Mechanisms of DNA helicases and their regulation

项目摘要/摘要 《DNA解旋酶的机制及其调控》 解旋酶是一组无处不在的、多样化的分子机器，用于分离核酸链。 它们在生命各个领域的许多基因组维护过程中都是必不可少的参与者，包括一些病毒。 因此，解旋酶是生物医学上重要的蛋白质，它们的病理与许多 人类疾病和癌症。由于不受控制的解离不利于基因组的完整性，解旋酶活性 必须在细胞内受到严格控制。此外，由于许多解旋酶能够扮演多种不同的角色 在各种细胞通路中，它们必须仅在正确的环境中被激活。这些不同的功能是如何实现的 被定义和监管的人仍然知之甚少。 在这个项目中，我们将研究DNA解旋酶被调控的分子机制。我们的 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、真核生物和古生物中对DNA损伤的细胞反应，也是典型的 两个最大的解旋酶结构超家族的成员。关于它们的机制的见解是 预计将扩展到许多结构和功能上相同的系统。 我们和其他人之前的工作表明，这些类型的解旋酶具有辅助结构域和/或使 与DNA的二次接触，扮演着调节--通常是自动抑制--的角色。解旋酶的蛋白质伙伴 因此，有人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 在细胞中扮演的角色。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： 了解与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) 以及量化如何与辅助蛋白相遇--这两种蛋白都是招募和激活的蛋白质伙伴 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--光学镊子结合荧光显微镜--与传统生物化学相结合 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 组装的合作者，并已成功地应用于我们的高分辨率测量 解旋酶的解离和构象动力学，它们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。除了提供关于解旋酶的见解之外 机制和它们参与的基因组维持途径，我们的研究将取得新的进展 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.