MUTATION OF TRYPTOPHAN-128 IN T4 ENDONUCLEASE-V DOES NOT AFFECT GLYCOSYLASE OR ABASIC SITE LYASE ACTIVITY

Mutation of various residues within the carboxy-terminal 11 amino acid s of endonuclease V, an enzyme made up of 138 amino acids that initiat es the repair of cyclobutane pyrimidine dimers in DNA, has demonstrate d the importance of this region in dimer-specific binding. In a previo us study, substitution of a serine residue for tryptophan 128 resulted in a protein with decreased abasic site lyase activity without a conc omitant decrease in DNA glycosylase activity [Nakabeppu, Y., et al. (1 982) J. Biol. Chem. 257, 2556-2562]. To assess the importance of the t ryptophan at position 128, six mutants were constructed by site-direct ed mutagenesis, including W128Y, W128V, W128I, W128G, W128S, and W128T . Upon characterization, these six mutants were found qualitatively to complement the repair deficiency of ultraviolet (UV) light irradiated Escherichia coli cells (recA(-), UVRA(-)) to levels comparable to tha t of wild-type endonuclease V. The activities of the mutant proteins w ere characterized using UV-irradiated plasmid DNA and oligonucleotides containing either a site-specific cyclobutane pyrimidine dimer or an abasic site. In all cases, the six mutants displayed glycosylase and a basic site lyase activities comparable to those of wild-type endonucle ase V, indicating that Trp-128 is not crucial for dimer-specific bindi ng or catalysis.