Structural Biology of XPB and XPD Helicases

XPB 和 XPD 解旋酶的结构生物学

• 批准号: 7563283
• 负责人: John A. Tainer
• 金额: $ 29.84万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7563283
• 关键词: ATP phosphohydrolase; Architecture; Biochemical; Biochemistry; Biological; Biological Process; Biophysics; Cell Death; Cockayne Syndrome; Complex; DNA; DNA Damage; DNA Repair; Defect; Disease; ERCC3 gene; Electrons; Eukaryota; Event; Figs - dietary; Gene Mutation; Genetic Transcription; Germ-Line Mutation; Homologous Gene; Human; In Vitro; Inherited; Knowledge; Laboratories; Lead; Length; Link; Malignant Neoplasms; Maps; Mediating; Mental Retardation; Microscopic; Molecular; Molecular Conformation; Molecular Structure; Mutation; Nerve Degeneration; Nucleotide Excision Repair; Outcome; Pathway interactions; Patients; Phenotype; Predisposition; Premature aging syndrome; Process; Proteins; Repair Complex; Resolution; Role; Skin Cancer; Specificity; Structure; Structure-Activity Relationship; Syndrome; Testing; Transcription Initiation; Transcription-Coupled Repair; Trichothiodystrophy; XPGC protein; Xeroderma Pigmentosum; base; cancer cell; clinical phenotype; disease phenotype; disease-causing mutation; helicase; human disease; in vivo; mutant; protein protein interaction; repaired; research study; structural biology; transcription factor TFIIH

Hereditary mutations in the DNA helicases XPB and XPD lead to human diseases with different phenotypes reflecting increased cancers or increased cell death: xeroderma pigmentosum (XP), XP- linked Cockayne syndrome (CS), and trichothiodystrophy (TTD). These diseases reflect the disruption of different cellular pathways: Defective nucleotide-excision repair (NER) results in XP, perturbed transcription-coupled repair (TCR) leads to CS, and transcription abnormalities combined with defective NER cause TTD. In humans, XPB and XPD helicases are part of the ten subunit TFIIH transcription/repair complex, but disease-causing mutations cluster in XPB and particularly XPD rather than in the other TFIIH proteins, excepting TFB5, so these XP helicases appear key to controlling coordination of transcription and repair. Furthermore, the repair proteins XPG and CSB interact with the XP helicases in TCR. However, there is little knowledge at the molecular level about XPB and XPD, their helicase and repair activities, or their interactions with TFB5, CSB and XPG. We aim to understand the molecular features underlying the specificity, activity, conformational controls and pathway coordination by the XPB and XPD helicases. Our hypothesis is that well-defined architectures, conformational states, and molecular interfaces of XPB and XPD helicases provide critical controls for transcription, NER, and TCR. We furthermore propose that characterizations of these features and their disruption by disease-causing mutations will provide a molecular basis to directly connect the inherited gene mutations to disease phenotypes. To test this, we herein propose to integrate structural and biophysical experiments (Tainer laboratory) with biochemical and biological experiments (Cooper laboratory). Our experiments on XPB and XPD domains and full-length proteins, their archaeal homologues, and their key assemblies will establish molecular architectures, conformational switching mechanisms, and allosteric interactions. We expect to characterize a prototypical set of helicase structures, their complexes with DNA and with protein partners, and to define the key interactions for their activities. The anticipated outcome of the proposed cross-disciplinary experiments is a molecular picture of the protein-DNA complexes, protein-protein interactions and functional states that orchestrate transcription and repair events mediated by XPB and XPD as components of TFIIH. These results will help provide a detailed molecular understanding of the processes that underlie the cancer and cell death disease phenotypes associated with XPB, XPD, TFB5, CSB and XPG patient mutations.

DNA 解旋酶 XPB 和 XPD 的遗传性突变会导致不同类型的人类疾病。 反映癌症增加或细胞死亡增加的表型：着色性干皮病 (XP)、XP- 与科凯恩综合征（CS）和毛发硫营养不良（TTD）相关。这些疾病反映了 不同的细胞途径：缺陷性核苷酸切除修复 (NER) 导致 XP，受到干扰 转录偶联修复 (TCR) 导致 CS，转录异常与缺陷相结合 NER 导致 TTD。在人类中，XPB 和 XPD 解旋酶是十个亚基 TFIIH 的一部分 转录/修复复合物，但致病突变集中在 XPB，尤其是 XPD 中 比其他 TFIIH 蛋白（TFB5 除外）中的解旋酶更重要，因此这些 XP 解旋酶似乎是控制 转录和修复的协调。此外，修复蛋白 XPG 和 CSB 与 TCR 中的 XP 解旋酶。然而，人们对XPB和XPD在分子水平上的了解却很少， 它们的解旋酶和修复活性，或它们与 TFB5、CSB 和 XPG 的相互作用。我们的目标是 了解特异性、活性、构象控制和基础的分子特征 XPB 和 XPD 解旋酶的途径协调。我们的假设是定义良好的架构， XPB 和 XPD 解旋酶的构象状态和分子界面为 转录、NER 和 TCR。我们进一步建议这些特征及其特征的表征 致病突变的破坏将为直接连接遗传性提供分子基础 基因突变导致疾病表型。为了测试这一点，我们在此建议整合结构和 生物物理实验（泰纳实验室）与生化和生物实验（库珀 实验室）。我们对 XPB 和 XPD 结构域以及全长蛋白质及其古菌的实验 同系物及其关键组装将建立分子结构、构象转换 机制和变构相互作用。我们期望表征一组典型的解旋酶 结构、它们与 DNA 和蛋白质伙伴的复合物，并定义关键的相互作用 他们的活动。拟议的跨学科实验的预期结果是分子 蛋白质-DNA 复合物、蛋白质-蛋白质相互作用以及协调的功能状态的图片 XPB 和 XPD 作为 TFIIH 的组成部分介导的转录和修复事件。这些结果将 帮助提供对癌症和细胞基础过程的详细分子理解 与 XPB、XPD、TFB5、CSB 和 XPG 患者突变相关的死亡疾病表型。