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，反映癌症增加或细胞死亡增加：色素干皮病（XP）， XP相关的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：有缺陷的核苷酸切除修复（NER）导致XP，转录偶联修复（TCR）紊乱导致CS，转录异常结合有缺陷的NER导致TTD。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。此外，修复蛋白XPG和CSB在TCR中与XP解旋酶相互作用。然而，在分子水平上对XPB和XPD及其解旋酶和修复活性，以及它们与TFB5、CSB和XPG的相互作用知之甚少。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。我们进一步提出，这些特征的表征及其由致病突变引起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。为了验证这一点，我们建议将结构和生物物理实验（Tainer实验室）与生化和生物学实验（Cooper实验室）相结合。我们对XPB和XPD结构域和全长蛋白、它们的古细菌同源物及其关键组件的实验将建立分子结构、构象开关机制和变构相互作用。我们期望描述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。

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.