Mechanisms of DNA helicases and their regulation

DNA解旋酶的机制及其调控

• 批准号: 10591506
• 负责人: Yann R. Chemla
• 金额: $ 37.15万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-15 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10591506
• 关键词: Archaea; Biochemistry; Cells; DNA; DNA Damage; DNA Repair; DNA Structure; Disease; Enzymes; Eukaryota; Fluorescence Microscopy; Genome; Goals; Human; Life; Maintenance; Malignant Neoplasms; Measurement; Modeling; Molecular; Molecular Conformation; Molecular Machines; Nucleic Acids; Organism; Pathology; Pathway interactions; Play; Process; Prokaryotic Cells; Proteins; RNA; Regulation; Research; Resolution; Role; Structural Models; System; Virus; Work; biophysical techniques; genome integrity; helicase; human disease; insight; laser tweezer; member; optic tweezer; recruit; response; single molecule

PROJECT SUMMARY / ABSTRACT “Mechanisms of DNA helicases and their regulation” Helicases are a ubiquitous and diverse group of molecular machines that separate the strands of nucleic acids. They are essential actors in many genome maintenance processes in all domains of life, including some viruses. As a result, helicases are biomedically important proteins, and their pathologies are associated with a number of human diseases and cancer. Since uncontrolled unwinding is detrimental to genomic integrity, helicase activity must be tightly regulated in the cell. Furthermore, since many helicases are able to play multiple, distinct roles in a variety of cellular pathways, they must be activated only in the correct contexts. How these different functions are defined and regulated remains poorly understood. In this project, we will investigate the molecular mechanisms by which DNA helicases are regulated. Our studies will focus on the model non-hexameric helicases UvrD, Rep, and XPD, which are critical components of the cellular response to DNA damage in prokaryotes, eukaryotes, and archaea and also serve as prototypical members of the two largest structural superfamilies of helicases. Insights gained on their mechanisms are expected to extend to a number of structurally and functionally homologous systems. Prior work by us and others has shown that these types of helicases have auxiliary domains and/or make secondary contacts with DNA that play regulatory—often, auto-inhibitory—roles. Protein partners to helicases have thus been proposed to activate helicase activity by controlling these mechanisms, thus defining helicase roles in the cell. To gain insights into these mechanisms, our studies will focus on two main research goals: understanding how interactions with DNA and non-canonical DNA structures control helicase activity (Goal 1), and quantifying how encounters with accessory proteins—both protein partners that recruit and activate helicases and proteins that compete for the same DNA substrates—regulate helicases (Goal 2). Our approach for achieving these research goals will integrate advanced single-molecule biophysical techniques—optical tweezers combined with fluorescence microscopy—together with traditional biochemistry and computational biophysics methods. These approaches leverage our group's expertise and that of the assembled collaborators, and have been successfully applied by us in our high-resolution measurements of helicase unwinding and conformational dynamics, their modulation by interactions with accessory proteins, and their connection to atomic-level structural models of helicases,. Beyond providing insights on helicase mechanism and the genome maintenance pathways in which they participate, our studies will advance new biophysical methods for investigating biomolecular dynamics.

项目摘要 /摘要 “ DNA解旋酶的机制及其调节” 解旋酶是一组无处不在的分子机器，可将核酸链分开。 它们是生命所有领域（包括某些病毒）的许多基因组维持过程中必不可少的参与者。 结果，解旋酶是生物医学上重要的蛋白质，它们的病理与数量有关 人类疾病和癌症。由于不受控制的放松剂对基因组完整性有害，因此解旋酶活性 必须在细胞中严格调节。此外，由于许多解旋酶能够扮演多个不同的角色 在多种细胞途径中，必须仅在正确的环境中激活它们。这些不同的功能如何 定义和监管仍然很少理解。 在这个项目中，我们将研究调节DNA解旋酶的分子机制。我们的 研究将重点放在模型的非尚面型解旋酶UVRD，REP和XPD上，它们是关键的组成部分 原核生物，真核生物和古细菌中对DNA损伤的细胞反应，也用作典型 两个最大的结构型超家族的成员。从他们的机制上获得的见解是 预计将扩展到许多结构和功能同源系统。 我们和其他人的先前工作表明，这些类型的解旋酶具有辅助域和/或制造 与播放调节性的DNA接触（通常是自动抑制性）。蛋白质伴侣的解旋酶 因此，已经提出通过控制这些机制来激活解旋酶活性，从而定义了解旋酶 在细胞中的作用。为了了解这些机制，我们的研究将重点介绍两个主要的研究目标： 了解与DNA和非典型DNA结构的相互作用如何控制解旋酶活性（目标1），， 并量化如何与辅助蛋白相遇 - 募集和激活的蛋白质伴侣 竞争相同DNA底物的解旋酶和蛋白质 - 调节解旋酶（目标2）。 我们实现这些研究目标的方法将整合先进的单分子生物物理 技术 - 光学镊子与荧光显微镜结合 - 与传统生物化学结合 和计算生物物理学方法。这些方法利用了我们小组的专业知识和 组装合作者，并已在我们的高分辨率测量中成功应用 解旋酶放松和构象动态，与附件蛋白质相互作用的调节，以及 它们与解旋酶的原子级结构模型的联系。除了提供有关解旋酶的见解之外 他们参与的机制和基因组维持途径，我们的研究将推进新的发展 研究生物分子动力学的生物物理方法。