Structural Biology of XPB and XPD Helicases

描述（由申请人提供）：DNA解旋酶XPB和XPD中的遗传突变导致具有不同表型的人类疾病，反映了癌症或衰老的增加：着色性干皮病（XP）、XP合并Cockayne综合征（CS）和甲状腺营养不良（TTD）。这些疾病反映了不同细胞途径的破坏：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解旋酶是十个亚基TFIIH转录/修复复合物的一部分，但致病突变聚集在XPB中，特别是XPD中，而不是在除TFB 5之外的其他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。我们的目的是了解潜在的特异性，活性，构象控制和途径协调的XPB和XPD解旋酶的分子特征。我们的假设是，明确定义的架构，构象状态，和XPB和XPD解旋酶的分子界面提供了关键的控制转录，NER和TCR。我们已经表明，这些特征的表征和致病突变对它们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。在我们的XPB和XPD晶体结构的基础上，我们建议整合结构和生物物理实验，包括小角X射线散射，以确定溶液中的构象和复合物，生物化学和生物学实验，以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。所提出的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像，所述功能状态协调由XPB和XPD作为TFIIH的组分介导的转录和修复事件。这些结果将有助于提供一个详细的分子理解的过程，基础上的癌症和细胞死亡疾病的表型与XP，XP/CS和TTD患者突变。 公共卫生相关性：XP解旋酶位于癌症和衰老的十字路口。作为TFIIH复合体的成员，它们参与维持遗传密码完整性的DNA修复和执行该密码的转录。这种机制的缺陷，小到单个氨基酸的变化，都会对人类健康产生影响。XP解旋酶因其在着色性干皮病（XP）中的作用而命名，着色性干皮病是一种以极端皮肤癌易感性为特征的疾病，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，沿着其他一些基因，会导致另外两种不会增加癌症风险的疾病。Cockayne综合征（CS）和甲状腺营养不良（TTD）是具有严重神经缺陷的过早衰老疾病。本研究的目的是了解正常XP解旋酶活性的分子基础，以及在与XP解旋酶缺陷相关的人类疾病表型中其分解的分子基础。

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.