MUTATIONAL ANALYSIS OF VACCINIA DNA-LIGASE DEFINES RESIDUES ESSENTIALFOR COVALENT CATALYSIS

DNA ligation entails AMP transfer from ATP to the 5' end of DNA to for m a DNA-adenylate structure, A(5')pp(5')N. A similar reaction involvin g GMP transfer occurs during 5' capping of eukaryotic mRNA. In both ca ses, nucleotidyl transfer occurs through a covalent lysyl-NMP intermed iate. There is local sequence conservation among ligases and capping e nzymes in the vicinity of the active site lysine (KxDG) and at three o ther collinear motifs. The role of these motifs in DNA ligation was te sted by mutating individual conserved residues in the vaccinia virus D NA ligase. Wild-type and mutated versions of vaccinia ligase were expr essed in bacteria as His-tagged fusion proteins and purified by Ni-aff inity and phosphocellulose chromatography steps. We found that Ala sub stitution for Lys-231 (the presumptive active site) abrogated enzyme-a denylate formation and DNA ligation activities. Ala mutations at conse rved residues Glu-283, Glu-377, and Lys-397 also resulted in loss of l igation activity, which correlated with a defect in ligase-AMP formati on. These results are concordant with mutational studies of yeast RNA capping enzyme and suggest a common structural basis for covalent nucl eotidyl transfer. (C) 1995 Academic Press, Inc.