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Mechanisms of DNA replication and maintenance in eukaryotes

真核生物中 DNA 复制和维持的机制

基本信息

批准号：
9281879
负责人：
PETER M BURGERS
金额：
$ 67.52万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
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/9281879
关键词：
ATR gene Affect Animal Model Aromatic Amino Acids Binding Proteins Biochemical Catalysis Catalytic Domain Cell Cycle Cell Cycle Checkpoint Cell division Closure by clamp 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 activation protein complex public health relevance response scaffold sensor

项目摘要

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.