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）：该申请专注于含铁硫的（FES）解旋酶，这是一种突出的DNA解旋酶家族，其缺乏或失调与从癌症倾向到高血压的人类疾病有关。除了超家族II运动芯外，FES解旋酶还具有两个家庭特异性辅助域：FES群集域和一个拱形域。辅助DNA结合位点借助辅助结构域形成，该辅助结构域将解旋酶定位在取向以解开双链体，控制解旋酶速率并验证转运链的完整性中。我建议在FES解旋酶中开放和关闭的拱形域的频率调节其活动。我们将首次使用该解旋酶家族来测试外源性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。我们的目标是确定域迁移率控制三个FES Helicases的活性的机制，即XPD，FANCJ和RTEL1。为了实现这一目标，我们将使用一组协同的生化重构组和我实验室中开发的新型单分子方法。 目标1：确定拱形域移动性在控制XPD活动中的作用。我们将基于初步数据，表明同源DNA病变稳定了弓形的封闭构型。使用单分子总内反射荧光显微镜（TIRFM），我们将观察到与DNA相互作用时单个荧光标记的XPD分子的域运动。我们将学习拱形域运动如何控制XPD解旋酶的活动及其在疾病中的故障。 AIM 2：确定Arch域迁移率在FANCJ和RTEL1介导的DNA介导的DNA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域运动相关。我们还将学习与乳腺癌和法科诺尼贫血相关的FANCJ突变扰动FANCJ活动，拱形域迁移率以及区分无损害和无损害DNA的能力。 AIM 3：确定蛋白质伴侣如何调整FANCJ和RTEL1的活动。我们将检验以下假设：与关键蛋白伴侣（BRCA1肿瘤抑制蛋白，HMLH1不匹配修复蛋白和PCNA夹）相互作用，通过修改FANCJ和RTEL1的域迁移率来控制解旋酶和易位酶活性。总之，提出的三个目标的预期结果不仅会缩小对解旋酶如何调节不同生物化学活动的机械理解的差距，而且还确定了明确的策略以选择性调节它们。该信息将为设计范围或RTEL1抑制剂的设计铺平道路，以针对癌症和衰老相关疾病的特定方面。