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 acids of endonuclease V, an enzyme made up of 138 amino acids that initiates the repair of cyclobutane pyrimidine dimers in DNA, has demonstrated the importance of this region in dimer-specific binding. In a previous study, substitution of a serine residue for tryptophan 128 resulted in a protein with decreased abasic site lyase activity without a concomitant decrease in DNA glycosylase activity [Nakabeppu, Y., et al. (1982) J. Biol. Chem. 257, 2556-2562]. To assess the importance of the tryptophan at position 128, six mutants were constructed by site-directed 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 that of wild-type endonuclease V. The activities of the mutant proteins were 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 abasic site lyase activities comparable to those of wild-type endonuclease V, indicating that Trp-128 is not crucial for dimer-specific binding or catalysis.
Latham
K A
KA
Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232.
Carmical
J R
JR
Lloyd
R S
RS
D001483:Base Sequence; D004249:DNA Damage / Q000528:radiation effects; D045647:DNA Glycosylases; D004260:DNA Repair; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D009841:Oligonucleotides; D014364:Tryptophan / Q000737:chemistry*; D014466:Ultraviolet Rays; D014764:Viral Proteins
D009841:Oligonucleotides; D014764:Viral Proteins; D014364:Tryptophan; D004706:Endodeoxyribonucleases; C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer); D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases