Regulation of DNA replication and repair

DNA复制和修复的调节

• 批准号: 7627423
• 负责人: MARK D. SUTTON
• 金额: $ 30.84万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7627423
• 关键词: Antibiotic Resistance; Antibiotics; Area; Binding; Biochemical; Biological Assay; Bypass; Cell Cycle; Class; Complement; Complex; DNA; DNA Polymerase III; DNA Polymerase beta; DNA Repair; DNA biosynthesis; DNA chemical synthesis; DNA-Directed DNA Polymerase; Depth; Development; E coli replicase; Escherichia coli; Failure; Family; Genetic; Genome Stability; Genomic Instability; Goals; Hand; Holoenzymes; Human; Immunoglobulin Somatic Hypermutation; Immunoglobulins; In Vitro; Laboratories; Lead; Life; Malignant Neoplasms; Mediating; Modeling; Mutagenesis; Mutation; Organism; Pathogenesis; Phenotype; Play; Polymerase; Process; Proteins; Reagent; Regulation; Replication Error; Replication Initiation; Research; Role; Slide; Stress; Surface; Testing; Time; Travel; Work; base; chromatin immunoprecipitation; genetic analysis; human disease; in vitro Assay; mutant; novel; pol genes; prevent; programs; repaired; research study

The long-term goal of this research program is to develop an integrated mechanistic view of how organisms coordinate the actions of their replication machinery with those of other cellular factors involved in DNA repair and damage tolerance. Failure to do so leads to a loss of genetic fidelity and contributes to human disease. Furthermore, inappropriate regulation of Y-family DNA polymerase function is proposed to contribute to mutations that lead to cancer. Work from our laboratory and others have demonstrated unambiguously that DNA polymerase processivity clamps (¿ or DnaN sliding clamps) play critically important roles in this complex process. The proposed research program utilizes an integrated genetic¿biochemical¿physical biochemical approach, placing particular emphasis on determining how the ¿ clamp helps to coordinate the actions of the E. coli replicase, DNA polymerase III holoenzyme (Pol III), with the dinB-encoded Pol IV, which acts in translesion DNA synthesis (TLS), and with the Hda protein, which helps to regulate initiation of DNA replication by inactivating the DnaA initiator protein. We will utilize in vitro assays to characterize interactions of Pol III and Pol IV with various mutant ¿ clamp proteins. As part of this work, we will purify heterodimeric clamp proteins bearing either a single mutation in one subunit, or different mutations in each subunit. Using these mutant clamps, we will dissect the mechanism by which the ¿ clamp mediates a switch between Pol III and Pol IV to coordinate high fidelity replication with TLS. We will complement these studies with genetic analyses. We anticipate that the model(s) for polymerase switching supported by our results will serve as a valuable paradigm for similar switch mechanisms in other organisms, including humans. In addition, since Y-family Pols are remarkably well conserved throughout all three branches of life, results from our studies will also contribute significantly to our understanding of mechanisms underlying mutagenesis under times of stress, thereby impacting on pathogenesis and antibiotic resistance, as well as mechanisms contributing to immunoglobulin diversity by error-prone replication during somatic hypermutation. We will also apply the approaches that we have developed to characterize polymerase switching to Hda protein in order to understand the role of the ¿ clamp in coordinating replication with Hda-dependent regulation of initiation of replication. Failure to properly regulate initiation leads to over-replication, genome instability, and can be lethal. We will distinguish between different models for Hda function, and will determine whether Hda and Pol III simultaneously bind to the same ¿ clamp. We will also determine whether Hda acts to limit access of Y-family Pols to the replication fork until such time as they are required for TLS. Finally, since replication errors contribute significantly to mutagenesis, and since the coordinate regulation of initiation and elongation of DNA replication is critically important for genome stability, our findings in these areas may also identify new classes of targets for the development of novel antibiotics.

该研究计划的长期目标是建立一个综合的机制观点，以了解如何 生物体协调其复制机制的行为与参与复制的其他细胞因子的行为 DNA 修复和损伤耐受性失败会导致遗传保真度的丧失，并对人类做出贡献。 此外，Y 家族 DNA 聚合酶功能的不当调节也可能导致疾病。 我们实验室和其他实验室的工作已经明确地证明了这一点。 DNA 聚合酶持续钳（¿ 或 DnaN 滑动钳）在此复合体中发挥着至关重要的作用 拟议的研究计划利用了综合遗传技术。生物化学??物理生物化学 方法，特别强调确定如何夹子有助于协调动作 大肠杆菌复制酶、DNA 聚合酶 III 全酶 (Pol III)，以及 dinB 编码的 Pol IV，其作用于 跨损伤 DNA 合成 (TLS)，以及 Hda 蛋白，有助于调节 DNA 复制的起始 通过灭活 DnaA 起始蛋白，我们将利用体外测定来表征 Pol III 和 Pol III 的相互作用。 Pol IV 与各种突变体 ¿作为这项工作的一部分，我们将纯化异二聚体钳蛋白。 使用这些突变体可以在一个亚基中携带单个突变，或者在每个亚基中携带不同的突变。 夹子，我们将剖析 ¿钳位介导 Pol III 和 Pol IV 之间的转换 我们将通过遗传分析来补充这些研究。 预计我们的结果支持的聚合酶转换模型将成为有价值的 包括人类在内的其他生物体中类似开关机制的范例。 令人惊讶的是，它们在生命的所有三个分支中都保存得很好，我们的研究结果也将做出贡献 对我们对压力下诱变机制的理解具有重要意义，从而 对发病机制和抗生素耐药性的影响，以及免疫球蛋白的机制 我们还将应用我们在体细胞超突变过程中容易出错的复制来实现多样性。 已经开发出表征聚合酶转换为 Hda 蛋白的特征，以了解 ¿ 钳制复制与 Hda 依赖性复制起始调节的协调失败。 调节启动会导致过度复制、基因组不稳定，并且可能是致命的。我们将区分它们。 Hda 功能的不同模型，并将确定 Hda 和 Pol III 是否同时结合到相同的 ¿我们还将确定 Hda 是否会限制 Y 家族 Pols 访问复制叉，直到 最后，由于复制错误会显着导致突变， 并且由于 DNA 复制起始和延伸的协调调节对于 DNA 复制至关重要 基因组稳定性，我们在这些领域的发现也可能确定用于开发的新靶标 新型抗生素。