Mechanisms of DNA helicases and their regulation

项目总结/摘要 “DNA解旋酶及其调控机制” 解旋酶是一种普遍存在的和多样化的分离核酸链的分子机器。 它们是生命所有领域（包括某些病毒）中许多基因组维持过程的重要参与者。 因此，解旋酶是生物医学上重要的蛋白质，并且它们的病理与许多疾病相关。 人类疾病和癌症。由于不受控制的解旋对基因组完整性有害，解旋酶活性 必须在细胞中受到严格的调控。此外，由于许多解旋酶能够发挥多种不同的作用， 在各种细胞途径中，它们必须仅在正确的环境中被激活。这些不同的功能 定义和监管仍然知之甚少。 在这个项目中，我们将研究DNA解旋酶调节的分子机制。我们 研究将集中在模型非六聚体解旋酶UvrD、Rep和XPD上，它们是 在原核生物、真核生物和古细菌中对DNA损伤细胞反应，也作为原型 解旋酶的两个最大的结构超家族的成员。对这些机制的了解如下 预计将扩展到许多结构和功能上同源的系统。 我们和其他人的先前工作已经表明，这些类型的解旋酶具有辅助结构域和/或使解旋酶的活性降低。 二次接触的DNA发挥调节作用，往往是自我抑制作用。解旋酶的蛋白质伴侣 因此提出通过控制这些机制来激活解旋酶活性，从而定义解旋酶 细胞中的角色。为了深入了解这些机制，我们的研究将集中在两个主要研究目标上： 理解与DNA和非规范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质相遇--这两种蛋白质伴侣都是招募和激活 解旋酶和竞争相同DNA底物的蛋白质调节解旋酶（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-光镊结合荧光显微镜-与传统的生物化学 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，并已成功地应用于我们的高分辨率测量， 解旋酶解旋和构象动力学，它们通过与辅助蛋白相互作用的调节，以及 它们与解旋酶的原子级结构模型的联系。除了提供解旋酶的见解 机制和它们参与的基因组维持途径，我们的研究将推进新的 研究生物分子动力学的生物物理方法。

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.