Structural Biology of XPB and XPD Helicases

描述（由申请人提供）：DNA解旋酶XPB和XPD的遗传突变导致不同表型的人类疾病，反映癌症增加或衰老：色素干皮病（XP）， XP合并Cockayne综合征（CS）和毛硫营养不良（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/修复复合体的10个亚基的一部分，但致病突变集中在XPB，特别是XPD中，而不是在除TFB5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。我们的目的是了解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。我们已经证明，这些特征的特征及其由致病突变引起的破坏为直接将遗传基因突变与疾病表型联系起来提供了分子基础。基于我们的XPB和XPD晶体结构，我们建议结合结构和生物物理实验，包括小角度x射线散射来确定溶液中的构象和复合物，以及生化和生物学实验来确定疾病相关突变体、蛋白质- dna复合物的结构，并确定其活性的关键相互作用。该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谱，这些蛋白质- dna复合物、蛋白质-蛋白质相互作用和功能状态是由XPB和XPD作为TFIIH的组成部分介导的转录和修复事件。这些结果将有助于对与XP、XP/CS和TTD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。

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.