Regulation of unwinding and remodeling activities in FeS-DNA helicases

FeS-DNA 解旋酶解旋和重塑活性的调节

• 批准号: 9222028
• 负责人: Maria Spies
• 金额: $ 28万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9222028
• 关键词: ATP phosphohydrolase; Affect; Aging; Antineoplastic Agents; BRCA1 gene; Binding; Binding Sites; Biochemical; Cell physiology; Chemistry; Closure by clamp; Complex; DNA; DNA Binding; DNA Damage; DNA Maintenance; DNA Repair; DNA Structure; DNA lesion; Data; Defect; Diagnosis; Disease; Drug Targeting; Enzymes; Exogenous Factors; Family; Fanconi' s Anemia; Fluorescence Microscopy; Foundations; Frequencies; G-Quartets; Generations; Genetic Recombination; Genetic Transcription; Genome; Genomic Instability; HDAC1 gene; Hereditary Disease; Human Activities; Hypertension; Individual; Iron; Label; Learning; Life; Link; Maintenance; Malignant Neoplasms; Mediating; Methodology; Mismatch Repair; Molecular Conformation; Molecular Motors; Motion; Motor; Mutation; Nucleoproteins; Nucleotide Excision Repair; Positioning Attribute; Predisposition; Process; Progeria; Proteins; Regulation; Role; Site; Sulfur; Symptoms; Testing; Time; Trichothiodystrophy; Tumor Suppressor Proteins; design; genome integrity; helicase; human disease; inhibitor/antagonist; innovation; malignant breast neoplasm; mutant; novel; protein protein interaction; public health relevance; reconstitution; single molecule; synthetic construct; targeted treatment; translocase

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.

描述（由申请人提供）：本申请重点关注含铁硫 (FeS) 解旋酶，这是一种重要的 DNA 解旋酶家族，其缺陷或失调与从癌症易感性到高血压等人类疾病有关。除了超家族 II 运动核心之外，FeS 解旋酶还具有两个家族特异性辅助结构域：FeS 簇结构域和 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解旋双链体的方向，控制解旋酶速率，并验证易位链的完整性。我认为 FeS 解旋酶中 ARCH 结构域打开和关闭的频率可以调节它们的活性。我们将使用该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移动性控制三种 FeS 解旋酶 XPD、FANCJ 和 RTEL1 活性的机制。为了实现这一目标，我们将使用我的实验室开发的一套协同生化重组和新颖的单分子方法。 目标 1：确定 ARCH 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的基础上，这些数据表明同源 DNA 损伤稳定了 ARCH 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 XPD 分子与 DNA 相互作用时的域运动。我们将了解 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。 目标 2：确定 ARCH 结构域移动性在 FANCJ 和 RTEL1 介导的 G-四链体 DNA 解旋和重塑中的作用。完成此目标后，我们将了解 FANCJ 和 RTEL1 的解旋酶和 G 四链体重塑活动如何与 ARCH 结构域运动相关。我们还将了解与乳腺癌和范可尼贫血相关的 FANCJ 突变如何扰乱 FANCJ 活性、ARCH 结构域移动性以及区分受损和无损伤 DNA 的能力。 目标 3：确定蛋白质伙伴如何调节 FANCJ 和 RTEL1 的活性。我们将测试以下假设：与关键蛋白伙伴（BRCA1 肿瘤抑制蛋白、hMLH1 错配修复蛋白和 PCNA 钳）的相互作用通过改变 FANCJ 和 RTEL1 的结构域迁移性来控制解旋酶和转位酶活性。总之，这三个拟议目标的预期结果不仅将缩小对解旋酶独特生化活性如何调节的机制理解的差距，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 RTEL1 抑制剂的设计铺平道路，这些抑制剂可用于针对癌症和衰老相关疾病的特定方面。