Structural Biology of XPB and XPD Helicases

描述(申请人提供)：DNA解旋酶XPB和XPD的遗传突变会导致人类疾病，这些疾病具有不同的表型，反映癌症或衰老的增加：色素性干皮病(XP)、XP合并Cockayne综合征(CS)和毛硫代营养不良(TTD)。这些疾病反映了不同细胞通路的破坏：核苷酸切除修复(NER)、转录偶联修复(TCR)或转录。在人类中，XPB和XPD解旋酶是TFIIH转录/修复复合体10个亚基的一部分，但致病突变聚集在XPB，特别是XPD，而不是TFB5以外的其他TFIIH蛋白中，因此这些XP解旋酶似乎是控制转录和修复协调的关键。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的分子特征。我们的假设是，XPB和XPD解旋酶明确的结构、构象状态和分子界面为转录、NER和TCR提供了关键的控制。我们已经证明，这些特征的特征及其被致病突变的破坏提供了直接将遗传基因突变与疾病表型联系起来的分子基础。在我们XPB和XPD晶体结构的基础上，我们建议将结构和生物物理实验(包括小角X射线散射)与生化和生物实验相结合，以定义溶液中的构象和复合体，以确定与疾病相关的突变体、蛋白质-DNA复合体的结构，并确定其活性的关键相互作用。拟议的跨学科实验的预期结果是蛋白质-DNA复合体、蛋白质-蛋白质相互作用和功能状态的分子图像，这些功能状态协调由XPB和XPD作为TFIIH的组成部分介导的转录和修复事件。这些结果将有助于详细的分子理解与XP、XP/CS和TTD患者突变相关的癌症和细胞死亡疾病表型的基础过程。

DESCRIPTION (provided by applicant): Hereditary mutations in the DNA helicases XPB and XPD lead to human diseases with different phenotypes reflecting increased cancers or aging: xeroderma pigmentosum (XP), XP combined with Cockayne syndrome (CS), and trichothiodystrophy (TTD). These diseases reflect the disruption of different cellular pathways: nucleotide-excision repair (NER), transcription-coupled repair (TCR), or transcription. 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. 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 have shown that characterizations of these features and their disruption by disease-causing mutations provide a molecular basis to directly connect the inherited gene mutations to disease phenotypes. Building on our crystal structures of XPB and XPD, we propose to integrate structural and biophysical experiments including small angle x-ray scattering to define conformations and complexes in solution with biochemical and biological experiments to determine structures of disease-relevant mutants, protein-DNA complexes, and define 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 XP, XP/CS, and TTD patient mutations.