Mechanisms of the 3'-5' deoxyribonucleases

描述(由申请人提供)： 在这项新的拨款申请中提出的研究将检查由TREX(三个主要修复核酸酶)基因编码的3‘--&gt；5’脱氧核糖核酸酶的结构、机制和功能。3‘-&gt；5’脱氧核糖核酸酶是DNA代谢中的重要酶，它催化从DNA的3‘端切除核苷酸，为DNA复制、修复和重组的后续步骤准备这些3’端。3‘脱氧核糖核酸酶剔除DNA 3’末端不匹配、修饰、断裂或正常的核苷酸，这些酶的作用在许多DNA代谢途径中都是关键的，这些代谢途径在所有生物中维持基因组的完整性。虽然真核生物中存在3‘脱氧核糖核酸酶活性的认识已有三十多年的历史，但编码这些脱氧核糖核酸酶的一些基因直到最近才被发现。在这个实验室中发现的TREX基因存在于后生动物中，编码的蛋白质是一个更大的核酸酶家族的成员，该家族包括脱氧核酸酶和核糖核酸外切酶。该家族中的脱氧核糖核酸酶包括大的多结构域蛋白和较小的单结构域蛋白。目前，关于3‘--&gt；5’脱氧核糖核酸酶在人类细胞中的功能还没有足够的信息来理解这些蛋白质识别和切除3‘核苷酸的机制，这使得我们很难区分这些蛋白质发挥作用的分子途径。这项提议中的实验的目的是寻求更好地了解TREX 3‘-&gt；5’外切酶的生物化学，并建立一个遗传系统来解决TREX蛋白在体内的功能。作为第一步，我们克隆了后生动物TREX基因，并建立了一个表达系统来生产这些蛋白质用于生化研究。该项目的目标1是为机械学和结构研究提供足够数量的重组TREX蛋白和定点突变体。在目标2中，酶的机理研究将与核磁共振研究结合使用，以量化酶与底物的相互作用，并确定TREX蛋白中底物识别和特异性的性质。目标3的实验重点是TREX蛋白的X射线结构研究。这些结构研究将提供对催化位置、二聚体界面和核酸结合表面的见解。目标4中的实验将通过识别TREX蛋白结合伙伴和在果蝇中建立遗传系统来解决功能问题。编码这些3‘-&gt；5’脱氧核糖核酸酶的TREX基因的鉴定使这些机制研究成为可能，这将为这些酶的生理功能提供新的见解。

DESCRIPTION (provided by applicant): The studies proposed in this new grant application will examine structure, mechanism, and function of the 3'-->5' deoxyribonucleases encoded by the TREX (Three prime Repair EXonuclease) genes. The 3'-->5' deoxyribonucleases are essential enzymes in DNA metabolism that catalyze excision of nucleotides from the 3' ends of DNA to prepare these 3' termini for subsequent steps during DNA replication, repair, and recombination. The 3' deoxyribonucleases excise mismatched, modified, fragmented, or normal nucleotides from DNA 3' termini, and the actions of these enzymes are critical in many DNA metabolic pathways that maintain genomic integrity in all organisms. While the existence of 3' deoxyribonuclease activities in eucaryotes has been recognized for more than thirty years, only recently have some of the genes encoding these deoxyribonucleases been identified. The TREX genes identified in this laboratory are present in metazoans and encode proteins that are members of a larger nuclease family that includes both deoxy- and ribo-exonucleases. The deoxyribonucleases in this family include large multiple-domain proteins as well as smaller single-domain proteins. There is currently insufficient information about the 3'-->5' deoxyribonucleases functioning in human cells to understand the mechanisms by which these proteins recognize and excise 3' nucleotides making it difficult to dicern the molecular pathways in which these proteins function. The goal of experiments in this proposal is to seek a better understanding of the biochemistry of the TREX 3'-->5' exonucleases and to establish a genetic system to address TREX protein function in vivo. As a first step, we have cloned the metazoan TREX genes and established an expression system to produce these proteins for biochemical studies. Aim 1 of this project is to generate the recombinant TREX proteins and site-directed mutants in sufficient quantities for mechanistic and structural studies. In aim 2, mechanistic studies of the enzymes will be used in conjunction with NMR studies to quantify enzyme-substrate interactions and to determine the nature of substrate recognition and specificity in the TREX proteins. Experiments in aim 3 focus on X-ray structural studies of the TREX proteins. These structural studies will provide insights into the catalytic site, dimer interface, and nucleic acid binding surfaces. Experiments in aim 4 will address issues of function by identifying TREX protein binding partners and by establishing a genetic system in Drosophila. Identification of the TREX genes encoding these 3'-->5' deoxyribonucleases has made possible these mechanistic studies that will provide new insights into the physiological function of these enzymes.