RAD51和RAD51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、生物物理和化学生物学的工具。 我们对核蛋白复合体的形成、活性和调控的独特观点 编排重组植根于我们对单个人类DNA修复蛋白进行分类的能力 原生的翻译后修饰，并探索和分离与不同表面相关的活动- 单分子水平上的拴系蛋白和核蛋白复合体。我们的目标是提供一个完整的 关于细胞如何平衡可修复的分子机器的组装和活动的新前景， 但也会破坏基因组的稳定，并能够用新的抗癌化疗药物改变这种平衡。