Regulation of unwinding and remodeling activities in FeS-DNA helicases

本申请集中于含铁-硫（FeS）解旋酶，其是一个突出的DNA解旋酶家族， 缺乏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。二级DNA结合位点是在 辅助结构域，其将解旋酶定位在解旋双链体的方向上，控制解旋酶速率， 并验证移位链的完整性。我建议， 而FeS解旋酶的关闭调节了它们的活性。我们将使用这个解旋酶家族来测试第一个 时间外源性因素如何影响机械化学的解旋酶通过调节 其核心和辅助域运动的频率。我们的目标是确定 结构域移动性控制三种FeS解旋酶XPD、FANCJ和RTEL 1的活性。实现这一 目的我们将使用一组协同的生化重组和新型单分子 在我的实验室里开发的方法。 目标1：确定XPD域移动性在控制XPD活动中的作用。我们将建立在我们的 初步数据显示同源DNA损伤稳定了ARCH的闭合构象。 利用单分子全内反射荧光显微镜（TIRFM）， 单个荧光标记的XPD分子与DNA相互作用时的运动。我们将学习如何 XPD解旋酶的结构域运动控制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解旋中的FANCJ结构域移动性的作用 和G-四链体的重塑完成这一目标后，我们将学习如何解旋酶和G- FANCJ和RTEL 1的四链体重塑活性与FANCJ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， 结构域迁移率和区分受损和无损伤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 假设与关键蛋白质伴侣（BRCA 1肿瘤抑制蛋白，hMLH 1错配）的相互作用 修复蛋白和增殖细胞核抗原钳）控制解旋酶和转位酶的活动，通过修改结构域的流动性， FANCJ和RTEL 1。 这三个拟议目标的预期结果不仅将缩小机械方面的差距， 了解解旋酶独特的生化活性是如何调节的，但也要确定明确的 有选择地调节它们的策略。这些信息将为设计抑制剂铺平道路。 FANCJ或RTEL 1用于靶向癌症和衰老相关疾病的特定方面。

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. Aim1: 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. Aim2: 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. Aim3: 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.