Abstract
The 3' --> 5'-exonucleases process DNA ends in many DNA repair pathways of human cells. Determination of the human TREX2 structure is the first of a dimeric 3'-deoxyribonuclease and indicates how this highly efficient nonprocessive enzyme removes nucleotides at DNA 3' termini. Symmetry in the TREX2 dimer positions the active sites at opposite outer edges providing open access for the DNA. Adjacent to each active site is a flexible region containing three arginines positioned appropriately to bind DNA and to control its entry into the active site. Mutation of these three arginines to alanines reduces the DNA binding capacity by approximately 100-fold with no effect on catalysis. The human TREX2 catalytic residues overlay with the bacterial DnaQ family of 3'-exonucleases confirming the structural conservation of the catalytic sites despite limited sequence identity, and mutations of these residues decrease the still measurable activity by approximately 10(5)-fold, confirming their catalytic role.
Highlights
During the multistep processes of DNA replication, repair, and recombination DNA ends are often remodeled by enzymes containing 3Ј 3 5Ј-exonuclease activities to remove mispaired, modified, fragmented, and normal nucleotides from DNA 3Ј termini
In an effort to understand better this concept of exonucleolytic proofreading by autonomous 3Ј-exonucleases to edit kinetically blocked DNA 3Ј termini we have determined the x-ray structure of the human TREX2 dimer with data extending to 2.47 Å resolution
The crystallographic model of the TREX2 dimer has been refined to an R factor of 21.6% (Rfree ϭ 26.1%) using all x-ray data extending to a resolution limit of 2.47 Å (Table I)
Summary
Enzyme Preparation—The plasmid construct used to express the human TREX2 enzyme and mutants in Escherichia coli and purification of the TREX2 used for structural studies has been described (7). The peak fraction containing the TREX2 protein eluted at ϳ250 mM NaCl at a concentration of ϳ0.5 mg/ml and was stored in aliquots at Ϫ80 °C. Crystallization and X-ray Data Collection—The purified TREX2 protein was dialyzed into 20 mM Tris, pH 7.6, 150 mM NaCl, 20 mM dithiothreitol. Prior to data collection crystals were soaked in 0.4 M ammonium phosphate, 25% PEG 400 for 2 min in preparation for freezing. Five ordered selenium sites were located in the crystallographic asymmetric unit using the program SOLVE (56). The model was refined with no noncrystallographic constraints against the peak wavelength data set by conjugate gradient minimization and torsion angle-restrained molecular dynamics using the program CNS (59). Model refinement converged with a final R factor of 21.6%
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