Mutational analysis of endonuclease V from Thermotoga maritima.
Endonuclease V nicks damaged DNA at the second phosphodiester bond 3' to inosine, uracil, mismatched bases, or abasic (AP) sites. Alanine scanning mutagenesis was performed in nine conserved positions of Thermotoga maritima endonuclease V to identify amino acid residues involved in recognition or endonucleolytic cleavage of these diverse substrates. Alanine substitution at D43, E89, and D110 either abolishes or substantially reduces inosine cleavage activity. These three mutants gain binding affinity for binding to double-stranded or single-stranded inosine substrates in the absence of a metal ion, suggesting that these residues may be involved in coordinating catalytic metal ion(s). Y80A, H116A, and, to a lesser extent, R88A demonstrate reduced affinities for double-stranded or single-stranded inosine substrates or nicked products. The lack of tight binding to a nicked inosine product accounts for the increased rate of turnover of inosine substrate since the product release is less rate-limiting. Y80A, R88A, and H116A fail to cleave AP site substrates. Their activities toward uracil substrates are in the following order: H116A > R88A > Y80A. These residues may play a role in substrate recognition. K139A maintains wild-type binding affinity for binding to double-stranded and single-stranded inosine substrate, but fails to cleave AP site and uracil substrate efficiently, suggesting that K139 may play a role in facilitating non-inosine substrate cleavage.
Huang
Jianmin
J
Department of Microbiology and Immunology, Hearst Microbiology Research Center and Strang Cancer Prevention Center, The Joan and Sanford I. Weill Medical College of Cornell University, 1300 York Avenue, Box 62, New York, New York 10021, USA.
Lu
Jing
J
Barany
Francis
F
Cao
Weiguo
W
D000409:Alanine; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001105:Archaea / Q000201:enzymology; D001419:Bacteria / Q000201:enzymology; D001483:Base Sequence; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D012568:Schizosaccharomyces / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D020124:Thermotoga maritima / Q000201:enzymology*
D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer); D000409:Alanine