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MECHANISM OF TRANSLESION REPLICATION IN EUKARYOTES

真核生物中的跨损伤复制机制

基本信息

批准号：
6651038
负责人：
Christopher W Lawrence
金额：
$ 34.1万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-09-01 至 2004-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6651038
关键词：
DNA binding protein DNA directed DNA polymerase DNA repair DNA replication Saccharomyces cerevisiae complementary DNA fungal genetics fungal proteins gene mutation genetically modified animals human genetic material tag laboratory mouse ligase nucleic acid sequence nucleotidyltransferase ubiquitin

项目摘要

The principle aim of this proposal is to gain an understanding of the molecular mechanisms of translesion replication in eukaryotes. This process promotes DNA chain elongation past sites of template damage, at which normal replication is stalled, and is chiefly significant because it is a major source of induced and spontaneous mutations. Information about this process is therefore likely to provide important insights into carcinogenesis and genotoxicity in humans. Much of the proposed work will employ the technically amenable model eukaryote, the budding yeast Saccharomyces cerevisiae. Specific aims include the isolation of new genes whose products are involved in translesion replication, by virtue of their association with the Rev1, Rev3, or Rev7 proteins; isolation of the REV6 and NGM2 genes, thought to be involved in translesion replication; and identification of other genes with related function, by fractioning cell extracts for proteins that enhance translesion replication by DNA polymerase zeta and Rev1 protein in vitro. Other aims include in vivo analysis of REV or related gene functions by transforming deletion strains with plasmids carrying site specific UV photoproducts or abasic sites; enzymatic studies of the gene products, particularly in conjunction with pol zeta and Rev1 protein; and a mutational analysis of REV gene function. Information from yeast will be used to identify the human homologs of yeast genes, and to investigate the enzymatic properties of human DNA polymerase zeta, human Rev1 protein, and other gene products identified that have a related function. A secondary aim of the proposal is to investigate the mechanism of the RAD6-dependent error-free DNA damage tolerance pathway in yeast, which diminishes mutagenesis by unrepaired DNA damage. For this purpose, excision defective mutant strains that carry rad5, rad6, rad18, and rad30 deletions, or the pol30-46 allele (all of which are believed to involved in this pathway), will be transformed with double-stranded vectors that carry mutagenic lesions in both strands at staggered, variable positions. These plasmids have proved useful in detecting different kinds of recombinant events, a likely mechanism for the pathway.

这个建议的主要目的是了解真核生物中跨病变复制的分子机制。这一过程促进DNA链延伸通过模板损伤位点，在模板损伤位点正常复制停止，并且主要是重要的，因为它是诱导和自发突变的主要来源。因此，有关这一过程的信息可能为人类致癌和遗传毒性提供重要的见解。许多拟议的工作将采用技术上可行的模型真核生物，芽殖酵母酿酒酵母。具体目的包括分离其产物参与跨损伤复制的新基因，这是由于它们与Rev 1、Rev 3或Rev 7蛋白质相关;分离REV 6和NGM 2基因，它们被认为参与跨损伤复制;以及鉴定具有相关功能的其他基因，通过分离细胞提取物中的蛋白质，这些蛋白质在体外通过DNA聚合酶zeta和Rev 1蛋白增强跨损伤复制。其他目标包括通过用携带位点特异性紫外光产物或无碱基位点的质粒转化缺失菌株来体内分析REV或相关基因功能;基因产物的酶研究，特别是与pol zeta和Rev 1蛋白结合;以及REV基因功能的突变分析。来自酵母的信息将用于鉴定酵母基因的人类同源物，并研究人类DNA聚合酶zeta、人类Rev 1蛋白和其他鉴定出的具有相关功能的基因产物的酶性质。该提案的第二个目的是研究酵母中RAD 6依赖性无错误DNA损伤耐受途径的机制，该途径通过未修复的DNA损伤减少诱变。为此，将用在交错可变位置的两条链中携带诱变损伤的双链载体转化携带rad 5、rad 6、rad 18和rad 30缺失或pol 30 -46等位基因（所有这些都被认为参与该途径）的切除缺陷突变株。这些质粒已被证明可用于检测不同类型的重组事件，这是该途径的可能机制。