Regulation of DNA replication and repair

DNA复制和修复的调节

• 批准号: 7653128
• 负责人: MARK D. SUTTON
• 金额: $ 27.28万
• 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 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7653128
• 关键词: Antibiotic Resistance; Antibiotics; Area; Bacteria; Binding; Biochemical; Biochemical Genetics; Biological Assay; Biological Models; Bypass; Cell Cycle; Cells; Complex; DNA; DNA Polymerase II; DNA Polymerase III; DNA Polymerase beta; DNA Repair; DNA biosynthesis; DNA-Directed DNA Polymerase; Development; E coli replicase; Escherichia coli; Event; Failure; Family; Genetic; Genome Stability; Goals; Hand; Holoenzymes; Human; Immunoglobulin Somatic Hypermutation; Immunoglobulins; In Vitro; Laboratories; Lead; Life; Literature; Malignant Neoplasms; Mediating; Modeling; Mutagenesis; Mutation; Organism; Pathogenesis; Phenotype; Play; Polymerase; Process; Proteins; Reagent; Recycling; Regulation; Replication Error; Replication Initiation; Research; Role; Slide; Stress; Structure; Surface; Testing; Time; Travel; Variant; Work; base; chromatin immunoprecipitation; experience; genetic analysis; human disease; in vitro Assay; in vivo; meetings; mutant; novel; prevent; programs; public health relevance; repaired; research study

DESCRIPTION (provided by applicant): 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. Work from our laboratory and others have demonstrated unambiguously that DNA polymerase (Pol) processivity clamps (? or DnaN sliding clamps) play multiple essential roles in this highly complex process. The proposed research program utilizes an integrated genetic-biochemical-physical biochemical approach, placing particular emphasis on determining how the ? clamp coordinates the actions of the E. coli replicase, DNA polymerase III holoenzyme (Pol III HE), with the polB-encoded Pol II and the dinB-encoded Pol IV, which act in replication and translesion DNA synthesis (TLS), as well as with the Hda protein, which regulates initiation of DNA replication by inactivating the DnaA initiator protein. Over the next progress period, we will utilize in vitro assays to characterize interactions of Pol III HE, Pol II, 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(s) by which the ? clamp mediates Pol switching to coordinate high fidelity replication with TLS. We will also utilize genetic approaches to define the mechanism(s) of Pol switching in vivo, and to determine whether additional cellular factors contribute to this critically important process. We anticipate that model(s) for Pol switching supported by our results will serve as a valuable paradigm for similar switch mechanisms in other organisms, including humans. Moreover, since TLS Pols are well conserved throughout all three branches of life, results from our studies will also contribute to our understanding of the mechanisms underlying mutagenesis under times of stress, thereby impacting on pathogenesis and antibiotic resistance, as well as the mechanism(s) by which TLS Pols contribute to immunoglobulin diversity during somatic hypermutation. We will also apply the approaches that we are developing to characterize Pol switching to the Hda protein in order to define the mechanism by which E. coli coordinates replication with Hda-dependent regulation of initiation of replication. Failure to properly regulate initiation can be lethal. We will distinguish between different models for Hda function, and will determine whether Hda and Pol III HE simultaneously bind to the same ? clamp. We will also utilize genetic and biochemical approaches to determine whether Hda acts to regulate access of TLS Pols to the replication fork until such time as they are required. 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. PUBLIC HEALTH RELEVANCE: Failure to coordinate the actions of the different replication and repair factors leads to a loss of genetic fidelity and contributes to human disease. Since mechanisms of replication and repair are remarkably well conserved from bacteria to humans, we will utilize Escherichia coli as a model system to understand how the actions of different replication and repair factors are coordinately regulated with each other. We anticipate that our results will serve as a framework for understanding similar control networks in humans, were the complexity of the events is far greater, and as such, will contribute to our understanding of mechanisms contributing to cancer and other human diseases.

描述（由申请人提供）：该研究计划的长期目标是开发一种综合机制观点，以了解生物体如何协调其复制机制的行为与涉及 DNA 修复和损伤耐受的其他细胞因子的行为。如果不这样做，就会导致遗传保真度的丧失，并导致人类疾病。我们实验室和其他实验室的工作已经明确证明 DNA 聚合酶 (Pol) 持续钳（或 DnaN 滑动钳）在这个高度复杂的过程中发挥着多种重要作用。拟议的研究计划采用综合遗传-生化-物理生化方法，特别强调确定如何？夹子协调大肠杆菌复制酶、DNA 聚合酶 III 全酶 (Pol III HE) 的作用，以及 polB 编码的 Pol II 和 dinB 编码的 Pol IV，它们也在复制和跨损伤 DNA 合成 (TLS) 中发挥作用与 Hda 蛋白一样，它通过灭活 DnaA 起始蛋白来调节 DNA 复制的起始。在下一个进展阶段，我们将利用体外测定来表征 Pol III HE、Pol II 和 Pol IV 与各种突变体 ? 的相互作用。钳蛋白。作为这项工作的一部分，我们将纯化在一个亚基中带有单一突变或在每个亚基中带有不同突变的异二聚体钳蛋白。使用这些突变钳，我们将剖析？ Clip 介导 Pol 切换，以协调 TLS 的高保真复制。我们还将利用遗传方法来定义体内 Pol 转换的机制，并确定其他细胞因素是否有助于这一至关重要的过程。我们预计我们的结果支持的 Pol 转换模型将成为其他生物体（包括人类）中类似转换机制的有价值的范例。此外，由于 TLS Pols 在生命的所有三个分支中都得到了很好的保存，因此我们的研究结果也将有助于我们了解应激时突变的机制，从而影响发病机制和抗生素耐药性，以及机制），TLS Pols 在体细胞超突变期间有助于免疫球蛋白多样性。我们还将应用我们正在开发的方法来表征 Pol 转换为 Hda 蛋白的特征，以便定义大肠杆菌通过依赖 Hda 的复制起始调节来协调复制的机制。未能正确监管启动可能是致命的。我们将区分 Hda 功能的不同模型，并确定 Hda 和 Pol III HE 是否同时结合到相同的 ?夹钳。我们还将利用遗传和生化方法来确定 Hda 是否起到调节 TLS Pols 访问复制叉的作用，直到需要它们为止。由于复制错误对诱变有显着影响，而且 DNA 复制起始和延伸的协调调节对于基因组稳定性至关重要，因此我们在这些领域的发现也可能为新型抗生素的开发确定新的靶标类别。公共健康相关性：如果不同的复制和修复因子的作用无法协调，就会导致遗传保真度的丧失，并导致人类疾病。由于复制和修复机制从细菌到人类都非常保守，我们将利用大肠杆菌作为模型系统来了解不同复制和修复因子的作用如何相互协调调节。我们预计，如果事件的复杂性要大得多，我们的结果将作为理解人类类似控制网络的框架，因此将有助于我们理解导致癌症和其他人类疾病的机制。