Mechanisms of DNA replication and maintenance in eukaryotes

真核生物中 DNA 复制和维持的机制

• 批准号: 9071509
• 负责人: PETER M BURGERS
• 金额: $ 48.15万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9071509
• 关键词: Affect; Amino Acid Motifs; Animal Model; Aromatic Amino Acids; Binding Proteins; Biochemical; Catalysis; Catalytic Domain; Cell Cycle; Cell Cycle Checkpoint; Cell division; Complex; DNA; DNA Damage; DNA Maintenance; DNA Replication Damage; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; Defect; Disease; Enzymes; Eukaryota; Experimental Models; Flap Endonucleases; Genetic; Genetic Materials; Human; Investigation; Iron; Kinetics; Malignant Neoplasms; Mammalian Cell; Mediating; Metabolic Pathway; Metabolism; Monitor; Mutagenesis; Mutation; Okazaki fragments; Organism; Orthologous Gene; Oxidation-Reduction; Pathway interactions; Patients; Phosphotransferases; Physiological; Polymerase; Post-Translational Protein Processing; Process; Protein Kinase; Proteins; Risk; Single-Stranded DNA; Sulfur; Surgical Flaps; Testing; Time; Yeasts; fascinate; genetic analysis; mutant; protein complex; public health relevance; response; scaffold; sensor

 DESCRIPTION (provided by applicant): This proposal focuses on several DNA metabolic pathways in eukaryotes, with an emphasis on DNA replication and DNA damage response mechanisms. DNA polymerases δ (Pol δ) and ζ (Pol ζ) carry out the elongation and maturation of Okazaki fragments on the lagging strand of the replication fork, and translesion synthesis (TLS), respectively. Our recent discovery that their catalytic subunits contain an essential iron-sulfur cluster makes new functional studies of these enzymes both timely and important. Thus, we will determine whether the redox state of the iron-sulfur cluster affects the biochemical parameters of these enzymes, either in catalysis or complex interactions. We will test the hypothesis that regulated strand displacement synthesis by Pol δ is a critical aspect of its function during Okazaki fragment maturation. During this process, which occurs millions of times during each mammalian cell division, strand displacement synthesis by Pol δ generates 5'-flaps that are cut by the flap endonuclease FEN1. The kinetic mechanism of this machinery will be determined, and their physiological relevance will be queried through genetic analysis of informative mutants. Our proposed studies of mutagenesis focus on Rev1 and Pol ζ, which through interactions with the replication clamp PCNA form a productive mutasome. The Rev1 protein serves as a scaffold onto which the TLS machinery is organized. We will test the hypothesis that posttranslational modifications act as on/off switches for mutagenesis by mediating functional interactions between Pol ζ and Rev1 with PCNA. The primary focus of our checkpoint studies will be on the sensor protein kinase Mec1, the ortholog of human ATR, that initiates the DNA damage and replication checkpoints. The initiating step in these pathways is the activation of Mec1/ATR kinase activity through interaction with cell-cycle specific activators on single-stranded DNA substrates coated with the single-stranded binding protein RPA. Key features of these activators is that the activating regions are structurally disordered and contain small essential aromatic amino acid motifs. Biochemical studies are aimed at understanding Mec1 kinase activation by these proteins and complexes. In keeping with the MIRA principle, we also look forward to pursuing other fascinating questions in DNA metabolism that may, and undoubtedly will arise during our investigations.

 描述(申请人提供)：这项建议集中在真核生物中的几种DNA代谢途径，重点是DNA复制和DNA损伤反应机制。δ聚合酶(POLδ)和ζ聚合酶(POLζ)分别执行复制叉滞后链上冈崎片段的延长和成熟以及跨损伤合成。我们最近发现，它们的催化亚基包含一个必要的铁-硫簇，这使得对这些酶的新功能研究变得及时和重要。因此，我们将确定铁-硫簇的氧化还原状态是否影响这些酶的生化参数，无论是在催化还是复杂的相互作用中。我们将检验这一假设，即POLδ调控的链置换合成是其在冈崎片段成熟过程中功能的关键方面。在这一过程中，在每个哺乳动物细胞分裂期间发生数以百万计的次数，由POLδ进行的链置换合成产生了被FEN1酶切割的5‘瓣。这一机制的动力学机制将被确定，并将通过对信息突变的遗传分析来询问它们的生理相关性。我们建议的突变研究集中在Rev1和PolPCNA上，它们通过与复制钳ζ的相互作用形成一个高产的突变酶体。Rev1蛋白作为组织TLS机制的支架。我们将通过调节Polζ和Rev1与增殖细胞核抗原之间的功能相互作用来检验这一假设，即翻译后修饰作为突变的开/关开关。我们检查点研究的主要焦点将是传感器蛋白激酶Mec1，它是人类ATR的同源基因，启动DNA损伤和复制检查点。这些途径的起始步骤是通过与单链DNA底物上的细胞周期特异性激活物相互作用激活Mec1/ATR激酶活性，底物上包裹着单链结合蛋白RPA。这些激活剂的主要特征是激活区结构混乱，含有 小的必需芳香氨基酸模体。生化研究的目的是了解这些蛋白质和复合体对Mec1激酶的激活。为了与米拉原则保持一致，我们也期待着在我们的研究过程中可能并毫无疑问会出现的DNA新陈代谢方面的其他有趣的问题。