Catalytic mechanism of endonuclease v: a catalytic and regulatory two-metal model.
The enzyme endonuclease V initiates repair of deaminated DNA bases by making an endonucleolytic incision on the 3' side one nucleotide from a base lesion. In this study, we have used site-directed mutagenesis to characterize the role of the highly conserved residues D43, E89, D110, and H214 in Thermotoga maritima endonuclease V catalysis. DNA cleavage and Mn(2+)-rescue analysis suggest that amino acid substitutions at D43 impede the enzymatic activity severely while mutations at E89 and D110 may be tolerated. Mutations at H214 yield enzyme that maintains significant DNA cleavage activity. The H214D mutant exhibits little change in substrate specificity or DNA cleavage kinetics, suggesting the exchangeability between His and Asp at this site. DNA binding analysis implicates the involvement of the four residues in metal binding. Mn(2+)-mediated cleavage of inosine-containing DNA is stimulated by the addition of Ca(2+), a metal ion that does not support catalysis. The effects of Mn(2+) on Mg(2+)-mediated DNA cleavage show a complexed initial stimulatory and later inhibitory pattern. The data obtained from the dual metal ion analyses lead to the notion that two metal ions are involved in endonuclease V-mediated catalysis. A catalytic and regulatory two-metal model is proposed.
Feng
Hong
H
Department of Genetics and Biochemistry, South Carolina Experiment Station, Clemson University, Clemson, South Carolina 29634, USA.
Dong
Liang
L
Cao
Weiguo
W
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D004247:DNA / Q000737:chemistry; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008670:Metals / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011485:Protein Binding; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D008670:Metals; D004247:DNA; D043224:Deoxyribonuclease (Pyrimidine Dimer)