CELLULAR FUNCTIONS OF EUKARYOTIC DNA LIGASES

真核 DNA 连接酶的细胞功能

基本信息

批准号：
2184634
负责人：
Alan E Tomkinson
金额：
$ 11.25万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-08-01 至 1998-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2184634
关键词：
DNA DNA repair DNA replication Saccharomyces cerevisiae cell cycle cell growth regulation enzyme activity fungal genetics gene mutation genetic recombination genetic strain genetic transcription hybridomas immunoprecipitation laboratory mouse laboratory rabbit meiosis molecular cloning northern blottings nucleic acid metabolism nucleic acid sequence phase contrast microscopy phenotype protein purification protein sequence

DNA DNA repair DNA replication Saccharomyces cerevisiae cell cycle cell growth regulation enzyme activity fungal genetics gene mutation genetic recombination genetic strain genetic transcription hybridomas immunoprecipitation laboratory mouse laboratory rabbit meiosis molecular cloning northern blottings nucleic acid metabolism nucleic acid sequence phase contrast microscopy phenotype protein purification protein sequence

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项目摘要

My long term research goal is to define at the molecular level, the linked pathways of DNA replication, DNA repair and genetic recombination in eukaryotes. Since alterations in these functions have been described in cancer-prone human diseases and in cancer cells, this information will be used to investigate the mechanisms of carcinogenesis. This proposal will focus on DNA joining, which is an essential common step in the replication, repair and recombination of DNA molecules. Mammalian cells contain three distinct DNA ligase activities. Genetic and biochemical studies on one of these enzymes, DNA ligase I, have implicated this enzyme in the joining of Okazaki fragments at the replication fork and in DNA repair. the cellular functions of mammalian DNA ligases II and III cannot be predicted from their biochemical properties. Therefore I am proposing to search for similar enzymes in a eukaryotic organism, S. cerevisiae, that is amenable to genetic analysis. Prior to initiating these studies, the product of the CDc9 gene, which is functionally homologous to mammalian DNA ligase I and is quantitatively the major DNA ligase activity in extracts of vegetatively growing S. cerevisiae cells, has been purified and characterized. In preliminary experiments, a second DNA ligase activity has been partially purified and characterized from these extracts. This activity, which is antigenically distinct from Cdc9 DNA ligase and has similar biochemical properties to mammalian DNA ligase II, will be further purified and characterized. It is possible that the relative distribution of the yeast DNA ligases will be significantly different in extracts prepared from cells undergoing meiosis or these extracts may contain a meiosis- specific DNA ligase or meiotic extracts are enriched for activity, which is present at low levels in vegetatively growing cells, then the DNA ligase activity will be purified and characterized from meiotic extracts. The goal of these studies is to purify sufficient quantities of these enzymes for amino acid sequencing studies and for raising polyclonal antiserum. The specific molecular probes generated by these studies will be used to isolate the gene from a yeast genomic library and the phenotypic consequence of gene disruption will be investigated. As an alternative approach to identifying S. cerevisiae DNA ligase genes, I will purify bovine DNA ligase II. Using the same strategy as outlined above, I will isolate mammalian DNA ligase II cDNA sequences. These sequences will be used as a probe to screen for the functionally homologous enzyme in S. cerevisiae. Having identified the yeast gene, the phenotypic consequences of gene disruption will be investigated. by this integrated biochemical and genetic approach, the cellular function(s) of DNA ligase(s) in eukaryotic DNA metabolism will be determined.

我的长期研究目标是在分子水平上定义 DNA复制，DNA修复和遗传重组的链接途径在真核生物中。由于已经描述了这些功能的改变在容易癌症的人类疾病和癌细胞中，此信息将用于研究癌变的机制。该建议将重点放在DNA加入上，这是必不可少的介入DNA分子的复制，修复和重组。哺乳动物细胞包含三种不同的DNA连接酶活性。遗传以及关于其中一种DNA连接酶I的生化研究，具有将这种酶牵涉到Okazaki碎片连接的复制叉和DNA修复。哺乳动物的细胞功能 DNA连接酶II和III无法从其生化中预测特性。因此，我建议在真核生物，酿酒酵母，可以适合遗传分析。在启动这些研究之前，CDC9的乘积基因，在功能上与哺乳动物DNA连接酶I同源，IS 定量营养提取物中的主要DNA连接酶活性生长的酿酒酵母细胞已被纯化和表征。在初步实验，第二个DNA连接酶的活性已部分从这些提取物中纯化和表征。这项活动，就是抗原上与Cdc9 DNA连接酶不同，具有相似的生化哺乳动物DNA连接酶II的特性将进一步纯化，并且特征。可能的相对分布酵母DNA连接酶在制备的提取物中将显着不同从经历减数分裂或这些提取物的细胞中可能包含减数分裂 - 特定的DNA连接酶或减数分裂提取物富含活性，这在营养生长的细胞中以低水平存在，然后是DNA 连接酶活性将被纯化并从减数分裂提取物中表征。这些研究的目的是净化足够数量用于氨基酸测序研究和升高多克隆的酶抗血清。这些研究产生的特定分子探针将用于将基因与酵母基因组文库和将研究基因破坏的表型后果。作为识别酿酒酵母DNA连接酶基因的替代方法，I 将纯化牛DNA连接酶II。使用与概述相同的策略上面，我将分离哺乳动物DNA连接酶II cDNA序列。这些序列将用作筛选功能的探针酿酒酵母中的同源酶。鉴定出酵母基因，基因破坏的表型后果将被研究。经过这种整合的生化和遗传方法，细胞 DNA连接酶在真核DNA代谢中的功能将是决定。