RECJ PROTEIN INTERACTIONS

在所有细胞中，基因重组的过程修复DNA损伤 否则可能会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 患癌症的倾向。基因重组在中国的研究进展 细菌大肠杆菌已经详细定义了许多蛋白质， 基因交换的中介步骤。我们之前的工作主要集中在 了解DNA核酸外切酶在基因重组中的作用 在大肠杆菌中。我们建议对该细胞的生化特性进行表征 参与DNA修复的几种途径的RECJ核酸外切酶。 我们将测定偶联的RecJ核酸外切酶的性质 与大肠杆菌RecA蛋白的体外反应。RecA扮演一个中心角色 在基因重组中的作用，并可以促进 同源DNA分子。我们将研究模拟的反应 被认为构成重组的生化步骤的类型 DNA修复。这些实验将提供一个生化框架，以 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 对RECJ的遗传和生化功能很重要。我们会 分离和鉴定突变形式的蛋白质通过遗传和 生化手段。我们将分离、测序和比较预测的 氨基酸序列或其他细菌RecJ蛋白来确定这些 在整个进化过程中被保守的蛋白质残基。 我们从这一分析中的目标将是开发一个在RecJ之间共享的主题 和其他5‘端核酸外切酶。尽管在许多物种中发现了5‘外切酶 基因重组系统，它们之间没有太大的相似性 初级序列中的蛋白质。因此，不可能确定 仅根据序列信息推测的5‘DNA核酸外切酶。 核酸外切酶蛋白的广泛突变分析尚未得到 从我们提议的分析中，我们将能够确定 可能参与催化的蛋白质区域；这些区域 最有可能在核酸外切酶中共享相似的结构。 我们将利用RecJ的结构信息来寻找RecJ-同源 来自真核生物酿酒酵母。分子遗传学 在酵母中，酿酒酵母是发育良好的，许多基因参与了 基因重组和DNA修复已经被确定。最近， 介导基因重组和DNA的几个基因的同源性 在酵母中发现了细菌的修复作用。酵母的同系物已经被 反过来，在寻找与这些基因对应的人类基因方面也很有用。这个 从细菌到酵母的DNA修复途径的保守表明 RECJ样蛋白应该存在于真核生物中。我们提议的实验 寻求鉴定酵母中的这些对应关系，并开始研究 它们在这种真核生物体内的遗传和生化特性。

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.