Oligonucleotide site directed mutagenesis of all histidine residues within the T4 endonuclease V gene: role in enzyme-nontarget DNA binding.
In order to evaluate the contributions that histidine residues might play both in the catalytic activities of endonuclease V and in binding to nontarget DNA, the technique of oligonucleotide site directed mutagenesis was used to create mutations at each of the four histidine residues in the endonuclease V gene. Although none of the histidines were shown to be absolutely required for the pyrimidine dimer specific DNA glycosylase activity or the apurinic lyase activity, conservative amino acid changes at His16 produced enzymes with little or no catalytic activity. In addition, the evaluation of conservative and radical amino acid substitutions at positions 34, 56, and 107 is consistent with the interpretation that each of these histidines may be involved in nontarget DNA binding. The data supporting this conclusion are that histidine changes to lysine at positions 34 and 107 enhance the nontarget DNA binding activity of the mutant enzymes while neutralization of charge at His56 reduces nontarget DNA binding.
Augustine
M L
ML
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Hamilton
R W
RW
Dodson
M L
ML
Lloyd
R S
RS
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair / Q000528:radiation effects; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000528:radiation effects; D005814:Genes, Viral; D006639:Histidine; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D011485:Protein Binding; D011740:Pyrimidine Dimers; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013604:T-Phages / Q000201:enzymology* / Q000235:genetics / Q000528:radiation effects; D014466:Ultraviolet Rays; D014764:Viral Proteins; D015678:Viral Structural Proteins / Q000235:genetics*
D015345:Oligonucleotide Probes; D011740:Pyrimidine Dimers; D011994:Recombinant Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; D006639:Histidine; D004247:DNA; D004706:Endodeoxyribonucleases; C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer)