Regulation of DNA replication and repair

DNA复制和修复的调节

• 批准号: 8269036
• 负责人: MARK D. SUTTON
• 金额: $ 29.8万
• 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 2013-09-26
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8269036
• 关键词: Antibiotic Resistance; Antibiotics; Area; Bacteria; Binding; Biochemical; Biochemical Genetics; Biological Assay; Biological Models; Bypass; Cell Cycle; Cells; Complex; DNA; DNA Damage; 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; Health; 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; 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的机理观点，即生物如何将复制机制的作用与参与DNA修复和损伤耐受性的其他细胞因素的作用进行协调。不这样做会导致遗传保真度的丧失，并导致人类疾病。我们实验室和其他人的工作明确证明了DNA聚合酶（POL）的加工性夹具（或DNAN滑动夹）在这个高度复杂的过程中起着多种基本作用。拟议的研究计划采用了综合的遗传生物化学物质生化方法，特别强调确定如何？ 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.在下一个进步期间，我们将利用体外测定法来表征Pol III He，Pol II和Pol IV与各种突变体的相互作用？夹具蛋白。作为这项工作的一部分，我们将净化一个亚基中一个突变或每个亚基中不同突变的异二聚体夹蛋白。使用这些突变夹，我们将剖析？夹子介导POL切换以协调与TLS的高保真复制。我们还将利用遗传方法来定义体内POL开关的机制，并确定其他细胞因子是否有助于这一至关重要的过程。我们预计，由我们的结果支持的POL开关的模型将成为其他生物（包括人类）类似开关机制的有价值的范式。此外，由于TLS POL在生命的所有三个分支中都充分保守，因此我们的研究的结果还将有助于我们理解压力时间下诱变的机制，从而影响发病机理和抗生素耐药性，以及在其中有助于在体内的pols pols pols pols pols pols pols symogunoglobullobullobloblobillys shem symartys sompary shormations shem shormainty。我们还将应用正在开发的方法来表征POL切换到HDA蛋白，以定义大肠杆菌与HDA依赖性复制启动的调节的机制。无法正确调节启动可能是致命的。我们将区分HDA函数的不同模型，并确定HDA和Pol III是否同时结合了同时？夹钳。我们还将利用遗传和生化方法来确定HDA是否会调节TLS POLS对复制叉的访问，直到需要为止。由于复制误差对诱变有显着贡献，并且由于坐标的启动调节和DNA复制的伸长对基因组稳定性至关重要，因此我们在这些领域中的发现也可能确定用于开发新型抗生素的新目标。公共卫生相关性：未能协调不同复制和修复因素的作用会导致遗传保真度的丧失，并导致人类疾病。由于从细菌到人类的复制和修复机制非常保守，因此我们将利用大肠杆菌作为模型系统来了解如何相互调节不同复制和修复因子的作用。我们预计，如果事件的复杂性要大得多，我们的结果将成为理解人类类似控制网络的框架，因此，将有助于我们对有助于癌症和其他人类疾病的机制的理解。