Regulation of unwinding and remodeling activities in FeS-DNA helicases

描述(申请人提供)：本申请重点研究含铁硫解旋酶(FES)解旋酶，这是一个重要的DNA解旋酶家族，其缺乏或失调与从癌症易感性到高血压等人类疾病有关。除了超家族II运动核心外，FES解旋酶还具有两个家族特有的辅助结构域：FES簇区和ARCH结构域。在辅助结构域的帮助下形成的二级DNA结合位点将解旋酶定位在一个方向以解开双链，控制解旋酶的速率，并验证转移链的完整性。我认为FES解旋酶的ARCH结构域的开启和关闭的频率调节了它们的活性。我们将利用这个解旋酶家族首次测试外源因素如何通过调节其核心和辅助结构域的运动频率来影响解旋酶的机械力化学。我们的目标是确定结构域迁移率控制三种FES解旋酶XPD、FANCJ和RTEL1活性的机制。为了实现这一目标，我们将使用一套协同的生化重组和在我的实验室开发的新颖的单分子方法。目的1：确定ARCH结构域迁移率在控制XPD活性中的作用。我们将以我们的初步数据为基础，表明同源DNA损伤稳定了拱门的闭合构象。使用单分子全内反射荧光显微镜(TIRFM)，我们将观察单个荧光标记的XPD分子与DNA相互作用时的结构域运动。我们将学习ARCH结构域运动如何控制XPD解旋酶的活性及其在疾病中的功能障碍。目的：探讨ARCH结构域迁移率在FANCJ和RTEL1介导的G-四链DNA解离和重塑中的作用。在完成这一目标后，我们将了解FANCJ和RTEL1的解旋酶和G-四链重塑活动如何与ARCH结构域运动相关。我们还将了解与乳腺癌和Fanconi贫血相关的FANCJ突变如何扰乱FANCJ的活性、ARCH结构域的流动性以及区分受损和无损伤DNA的能力。目的3：确定蛋白质伙伴如何调节FANCJ和RTEL1的活性。我们将检验这一假设，即与关键蛋白质伙伴(BRCA1肿瘤抑制蛋白、hMLH1错配修复蛋白和增殖细胞核抗原钳)的相互作用通过改变FANCJ和RTEL1的结构域迁移率来控制解旋酶和易位酶的活性。总之，这三个拟议目标的预期结果不仅将弥合对解旋酶不同生化活动如何调节的机械性理解方面的差距，而且还将确定有选择地调节它们的明确策略。这些信息将为FANCJ或RTEL1抑制剂的设计铺平道路，以用于针对癌症和衰老相关疾病的特定方面。

DESCRIPTION (provided by applicant): This application focuses on iron-sulfur containing (FeS) helicases, a prominent DNA helicase family whose deficiency or dysregulation is linked to human diseases ranging from cancer predisposition to hypertension. In addition to the Superfamily II motor core, FeS helicases possess two family specific auxiliary domains: an FeS cluster domain and an ARCH domain. The secondary DNA binding site formed with the help of the auxiliary domains which positions the helicase in an orientation to unwind duplex, controls the helicase rate, and verifies the integrity of the translocating strand. I propose that the frequency of ARCH domain opening and closing in FeS helicases modulates their activities. We will use this helicase family to test for the first time how the exogenous factors affect the mechano-chemistry of the helicases through modulating the frequency of its core and auxiliary domains motions. Our objective is to determine the mechanism by which the domain mobility controls the activities of three FeS helicases, XPD, FANCJ and RTEL1. To achieve this objective we will use a synergistic set of biochemical reconstitutions and novel single-molecule methodologies developed in my lab. Aim 1: Determine the role of ARCH domain mobility in controlling XPD activities. We will build on our preliminary data showing that the cognate DNA lesions stabilize the closed conformation of the ARCH. Using single-molecule total internal reflection fluorescence microscopy (TIRFM), we will observe domain motions of individual fluorescently labeled XPD molecules as they interact with DNA. We will learn how ARCH domain motions control activities of XPD helicase and its malfunction in disease. Aim 2: Determine the role of ARCH domain mobility in FANCJ and RTEL1 mediated DNA unwinding and remodeling of G-quadruplexes. Upon completion of this aim we will learn how the helicase and G-quadruplex remodeling activities of FANCJ and RTEL1 correlate with ARCH domain motions. We will also learn how FANCJ mutations associated with breast cancer and Fanconi Anemia perturb FANCJ activities, ARCH domain mobility and the ability to discriminate between damaged and damage-free DNA. Aim 3: Determine how protein partners tune the activities of FANCJ and RTEL1. We will test the hypothesis that interactions with key protein partners (BRCA1 tumor suppressor protein, hMLH1 mismatch repair protein and PCNA clamp) govern helicase and translocase activities by modifying domain mobility of FANCJ and RTEL1. Together, the anticipated results of the three proposed aims will not only close the gaps in the mechanistic understanding of how helicases' distinct biochemical activities are regulated, but also identify explicit strategies to selectively modulate them. This information will pave the way for the design of inhibitors of FANCJ or RTEL1 to be used to target specific aspects of cancer and aging related diseases.