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 高效 DNA 修复是一把双刃剑，可准确修复双刃剑等有害 DNA 损伤。 链断裂、链间交联和受损的复制叉也能促进基因组稳定性。 使癌细胞获得更具攻击性的特征并产生对辐射和 DNA 的抵抗力 此外，DNA 修复的不及时部署和/或错误调节。 机器可能会进一步破坏基因组的稳定性（这可能导致癌症）或可能导致积累 我们对有毒修复中间体（可能导致细胞死亡）的理解仍然存在重大差距。 将原本精确修复的中间产物汇集到“流氓”、基因组中的分子事件 不稳定机制。 该研究计划强调同源重组的分子机制，它是如何进行的 整合到 DNA 复制、修复和重组（基因组稳定性的 3R）中，以及它是如何实现的 在处理停滞的 DNA 复制事件和 DNA 断裂的分子途径中被盗用 通过高度诱变、基因组不稳定机制。 我们的中心假设是 RAD51 重组酶、ssDNA 结合蛋白 RPA 的活性， 重组介质 BRCA2（人类）和 Rad52（酵母）以及 DNA 修复解旋酶均经过微调 通过多种因素相互作用，包括翻译后修饰、伴侣蛋白、特定 DNA 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。 我们正在利用和构建单分子生物化学、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、活性和调节的独特视角 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 天然翻译后修饰，并探测和分离与不同表面相关的活性 我们的目标是在单分子水平上提供完整的束缚蛋白和核蛋白复合物。 关于细胞如何平衡可修复分子机器的组装和活动的新观点， 但也会破坏基因组的稳定，并能够通过新的抗癌化疗药物改变这种平衡。