Mechanisms of the 3'-5' deoxyribonucleases

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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.