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Homology Directed Repair

同源定向修复

基本信息

批准号：
8327274
负责人：
SCOTT W MORRICAL
金额：
$ 19.64万
依托单位：
UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-09-03 至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8327274
关键词：
ATP Hydrolysis ATP phosphohydrolase Active Sites Amino Acids Base Excision Repairs Binding Biochemical Biological Assay Biological Process Cells Chromatin Chromosomes DNA DNA Binding DNA Polymerase beta DNA Repair DNA Repair Enzymes DNA Repair Pathway DNA-Directed DNA Polymerase Defect Diagnosis Enzymes Family Filament Funding Genome Stability Genomic Instability Germ Lines Glean Goals Human Knowledge Lead Link Malignant Neoplasms Methods Modeling Molecular Mutagenesis Mutation Orthologous Gene Pathway interactions Phenotype Play Polymerase Predisposition Property Proteins Rad51 recombinase Reaction Recruitment Activity Roentgen Rays Role Single-Stranded DNA Sister Site Site-Directed Mutagenesis Structure Testing Transferase Tumor-Derived Variant Yeasts base cancer cell cancer therapy cell transformation design in vivo interest mutant neoplastic cell novel presynaptic programs protein protein interaction recombinational repair repaired structural biology treatment strategy tumor

项目摘要

Rad51 protein is the eukaryotic representative of the RecA/Rad51 fannlly of DNA strand transferase enzymes. Homologous DNA strand exchanges catalyzed by RadSI are critical for Homology-Dlrected DNA Repair (HDR) and therefore for genome stability. To promote HDR, RadSI must first assemble onto single stranded DNA In the form of a presynaptic filament. Filament assembly allosterically activates RadSI to catalyze ATP hydrolysis, to search for homology in a sister chromosome, and to perform DNA strand exchange reactions. There Is compelling evidence that defects in the assembly and activity of RadSI presynaptic filaments are linked to human cancer. The overall goal of Project 3 Is to understand how specific changes in the structure, function, and molecular interactions of RadSI can lead to genomic instability and cancer. The SPECIFIC AIMS of Project 3 are: (1) To test the hypothesis that key amino acid residues at the filament interface and in the ATPase active site of RadSI control the allosteric transitions that couple the ATPase catalytic cycle to DNA strand exchange. Using yeast RadSI as a model, the catalytic and allosteric mechanisms of RadSI will be probed using a combination of site-directed mutagenesis, biochemical and biophysical analyses, and structural biology methods. (2) To test the hypothesis that tumor-derived variants of human hRADSI protein have altered biochemical and/or regulatory properties. hRADSI variants identified in Project 1 will be characterized biochemically alongside wild-type hRADSI to identify any changes in DNA binding or catalytic properties, or in key protein-protein interactions. (3) To test the hypothesis that interactions between hRADSI and DNA polymerase beta (Pol-beta) help recruit hRADSI onto ssDNA generated as a result of abortive base excision repair (BER). hRADSI:Pol-beta interactions will be characterized biochemically and disrupted by mutagenesis to assess their importance for DNA repair functions. Interesting mutants from Aims 2-3 will be exported to Projects 1 and 4 for in vivo and chromatin studies. This project will provide rigorous models for the structure, function, and assembly of RadSI presynaptic filaments, and for potential cross-talk between HDR and BER pathways, in normal vs, tumor cells, which will be useful for predicting cancer susceptibility and for developing new cancer treatments.

Rad51蛋白是DNA链转移酶RecA/Rad51系列的真核代表