Structural Biology of XPB and XPD Helicases

DNA解旋酶XPB和XPD的遗传性突变导致不同的人类疾病 反映癌症增加或细胞死亡增加的表型：着色性干皮病(XP)，XP- 连锁Cockayne综合征(CS)和毛发硫代营养不良(TTD)。这些疾病反映了 不同的细胞通路：核苷酸切除修复缺陷(NER)导致XP，心烦意乱 转录偶联修复(TCR)导致CS，转录异常合并缺陷 NER导致TTD。在人类中，XPB和XPD解旋酶是TFIIH十个亚基的一部分 转录/修复复合体，但致病突变聚集在XPB，特别是XPD 比在除TFB5以外的其他TFIIH蛋白中更多，因此这些XP解旋酶似乎是控制 转录和修复的协调。此外，修复蛋白XPG和CSB与 TCR中的XP解旋酶。然而，在分子水平上关于XPB和XPD的知识很少， 它们的解旋酶和修复活性，或它们与TFB5、CSB和XPG的相互作用。我们的目标是 了解潜在的特异性、活性、构象控制和分子特征 XPB和XPD解旋酶的途径协调。我们的假设是定义良好的架构， XPB和XPD解旋酶的构象状态和分子界面提供了关键的控制 转录、NER和TCR。此外，我们还提出，这些特征及其特征的表征 致病突变的破坏将提供直接连接遗传基因的分子基础 基因突变导致疾病表型。为了测试这一点，我们在此建议将结构和 生物物理实验(Tainer实验室)和生化和生物实验(库珀 实验室)。我们对XPB和XPD结构域以及它们的古细菌全长蛋白的实验 同系物及其关键组件将建立分子结构、构象交换 机理和变构相互作用。我们希望描述一组典型的解旋酶 结构，它们与DNA和蛋白质伙伴的络合物，并定义 他们的活动。拟议的跨学科实验的预期结果是一种分子 蛋白质-DNA复合体、蛋白质-蛋白质相互作用和协调的功能状态的图片 作为TFIIH组分的XPB和XPD介导的转录和修复事件。这些结果将 帮助提供对癌细胞和细胞基础过程的详细的分子理解 与XPB、XPD、TFB5、CSB和XPG患者突变相关的死亡疾病表型。

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.