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中的遗传突变导致具有不同的DNA水平的人类疾病。 反映癌症增加或细胞死亡增加的表型：着色性干皮病（XP），XP- 连锁Cockayne综合征（CS）和甲状腺营养不良（TTD）。这些疾病反映了 不同的细胞途径：缺陷核苷酸切除修复（NER）导致XP，干扰 转录偶联修复（TCR）导致CS，而转录异常与缺陷性 NER导致TTD。在人类中，XPB和XPD解旋酶是TFIIH十个亚基的一部分， 转录/修复复合物，但致病突变聚集在XPB，特别是XPD，而不是 比在其他TFIIH蛋白质中，除了TFB 5，所以这些XP解旋酶似乎是控制TFIIH蛋白质的关键。 转录和修复的协调。此外，修复蛋白XPG和CSB与蛋白质相互作用。 TCR中的XP解旋酶。然而，关于XPB和XPD在分子水平上的知识很少， 它们的解旋酶和修复活性，或它们与TFB 5、CSB和XPG的相互作用。我们的目标是 了解潜在的特异性，活性，构象控制和 通过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提供了关键的控制， 转录、NER和TCR。我们还提出，这些特征的表征及其 致病突变的破坏将提供一个分子基础， 基因突变到疾病表型。为了验证这一点，我们在此建议将结构和 生物物理实验（泰纳实验室）与生物化学和生物学实验（库珀 实验室）。我们对XPB和XPD结构域以及全长蛋白质、其古细菌的实验， 同源物及其关键组件将建立分子结构，构象转换， 机制和变构相互作用。我们希望能描述一组典型的解旋酶 结构，它们与DNA和蛋白质伴侣的复合物，并定义关键的相互作用， 他们的活动。所提出的跨学科实验的预期结果是一种分子 蛋白质-DNA复合物的图片，蛋白质-蛋白质相互作用和协调的功能状态 作为TFIIH组分的XPB和XPD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、TFB 5、CSB和XPG患者突变相关的死亡疾病表型。