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 processing. The long term goal of this project is to understand the biochemistry of the 3'??5'deoxyribonucleases encoded by the TREX genes that function in human cells and to elucidate the DNA metabolic pathways in which these enzymes function. The existence of 3'deoxyribonucleases in human cells has been recognized for almost forty years, but the biological functions of these enzymes are not well described. In the previous funding period we determined the structures of the TREX1 and TREX2 enzymes allowing us to begin to describe the biochemical properties of these 3'deoxyribonucleases. The TREX1 and TREX2 genes encode two structurally similar dimeric 3'deoxyribonucleases. The TREX1 enzyme contains a C-terminal region that is not found in TREX2, and the presence of two TREX genes is only found in mammals. Our biochemical studies to define the precise molecular characteristics of TREX1 have allowed us to quantify the activities of mutant TREX1 enzymes identified in patients diagnosed with the autoimmune diseases Aicardi- Goutieres syndrome, familial chilblain lupus, and in some cases of systemic lupus erythematosus. The finding of TREX1 mutations in autoimmune disease patients parallels the finding of TREX1 participation in apoptotic cell death. In this competitive renewal we will utilize the molecular tools we have developed in the previous funding period to focus our studies on the mechanisms of TREX1 action within cell death pathways. These studies will further our understanding of TREX1 exonuclease processing of DNA in cells by identifying the physiological substrate of TREX1 and will inform us of the mechanisms of TREX1 dysfunction that cause the pathological findings associated with autoimmune disease. Our efforts to understand the mechanisms of TREX1 biochemistry and cellular function are addressed according to the following aims.
In Aim 1 we will determine the mechanism of TREX1 action within the catalytic core.
In Aim 2, we will determine the effects of TREX1 on chromatin disassembly in cell death pathways.
In Aim 3 we will determine the mechanism of TREX1 targeting to the ER. PUBLIC HEALTH REVELANCE: This grant proposal is to study how enzymes process DNA and to determine how dysfunction of these enzymes causes autoimmune disease. The outcome of the proposed research will have a significant impact on the medical treatment of complex autoimmune diseases such as systemic lupus erythematosus.

Public Health Relevance

This grant proposal is to study how enzymes process DNA and to determine how dysfunction of these enzymes causes autoimmune disease. The outcome of the proposed research will have a significant impact on the medical treatment of complex autoimmune diseases such as systemic lupus erythematosus.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM069962-08
Application #
8245781
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Janes, Daniel E
Project Start
2004-09-01
Project End
2014-03-31
Budget Start
2012-04-01
Budget End
2014-03-31
Support Year
8
Fiscal Year
2012
Total Cost
$356,658
Indirect Cost
$115,673
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
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-Goutieres 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-Goutieres syndrome and lupus reveals dimeric structure requirements for DNA degradation activity. J Biol Chem 286:40246-54
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
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
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
Fye, Jason M; Orebaugh, Clinton D; Coffin, Stephanie R et al. (2011) Dominant mutation of the TREX1 exonuclease gene in lupus and Aicardi-Goutieres syndrome. J Biol Chem 286:32373-82

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