Regulation of unwinding and remodeling activities in FeS-DNA helicases

描述（由申请人提供）：本申请关注含铁硫（FeS）解旋酶，这是一个重要的DNA解旋酶家族，其缺陷或失调与从癌症易感性到高血压的人类疾病有关。除了超家族II马达核心之外，FeS解旋酶具有两个家族特异性辅助结构域：FeS簇结构域和FeS簇结构域。在辅助结构域的帮助下形成的二级DNA结合位点，其将解旋酶定位在解旋双链体的方向上，控制解旋酶速率，并验证易位链的完整性。我建议，在FeS解旋酶中的α-结构域的开放和关闭的频率调节它们的活动。我们将首次使用这个解旋酶家族来测试外源因素如何通过调节其核心和辅助结构域运动的频率来影响解旋酶的机械化学。我们的目标是确定结构域的流动性控制三个FeS解旋酶，XPD，FANCJ和RTEL 1的活动的机制。为了实现这一目标，我们将使用一套协同的生化重组和新的单分子方法在我的实验室开发。 目的1：确定XPD结构域迁移率在控制XPD活性中的作用。我们将建立在我们的初步数据显示，同源DNA病变稳定的ARCH. Using单分子全内反射荧光显微镜（TIRFM）的封闭构象，我们将观察域运动的个别荧光标记的XPD分子，因为它们与DNA相互作用。我们将了解XPD解旋酶的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2：确定FANCJ和RTEL 1介导的DNA解旋和G-四链体重构中的FANCJ和RTEL 1结构域迁移的作用。在完成这一目标后，我们将了解FANCJ和RTEL 1的解旋酶和G-四链体重塑活动如何与β结构域运动相关。我们还将了解与乳腺癌和范可尼贫血相关的FANCJ突变如何干扰FANCJ活性，FANCJ结构域移动性以及区分受损和无损伤DNA的能力。 目的3：确定蛋白质伴侣如何调节FANCJ和RTEL 1的活性。我们将测试的假设，即与关键蛋白质合作伙伴（BRCA 1肿瘤抑制蛋白，hMLH 1错配修复蛋白和PCNA钳）的相互作用，通过修改FANCJ和RTEL 1的结构域的流动性管理解旋酶和转位酶的活动。总的来说，这三个目标的预期结果不仅将缩小解旋酶独特的生物化学活性是如何调节的机械理解方面的差距，而且还将确定选择性调节它们的明确策略。这些信息将为FANCJ或RTEL 1抑制剂的设计铺平道路，用于靶向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.