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TREX（三素修复核酸外切酶）基因编码的3 '->5'脱氧核糖核酸酶的结构，机制和功能。3 '->5'脱氧核糖核酸酶是DNA代谢中的必需酶，其催化从DNA的3'末端切除核苷酸以制备这些3'末端用于DNA复制、修复和重组期间的后续步骤。3'脱氧核糖核酸酶从DNA 3'末端切除错配的、修饰的、片段化的或正常的核苷酸，并且这些酶的作用在维持所有生物体中基因组完整性的许多DNA代谢途径中是关键的。虽然真核生物中3'脱氧核糖核酸酶活性的存在已经被认识了30多年，但直到最近才鉴定出编码这些脱氧核糖核酸酶的一些基因。本实验室鉴定的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以及较小的单结构域蛋白。目前关于在人类细胞中起作用的3 '->5'脱氧核糖核酸酶的信息不足以理解这些蛋白质识别和切除3'核苷酸的机制，这使得难以确定这些蛋白质起作用的分子途径。本提案中的实验目标是寻求对TREX 3 '->5'外切核酸酶的生物化学的更好理解，并建立遗传系统以解决体内TREX蛋白功能。作为第一步，我们已经克隆了后生动物TREX基因，并建立了一个表达系统，以产生这些蛋白的生化研究。本项目的目标1是产生足够数量的重组TREX蛋白和定点突变体用于机制和结构研究。在目标2中，酶的机理研究将与NMR研究结合使用，以定量酶-底物相互作用，并确定TREX蛋白中底物识别和特异性的性质。目标3中的实验集中于TREX蛋白的X射线结构研究。这些结构的研究将提供深入了解的催化位点，二聚体接口，和核酸结合表面。目标4的实验将通过鉴定TREX蛋白结合伴侣和在果蝇中建立遗传系统来解决功能问题。编码这些3 '->5'脱氧核糖核酸酶的TREX基因的鉴定使得这些机制研究成为可能，这些机制研究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.