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中的遗传突变导致人类疾病，具有不同表型的人类疾病，反映了癌症增加或增加细胞死亡：Xeroderma chigmentosum（XP），XP连接的Cockayne综合征（CS）和Trichothiothiothiododododododyododydodyododydodydostrophy（TTD）。这些疾病反映了不同细胞途径的破坏：有缺陷的核苷酸 - 分离修复（NER）导致XP导致XP，扰动的转录耦合修复（TCR）导致CS，转录异常以及有缺陷的NER引起的TTD。在人类中，XPB和XPD解放酶是十个亚基TFIIH转录/修复复合物的一部分，但是引起疾病的突变集群在XPB，尤其是XPD中，而不是在其他TFB5蛋白中，除了TFB5以外的其他TFIIH蛋白质，因此这些XP Helicass似乎是控制转录和维修均衡的关键。此外，修复蛋白XPG和CSB与TCR中的XP解旋酶相互作用。但是，关于XPB和XPD，它们的解旋酶和修复活动或与TFB5，CSB和XPG的相互作用的分子水平上，知识很少。我们旨在了解XPB和XPD解旋酶的特异性，活性，构象控制和途径协调的基础分子特征。我们的假设是，XPB和XPD解酶的定义明确的结构，构象状态和分子界面为转录，NER和TCR提供了关键的控制。我们进一步提出，这些特征的特征及其因疾病突变而被破坏将提供分子基础，以将遗传基因突变与疾病表型直接连接起来。为了测试这一点，我们在此提议将结构和生物物理实验（Tainer Laboratory）与生化和生物学实验（Cooper Laboratory）相结合。我们对XPB和XPD结构域以及全长蛋白的实验，其古细菌同源物以及其关键组件将建立分子体系结构，构象切换机制和变构相互作用。我们希望表征一组原型的解旋酶结构，与DNA和蛋白质伴侣的复合物，并为其活动定义关键相互作用。拟议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，这些状态策划了由XPB和XPD作为TFIIH组成部分的转录和修复事件。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的过程提供详细的分子理解。