Investigating the role of XPB helicase in DNA nucleotide excision repair

研究 XPB 解旋酶在 DNA 核苷酸切除修复中的作用

• 批准号: 9544993
• 负责人: Li Fan
• 金额: $ 26.46万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9544993
• 关键词: Amino Acid Substitution; Amino Acids; Archaeoglobus fulgidus; B-DNA; Bacteria; Base Pairing; Biochemical; Biological Assay; Biological Process; C-terminal; Cell Culture Techniques; Cells; Chemicals; Chemistry; Cockayne Syndrome; Collaborations; Complex; Crystallization; DNA; DNA Damage; DNA Repair; DNA Repair Pathway; DNA lesion; Defect; Disease; ERCC1 gene; Frameshift Mutation; Genes; Genetic Transcription; Genomic Instability; Goals; Healthcare; Hereditary Disease; Homologous Gene; Human; Image; Imaging technology; In Vitro; Inherited; Lead; Lesion; Letters; Light; Link; Literature; Medical; Methods; Molecular; Molecular Conformation; Monitor; Mutagenesis; Mutation; N-terminal; Nerve Degeneration; Nucleotide Excision Repair; Pathogenicity; Phenotype; Phosphorylation; Play; Postdoctoral Fellow; Predisposition; Premature aging syndrome; RNA chemical synthesis; Reaction; Recombinant Proteins; Recovery; Regulation; Research; Roentgen Rays; Role; Site; Skin Cancer; Solubility; Structural Models; Structure; Structure-Activity Relationship; Surgical incisions; TYRP1 gene; Testing; Transcription Initiation; Trichothiodystrophy; Universities; Work; Xeroderma Pigmentosum; base; biophysical analysis; disease phenotype; ds-DNA; endonuclease; experimental study; helicase; high risk; human disease; insight; medical schools; melting; mutant; novel; promoter; protein B; public health relevance; pyrimidine-pyrimidone dimer; recruit; response; transcription factor; transcription factor TFIIH

 DESCRIPTION (provided by applicant): Xeroderma pigmentosum (XP) group B (XPB) protein is a DNA helicase required for both transcription and nucleotide excision repair (NER), a major DNA repair pathway that removes a variety of DNA lesions. Mutations in the XPB gene are associated with skin cancer predisposition, premature aging, and neurodegeneration. In order to solve two long-standing problems concerning NER: how XPB initiates dsDNA unwinding at the damage site and how the opening of damaged DNA is coordinated with the dual incision, we plan to achieve the following three specific aims: Aim1: To dissect the mechanism of DNA unwinding by XPB during NER. We will determine the crystal structures of human XPB in complex with dsDNA and DNA with a bubble containing a lesion such as (6-4) pyrimidine-pyrimidone dimer. The dynamic XPB-DNA interactions and functional conformations of XPB will be further analyzed by small angle X-ray scattering (SAXS) experiments. These structural models will be validated by selective mutagenesis for in vitro biochemical assays and cell-based imaging experiments to test the role of XPB during NER in living cells. Aim2: To illustrate the XPB-XPF interaction that regulates the 5'-incison by endonuclease ERCC1-XPF. XPB plays a role in regulating the dual incision since phosphorylation at residue Ser751 of XPB inhibits the 5'- incison of the damaged DNA by ERCC1-XPF. We have purified a complex of human XPB C-terminal half with the N-terminal half of XPF for structural and functional analyses. A novel hypothesis how phosphorylation at Ser751 of XPB inhibits 5'-incision by XPF will be tested by selective mutagenesis for in vitro biochemical assays and cell-based imaging experiments for damage incision in the living cells. Aim3: To illustrate the structural and functional changes induced by disease-causing xpb mutations. There are only three amino acid substitution mutations identified so far in the literatures: F99S mutation causes XP, T119P mutation causes Trichothiodystrophy (TTD), and XP11BE mutation causes XP/Cockayne Syndrome (CS) complex. XP11BE is a frame-shift mutation that changes the last 42 residues at the C-terminus of XPB. It has been known that XP11BE mutation abolishes the interaction motif of XPB with XPF leading to XP phenotype. Our preliminary results indicate that change of the last 42 amino acid residues reduces the solubility of XPB mutant compared to the wild type XPB, therefore leading to limited cellular level of TFIIH which in turn causes reduced recovery RNA synthesis, the hall mark of CS phenotype. There is little information about why the adjacent mutations F99S and T119P cause different phenotypes. In order to examine these two mutations at chemical and atomic level, XPB recombinant proteins bearing F99S and T119P mutations will be prepared for structural and functional analysis. These results will be very important for understanding the pathogenic mechanism of these mutations and will have significant medical implications.

 描述（由申请人提供）：Xeroderma色素（XP）蛋白是转录和DE切除修复（NER）所需的DNA解旋酶，这是一种去除多种DNA病变的主要DNA修复途径。 OW XPB启动DSDNA在损坏部位放弃，以及损坏的DNA的打开方式与双重配合，我们计划实现三个特定的目标：AIM1：剖析XPB在NER期间XPB的DNA机制。 DSDNA和带有气泡的DNA为（6-4）嘧啶 - 吡啶酮二聚体。用于体外生化测定和细胞凸起的成像实验的mutajenesis，在活细胞中测试XPB的作用通过ERCC1-XPF，我们纯化了XPB C端的N端的XPF的一半。基于成像为活细胞中的损伤切口：说明引起疾病的XPB突变引起的结构和功能变化。 XP11BE突变导致XP/Cockayne综合征（CS）复合物。与野生型XPB合成相比，酸性降低了XPB突变体的溶解度，CS表型的HALL标记很少。 XPB重组轴承F99S和T119P这些结果对于理解这些突变的致病型武器非常重要，并且具有ANT医学意义。