Structural Biology of XPB and XPD Helicases

XPB 和 XPD 解旋酶的结构生物学

• 批准号: 7096103
• 负责人: John A. Tainer
• 金额: $ 30.73万
• 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 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7096103
• 关键词: DNA; gene mutation; helicase; human; phenotype; proteins; structural biology

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：DNA 解旋酶 XPB 和 XPD 中的遗传性突变会导致具有不同表型的人类疾病，反映癌症增加或细胞死亡增加：着色性干皮病 (XP)、XP 相关科凯恩综合征 (CS) 和毛发硫营养不良 (TTD) ）。这些疾病反映了不同细胞途径的破坏：缺陷性核苷酸切除修复 (NER) 导致 XP，扰动的转录偶联修复 (TCR) 导致 CS，转录异常与缺陷性 NER 相结合导致 TTD。在人类中，XPB 和 XPD 解旋酶是 10 个亚基 TFIIH 转录/修复复合体的一部分，但致病突变集中在 XPB，特别是 XPD，而不是其他 TFIIH 蛋白（TFB5 除外），因此这些 XP 解旋酶似乎是控制协调的关键转录和修复。此外，修复蛋白 XPG 和 CSB 与 TCR 中的 XP 解旋酶相互作用。然而，在分子水平上，人们对 XPB 和 XPD、它们的解旋酶和修复活性或它们与 TFB5、CSB 和 XPG 的相互作用知之甚少。我们的目标是了解 XPB 和 XPD 解旋酶的特异性、活性、构象控制和途径协调背后的分子特征。我们的假设是 XPB 和 XPD 解旋酶的明确结构、构象状态和分子界面为转录、NER 和 TCR 提供了关键控制。我们还提出，这些特征的表征以及致病突变对它们的破坏将为直接将遗传基因突变与疾病表型联系起来提供分子基础。为了测试这一点，我们在此建议将结构和生物物理实验（泰纳实验室）与生化和生物实验（库珀实验室）结合起来。我们对 XPB 和 XPD 结构域以及全长蛋白质、它们的古细菌同源物以及它们的关键组装体的实验将建立分子结构、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，并定义它们活性的关键相互作用。所提出的跨学科实验的预期结果是蛋白质-DNA 复合物、蛋白质-蛋白质相互作用和功能状态的分子图景，这些功能状态协调由 XPB 和 XPD 作为 TFIIH 组成部分介导的转录和修复事件。这些结果将有助于对与 XPB、XPD、TFB5、CSB 和 XPG 患者突变相关的癌症和细胞死亡疾病表型背后的过程提供详细的分子理解。