Regulation of the E. coli Y-family polymearse DinB

大肠杆菌 Y 家族聚合酶 DinB 的调控

• 批准号: 7689333
• 负责人: James J FOTI
• 金额: $ 5.01万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-15 至 2010-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7689333
• 关键词: Active Sites; Affect; Aneuploidy; B lymphoid malignancy; Base Pairing; Binding; Biochemical; Biological Assay; Biological Models; Bypass; Cells; Collaborations; Complement; Complex; DNA; DNA Binding; DNA Damage; DNA Primers; DNA Sequence Rearrangement; DNA-Directed DNA Polymerase; Data; Disease; Employee Strikes; Escherichia coli; Etiology; Event; Family; Frequencies; Future; Genetic; Genetic Recombination; Genome; Genome Stability; Genomics; Genotoxic Stress; Goals; Human; Immunoglobulin Genes; Immunoglobulin Somatic Hypermutation; Kidney; Kinetics; Lesion; Life; Link; Loss of Heterozygosity; Lung; MSH6 gene; Maintenance; Malignant Neoplasms; Methods; Modeling; Molecular; Mutagenesis; Mutation; N-terminal; Operon; Pathway interactions; Phenotype; Play; Polymerase; Primer Extension; Process; Proteins; Reaction; Rectal Cancer; Regulation; Research Design; Role; Site; Skin; Structure; System; Techniques; X-Ray Crystallography; Xeroderma Pigmentosum; Yeasts; base; design; dimer; genetic regulatory protein; in vivo; insight; leukemia; member; overexpression; prevent; protein complex; public health relevance; repaired; research study; response

DESCRIPTION (provided by applicant): Somatic hypermutation (SHM) requires the mutagenic potential of the Y-family of DNA polymerases to increase the mutation frequency in immunoglobulin genes. Mistargeted SHM decreases genomic stability and is thought to contribute to B-cell malignancies. However, the absence of function of Y-family polymerases can also result in xeroderma pigmentosum suggesting that properly regulated function is critical for cancer avoidance. The mechanisms targeting Y-family polymerases function are mysterious, but may require protein accessory factors to regulate DNA binding or alter the kinetics of extension from a DNA primer terminus. Using the experimentally tractable Escherichia coli model system, I plan to elucidate the structural and biochemical mechanisms that regulate Y-family polymerase access to or extension from a primer terminus. In E. coli, genetic data suggests that the protein UmuD plays a critical role in regulating DinB, a Y family polymerase member. Specific Aims: Aim 1: Determine the structure of DinB"UmuD2 and DNA"DinB"UmuD2 by X-ray crystallography. Aim 2: Study the effect of UmuD and RecA on the DinB reaction pathway for the creation and extension from properly paired and bulged primer termini. Study Design: Using structural and fluorescent methods I will determine mechanisms by which protein regulatory factors alter Y-family polymerase activity. A structure of the DinB"UmuD2 complex bound to DNA will offer key insights into protein"protein and protein"DNA contacts required to activate or prevent DinB-dependent mutagenesis. Furthermore, a detailed structural analysis of the DinB"UmuD2 complex will identify protein conformational changes induced by protein and DNA contacts. Fluorescent pre-steady biochemical studies will complement structural data, allowing a detailed examination of DinB-reaction progression within the context of structural rearrangements induced by protein and DNA contacts. Public Health Relevance Genomic stability requires a specialized set of DNA polymerases that facilitate tolerance of DNA damage until it can be properly repaired. Improper regulation of these polymerases can often result in cancer. This study is designed to elucidate the structural and biochemical mechanisms necessary to regulate one of these specialized DNA polymerases, DinB.

描述（由申请人提供）：体细胞超突变（SHM）需要DNA聚合酶Y家族的诱变潜力，以增加免疫球蛋白基因的突变频率。错误定位的 SHM 会降低基因组稳定性，并被认为会导致 B 细胞恶性肿瘤。然而，Y 家族聚合酶功能的缺失也会导致着色性干皮病，这表明适当调节功能对于避免癌症至关重要。针对 Y 家族聚合酶功能的机制是神秘的，但可能需要蛋白质辅助因子来调节 DNA 结合或改变 DNA 引物末端的延伸动力学。我计划使用实验上易于处理的大肠杆菌模型系统来阐明调节 Y 家族聚合酶进入引物末端或从引物末端延伸的结构和生化机制。在大肠杆菌中，遗传数据表明蛋白质 UmuD 在调节 DinB（Y 家族聚合酶成员）中发挥着关键作用。具体目标： 目标 1：通过 X 射线晶体学确定 DinB"UmuD2 和 DNA"DinB"UmuD2 的结构。目标 2：研究 UmuD 和 RecA 对 DinB 反应途径的影响，以从正确配对和凸出产生和延伸研究设计：使用结构和荧光方法，我将确定蛋白质调节因子改变 Y 家族聚合酶 A 结构的机制。与DNA结合的DinB”UmuD2复合物将为激活或防止DinB依赖性诱变所需的蛋白质“蛋白质和蛋白质”DNA接触提供重要的见解。此外，对 DinB"UmuD2 复合物的详细结构分析将识别蛋白质和 DNA 接触引起的蛋白质构象变化。荧光预稳定生化研究将补充结构数据，从而可以在结构重排的背景下详细检查 DinB 反应进程由蛋白质和 DNA 接触引起的基因组稳定性需要一组专门的 DNA 聚合酶来促进 DNA 损伤的耐受性，直到它能够被正确修复。这项研究旨在阐明调节这些特殊 DNA 聚合酶 DinB 所必需的结构和生化机制。