Mutational analysis of vaccinia DNA ligase defines residues essential for covalent catalysis.
DNA ligation entails AMP transfer from ATP to the 5' end of DNA to form a DNA-adenylate structure, A(5')pp(5')N. A similar reaction involving GMP transfer occurs during 5' capping of eukaryotic mRNA. In both cases, nucleotidyl transfer occurs through a covalent lysyl-NMP intermediate. There is local sequence conservation among ligases and capping enzymes in the vicinity of the active site lysine (KxDG) and at three other collinear motifs. The role of these motifs in DNA ligation was tested by mutating individual conserved residues in the vaccinia virus DNA ligase. Wild-type and mutated versions of vaccinia ligase were expressed in bacteria as His-tagged fusion proteins and purified by Ni-affinity and phosphocellulose chromatography steps. We found that Ala substitution for Lys-231 (the presumptive active site) abrogated enzyme-adenylate formation and DNA ligation activities. Ala mutations at conserved residues Glu-283, Glu-377, and Lys-397 also resulted in loss of ligation activity, which correlated with a defect in ligase-AMP formation. These results are concordant with mutational studies of yeast RNA capping enzyme and suggest a common structural basis for covalent nucleotidyl transfer.
Shuman
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Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017124:Conserved Sequence; D011088:DNA Ligases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004926:Escherichia coli; D006639:Histidine; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D016324:Sequence Tagged Sites; D013379:Substrate Specificity; D013816:Thermodynamics; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D006639:Histidine; D011088:DNA Ligases