ERROR CORRECTION IN DNA SYNTHESIS--A BIOCHEMICAL STUDY

DNA 合成中的错误纠正——一项生化研究

• 批准号: 7314653
• 负责人: MYRON GOODMAN
• 金额: $ 39.23万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-09-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7314653
• 关键词: ATP Hydrolysis; Active Sites; Applications Grants; Base Pairing; Biochemical; Biological Assay; Biological Models; Biological Sciences; California; Childhood; Cities; Classification; Complement; Complex; DNA; DNA Polymerase II; DNA Polymerase III; DNA biosynthesis; DNA chemical synthesis; DNA-Directed DNA Polymerase; Disease; Enzymes; Escherichia coli; Excision; Exonuclease; Face; Fission Yeast; Fluorescence; Gel; Goals; Grant; Hereditary Disease; Holoenzymes; Human Resources; Hydrogen Bonding; Individual; Inherited; Instruction; Kinetics; Lesch-Nyhan Syndrome; Los Angeles; Malignant Neoplasms; Measurement; Measures; Mismatch Repair; Modeling; Monitor; Mutation; Myron; Names; Nucleotides; Numbers; Object Attachment; Okazaki fragments; Oncogene Activation; Pathway interactions; Performance; Plant Roots; Point Mutation; Polymerase; Principal Investigator; Printing; Process; Proliferating Cell Nuclear Antigen; Proteins; Reaction; Research Project Grants; Role; Site; Spottings; System; Testing; Thymine; Time; Tumor-Suppressor Gene Inactivation; Universities; activator 1 protein; analog; base; cancer type; design; difluorotoluene; dimer; inorganic phosphate; mathematical model; millisecond; mutant; novel; nucleotide analog; pol genes; programs; repaired; research study; time use

The broad objective of this grant proposal is to study the mechanisms responsible for the fidelity of DMA synthesis. DMA polymerases are the key enzymes involved in replication and repair of DMA. An analysis of how polymerases control fidelity is central to understanding the biochemical basis of a wide variety of genetic diseases. Lesch-Nyhan syndrome and ADA deficiency are two examples of inherited childhood diseases that can arise from a single point mutation. Activation of oncogenes and inactivation of tumor suppressor genes leading to cancer can result from single base changes in DNA. Genetic defects in post-replication mismatch repair of DNA polymerase errors are a root cause of hereditary nonpolyposis colin cancer, along with a variety of other types of cancer. Previous fidelity studies have focused on individual DNA polymerases in the absence of polymerase accessory proteins required to sustain processive synthesis. This grant investigates the fidelity of purified procaryotic and eucaryotic DNA polymerase holoenzymes, pol III and pol IIfrom Escherichia coli, and pol delta from Schizosaccharomyces pombe. A thorough understanding of fidelity mechanisms of DNA polymerases requires an analysis of the effects of sequence context and replication assessory proteins on fidelity. The proposed experiments, which include a full complement of polymerase subunits, are among the first of its kind, and make use of a mathematical model of polymerase fidelity and a gel fidelity assay that we've developed previously. The model is used to predict the effect of polymerase processivity subunits on base substitution fidelity. These predictions will be tested experimentally. Steady state kinetic experiments are designed to investigate the biochemical basis of mutational "hot" and "cold" spots. We propose to test the importance of hydrogen bonds between Watson-Crick base pairs on polymerase fidelity, by measuring the effects of base stacking on polymerase fidelity, in the absence of hydrogen bonding. Presteady state kinetic experiments, using fluorescent nucleotide analogs, are proposed to measure switching between polymerase and exonuclease active sites in "real-time". A second set of presteady state experiments are designed to determine the mechanism for loading and unloading the polymerase processivity clamp subunit onto DNA and toanalyze the requirements for ATP hydrolysis for each step in the clamp-loading pathway. This pathway is required for Okazaki fragment formation during discontinuous lagging-strand DNA synthesis.

这项拨款提案的广泛目标是研究负责DMA合成保真度的机制。DMA聚合酶是参与DMA复制和修复的关键酶。的分析