CELLULAR FUNCTIONS OF EUKARYOTIC DNA LIGASES

真核 DNA 连接酶的细胞功能

• 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
• 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

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 修复和基因重组的相关途径 在真核生物中。 由于已经描述了这些功能的改变 在易患癌症的人类疾病和癌细胞中，这些信息将 可用于研究致癌机制。 该提案将重点关注 DNA 连接，这是一种重要的共同点 DNA分子的复制、修复和重组的步骤。 哺乳动物细胞含有三种不同的 DNA 连接酶活性。 遗传 对其中一种酶 DNA 连接酶 I 的生化研究已经 表明该酶参与了冈崎片段的连接 复制叉和 DNA 修复。 哺乳动物的细胞功能 DNA 连接酶 II 和 III 无法从其生化指标预测 特性。 因此我建议寻找类似的酶 一种真核生物，酿酒酵母，易于遗传 分析。 在开始这些研究之前，CDc9 的产品 基因，其与哺乳动物 DNA 连接酶 I 功能同源， 定量分析植物提取物中的主要 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连接酶活性已部分 从这些提取物中纯化并表征。 此次活动，即 抗原性与 Cdc9 DNA 连接酶不同，并且具有相似的生化特性 哺乳动物 DNA 连接酶 II 的特性，将被进一步纯化和 特点。 可能是相对分布 酵母 DNA 连接酶在制备的提取物中会有显着差异 来自正在进行减数分裂的细胞或这些提取物可能含有减数分裂- 特定 DNA 连接酶或减数分裂提取物的活性得到富集， 在营养生长细胞中含量较低，那么 DNA 连接酶活性将从减数分裂提取物中纯化和表征。 这些研究的目的是纯化足够数量的这些 用于氨基酸测序研究和培养多克隆的酶 抗血清。 这些研究产生的特定分子探针将 用于从酵母基因组文库中分离基因并且 将研究基因破坏的表型后果。 作为一个 鉴定酿酒酵母 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，我将分离出哺乳动物 DNA 连接酶 II cDNA 序列。 这些 序列将用作探针来筛选功能 酿酒酵母中的同源酶。 鉴定出酵母基因后， 将研究基因破坏的表型后果。 经过 这种综合生化和遗传方法，细胞 DNA 连接酶在真核 DNA 代谢中的功能是 决定。