Oligonucleotide Site Directed Mutagenesis of All Histidine Residues within the T4 Endonuclease V Gene: Role in Enzyme-Nontarget DNA Binding

Mary Lou Augustine, Richard W. Hamilton, M. L. Dodson, R. Stephen Lloyd

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

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 His 16 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.

Original languageEnglish (US)
Pages (from-to)8052-8059
Number of pages8
JournalBiochemistry
Volume30
Issue number32
DOIs
StatePublished - Aug 1 1991

ASJC Scopus subject areas

  • Biochemistry

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