? 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. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM069962-01A1
Application #
6824434
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Portnoy, Matthew
Project Start
2004-09-01
Project End
2008-08-31
Budget Start
2004-09-01
Budget End
2005-08-31
Support Year
1
Fiscal Year
2004
Total Cost
$287,000
Indirect Cost
Name
Wake Forest University Health Sciences
Department
Biochemistry
Type
Schools of Medicine
DUNS #
937727907
City
Winston-Salem
State
NC
Country
United States
Zip Code
27157
Manils, Joan; Gómez, Diana; Salla-Martret, Mercè et al. (2015) Multifaceted role of TREX2 in the skin defense against UV-induced skin carcinogenesis. Oncotarget 6:22375-96
Fye, Jason M; Coffin, Stephanie R; Orebaugh, Clinton D et al. (2014) The Arg-62 residues of the TREX1 exonuclease act across the dimer interface contributing to catalysis in the opposing protomers. J Biol Chem 289:11556-65
Rice, Gillian I; Reijns, Martin A M; Coffin, Stephanie R et al. (2013) Synonymous mutations in RNASEH2A create cryptic splice sites impairing RNase H2 enzyme function in Aicardi-Goutières syndrome. Hum Mutat 34:1066-70
Orebaugh, Clinton D; Fye, Jason M; Harvey, Scott et al. (2013) The TREX1 C-terminal region controls cellular localization through ubiquitination. J Biol Chem 288:28881-92
Bailey, Suzanna L; Harvey, Scott; Perrino, Fred W et al. (2012) Defects in DNA degradation revealed in crystal structures of TREX1 exonuclease mutations linked to autoimmune disease. DNA Repair (Amst) 11:65-73
Orebaugh, Clinton D; Fye, Jason M; Harvey, Scott et al. (2011) The TREX1 exonuclease R114H mutation in Aicardi-Goutières syndrome and lupus reveals dimeric structure requirements for DNA degradation activity. J Biol Chem 286:40246-54
Namjou, B; Kothari, P H; Kelly, J A et al. (2011) Evaluation of the TREX1 gene in a large multi-ancestral lupus cohort. Genes Immun 12:270-9
Pence, Matthew G; Blans, Patrick; Zink, Charles N et al. (2011) Bypass of Nýý-ethylguanine by human DNA polymerase ýý. DNA Repair (Amst) 10:56-64
Powell, Rebecca D; Holland, Paul J; Hollis, Thomas et al. (2011) Aicardi-Goutieres syndrome gene and HIV-1 restriction factor SAMHD1 is a dGTP-regulated deoxynucleotide triphosphohydrolase. J Biol Chem 286:43596-600
Coffin, Stephanie R; Hollis, Thomas; Perrino, Fred W (2011) Functional consequences of the RNase H2A subunit mutations that cause Aicardi-Goutieres syndrome. J Biol Chem 286:16984-91

Showing the most recent 10 out of 25 publications