Functions of DNA helicases at hard-to-replicate sites and telomere regulation

难以复制位点 DNA 解旋酶的功能和端粒调控

• 批准号: 10488160
• 负责人: Roberto Galletto
• 金额: $ 39.29万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-13 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10488160
• 关键词: Address; Aging; Behavior; Biochemical; Cell Nucleus; Chromosomal Stability; Chromosomes; Complex; DNA; DNA Structure; DNA biosynthesis; DNA metabolism; DNA-Binding Proteins; Discrimination; Eukaryota; Excision; Family; Genomic Instability; Goals; Human; Individual; Length; Malignant Neoplasms; Methods; Mitochondria; Mutation; Nucleoproteins; Physical condensation; Play; Polymerase; Process; Property; Proteins; Regulation; Role; Site; Syndrome; System; Testing; Time; Work; Yeasts; base; biophysical techniques; helicase; novel; reconstitution; single molecule; synergism; telomere

Abstract The overall objective of this proposal is to answer two fundamental questions on DNA metabolism: 1) how DNA replication progresses timely and efficiently at hard-to-replicate sites; and 2) how the large nucleoprotein complexes that comprise telomeres, the end of linear chromosomes, are assembled and regulated. The first question emerges from our ongoing mechanistic studies on the conserved Pif1-family of helicases. Our work highlights the fundamental role that these helicases have in facilitating DNA replication at both secondary DNA structures and protein barriers. Going forward, we seek to address the following: what mechanisms couple the helicase activity to DNA synthesis by polymerases; how this synergy leads to removal of obstacles imparted by protein barriers encountered in the nucleus or in mitochondria; whether removal of obstacles is a general property of this class of helicases. The second question is novel and is based on our recent finding of new properties of both yeast and human telomere DNA binding proteins; namely, the ability of these proteins to interact with telomeric DNA repeats in alternative modes and to facilitate DNA condensation. Our goal is to test whether these properties play a role in establishing telomere length discrimination and accessibility. We tackle these problems by using biochemically reconstituted systems and employing a multipronged approach that integrates ensemble biochemical and biophysical techniques with single-molecule methods, thus providing unparalleled access to the behavior of individual molecules.

抽象的 该提议的总体目的是回答有关DNA的两个基本问题 代谢：1）DNA复制如何在难以复制的位点及时进展； 2）如何包含端粒的大核蛋白复合物，线性末端 染色体组装和调节。第一个问题来自我们正在进行的 关于保守的PIF1家庭解旋酶的机械研究。我们的工作突出了 这些解旋酶在促进两个次级的DNA复制中具有的基本作用 DNA结构和蛋白质屏障。展望未来，我们寻求解决以下内容：什么 机理将解旋酶活性与聚合酶合成DNA合成；这种协同作用 导致去除由核或中遇到的蛋白质屏障所赋予的障碍物 线粒体；消除障碍物是否是这类解旋酶的一般特性。这 第二个问题是新颖的，是基于我们最近发现的酵母新特性的发现 人端粒DNA结合蛋白；即，这些蛋白质与 端粒DNA在替代模式中重复并促进DNA缩合。我们的目标是 测试这些属性是否在建立端粒长度歧视和 可访问性。我们通过使用生化重构系统来解决这些问题， 采用一种多收缩的方法，该方法整合了集合生化和生物物理 具有单分子方法的技术，从而提供了对行为的无与伦比的访问 单个分子。