Site-directed mutagenesis of the NH₂ terminus of T4 endonuclease V: The position of the αNH₂ moiety affects catalytic activity

Reductive methylation of the αNH₂ moiety of the DNA repair enzyme T4 endonuclease V has been shown previously to eradicate both the N-glycosylase and apyrimidinic/apurinic lyase activities of the enzyme (Schrock, R. D., III, and Lloyd, R. S. (1991) J. Biol. Chem. 266, 17631-17639). The present study uses the technique of site-directed mutagenesis to investigate the important parameters involved in the cleavage mechanism. The prediction was that the addition of an amino acid in the immediate NH₂-terminal region of the protein would alter the proximity of the αNH₂ moiety of Thr² to its target, thereby severely compromising the enzyme's catalytic activity. However, substitutions in this region generally should be tolerated. To test this hypothesis, three substitutions of the NH₂-terminal amino acid were produced: Ser² (T2S), Val² (T2V), and Pro² (T2P). An addition mutant was also produced by adding a glycine between the first and second amino acids of the protein (Thr²-Gly-Arg³) (+Gly). The T2P and +Gly mutants had negligible pyrimidine dimer-specific N-glycosylase activity as well as negligible pyrimidine dimer-specific nicking activity in vitro. Conversely, the T2S enzyme exhibited wild type levels of activity and the T2V exhibited intermediate levels of activity in vitro. Results from ultraviolet (UV) survival studies of the mutant enzymes indicated that the in vivo activities of these enzymes were directly correlated to the enzymes' ability to cleave at pyrimidine dimers in vitro. These results indicate that a critical parameter for the functionality of endonuclease V is the relative distance between the primary αNH₂ group in the active site of the enzyme and those elements responsible for DNA binding and pyrimidine dimer recognition.