RECJ PROTEIN INTERACTIONS

RECJ 蛋白质相互作用

• 批准号: 2684933
• 负责人: SUSAN THOMAS LOVETT
• 金额: $ 23.15万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-04-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2684933
• 关键词: DNA binding protein; DNA repair; Escherichia coli; Saccharomyces cerevisiae; bacterial DNA; bacterial genetics; bacterial proteins; enzyme mechanism; enzyme substrate; exonuclease; gel mobility shift assay; gene expression; genetic recombination; immunochemistry; mutant; nucleic acid sequence; phosphodiesterase I; plasmids; polymerase chain reaction; protein sequence; protein structure function

In all cells, the process of genetic recombination repairs DNA damage that might otherwise lead to mutations or cell death. The loss of various DNA repair mechanisms in human cells has, in some cases, been correlated with a predisposition to cancer. Studies of genetic recombination in the bacterium Escherichia coli has defined in detail many of the proteins that mediate steps of genetic exchange. Our previous work has focused on understanding the role that DNA exonucleases play in genetic recombination in E. coli. We propose to characterize the biochemical properties of the RecJ exonuclease that is involved in several pathways for DNA repair. We will determine the properties of the RecJ exonuclease in coupled in vitro reactions with the RecA protein of E. coli. RecA plays a central role in genetic recombination and can promote strand exchange between homologous DNA molecules. We will investigate the reactions that mimic the types of biochemical steps that are thought to constitute recombinational DNA repair. These experiments will provide a biochemical framework to understand the role of RecJ in the bacterial cell. We also propose to define those regions of the RecJ protein that are important for the genetic and biochemical functions of RecJ. We will isolate and characterize mutant forms of the protein by genetic and biochemical means. We will isolate, sequence and compare the predicted amino acid sequence or other bacterial RecJ proteins to determine those residues of the protein that have been conserved throughout evolution. Our goal from this analysis will be to develop a motif shared among RecJ and other 5' exonucleases. Although 5' exonucleases are found in many genetic recombination systems, there is no great similarity among these proteins in primary sequence. It is therefore not possible to identify putative 5' DNA exonucleases based on sequence information alone. Extensive mutational analysis of exonuclease proteins has not been performed.From our proposed analysis, we will be able to identify the regions of the protein likely to be involved in catalysis; these regions are most likely to share similar structure among exonucleases. We will use the structural information about RecJ to seek RecJ-homologs from the eukaryotic organism Saccharomyces cerevisiae. Molecular genetics in the yeast S. cerevisiae is well-developed and many genes involved in genetic recombinationand DNA repair have been identified. Recently, homologs of several genes which mediate genetic recombination and DNA repair in bacteria have been found in yeast. The yeast homologs have been useful, in turn, in finding human counterparts to these genes. The conservation of DNA repair pathways from bacteria to yeast suggests that RecJ-like proteins should be found in eukaryotes. Our proposed experiments seek to identify these counterparts in yeast and to begin to investigate their genetic and biochemical properties in this eukaryotic organism.

在所有细胞中，基因重组的过程修复DNA损伤， 否则可能导致突变或细胞死亡。各种DNA的丢失 在某些情况下，人类细胞的修复机制与 易患癌症的体质基因重组的研究 大肠杆菌已经详细定义了许多蛋白质， 基因交换的步骤。我们以前的工作集中在 了解DNA外切核酸酶在基因重组中的作用 在大肠杆菌我们建议表征的生化特性的 RecJ外切核酸酶参与DNA修复的几种途径。 我们将确定RecJ核酸外切酶在偶联的条件下的性质。 体外与E.杆菌RecA扮演着一个中心角色， 在基因重组中的作用，并可促进 同源DNA分子我们将研究模拟 被认为构成重组的生化步骤类型 DNA修复这些实验将提供一个生化框架， 了解RecJ在细菌细胞中的作用。 我们还建议定义RecJ蛋白的那些区域， 重要的遗传和生化功能的RecJ。我们将 通过遗传学方法分离和表征蛋白质的突变形式， 生化手段。我们会分离测序并比较 氨基酸序列或其他细菌RecJ蛋白，以确定那些 在整个进化过程中一直保守的蛋白质残基。 我们的目标是从这个分析将开发一个主题之间共享RecJ 和其它5 ′核酸外切酶。虽然5'核酸外切酶在许多 基因重组系统，它们之间没有很大的相似性， 一级序列中的蛋白质。因此，无法确定 仅基于序列信息推定的5 ′ DNA外切核酸酶。 核酸外切酶蛋白的广泛突变分析尚未被证实。 执行。从我们提出的分析，我们将能够确定 可能参与催化的蛋白质区域;这些区域 在核酸外切酶中最有可能共享相似的结构。 我们将使用RecJ的结构信息来寻找RecJ同源物 来自真核生物酿酒酵母。分子遗传学 在酵母S.酿酒酵母是发达的，许多基因参与 基因重组和DNA修复已经被确认。最近， 介导遗传重组和DNA的几个基因的同源物 在酵母中发现了细菌的修复作用。酵母同系物已经被 反过来，在寻找这些基因的人类对应物方面也很有用。的 从细菌到酵母的DNA修复途径的保守性表明， RecJ样蛋白应该存在于真核生物中。我们提出的实验 试图在酵母中识别这些对应物，并开始研究 它们的遗传和生化特性。