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。 与公共健康相关：XP解旋酶处于癌症和老龄化的十字路口。作为TFIIH复合体的成员，它们既参与维持遗传密码完整性的DNA修复，也参与执行该密码的转录。这种机器的缺陷，小到一个氨基酸的变化，就会对人类健康造成后果。XP解旋酶的命名是因为它们在着色性干皮病(XP)中所起的作用，这种疾病的特征是极端的皮肤癌易感性，患有这种疾病的儿童被称为“月亮的孩子”。虽然XP是最常见的，但XPB和XPD的缺陷以及其他几个基因会导致另外两种不会增加癌症风险的疾病。Cockayne综合征(CS)和毛发硫代营养不良(TTD)是一种具有严重神经功能缺陷的早衰性疾病。这项研究的目的是了解XP解旋酶正常活性的分子基础以及它们在与XP解旋酶缺陷相关的人类疾病表型中的分解。

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. PUBLIC HEALTH RELEVANCE: The XP helicases sit at the crossroads of cancer and aging. As members of the TFIIH complex, they participate in both DNA repair that maintains the integrity of the genetic code, and transcription that executes that code. Defects in this machinery, as small as a single amino acid change, have consequences for human health. The XP helicases are named for their role in Xeroderma pigmentosum (XP) a disease characterized by such extreme skin cancer predisposition that children with this disease are called "children of the moon." Although XP is the most common, defects in XPB and XPD, along with a handful of other genes, cause two other diseases that do not have increased cancer risk. Cockayne Syndrome (CS) and trichothiodystrophy (TTD) are premature aging diseases with profound neurological defects. The goal of this research is to understand the molecular basis for the normal XP helicase activities and for their breakdown in human disease phenotypes associated with defects in the XP helicases.