The mechanism of mutation induction by a hydrogen bond ambivalent, bicyclic N4-oxy-2'-deoxycytidine in Escherichia coli.
The triphosphate of the nucleoside deoxyribosyl dihydropyrimido[4,5-c][1,2]oxazin-7-one (dP) is known to be incorporated into DNA efficiently by Taq polymerase and is a useful tool for polymerase-mediated in vitro mutagenesis. It is shown here that dP is a potent mutagen in Escherichia coli and Salmonella typhimurium . In E.coli , this deoxycytidine analog induces both GC-->AT and AT-->GC transitions. No induced transversions are observed. It is highly mutagenic in wild-type E.coli, but this is much reduced in a strain lacking thymidine kinase. Mutagenesis induced by dP is efficiently inhibited by the addition of thymidine. Partially purified thymidine kinase from E.coli catalyzes phosphorylation of dP to its 5'-monophosphate. When E.coli was grown in the presence of dP, the nucleoside analog was incorporated into its DNA. The content of dP in DNA was dependent on the concentration of dP added to the medium. The incorporation characteristics of the 5'-triphosphate of dP (dPTP) were also studied using E.coli DNA polymerase I large fragment. The results confirm that this triphosphate can be incorporated opposite A and G in the template with similar efficiencies. This indicates that dP is metabolized as a thymidine analog and that the resulting triphosphate induces a high rate of mutagenesis through replicational errors.
Negishi
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K
Gene Research Center, Okayama University, Tsushima, Okayama 700, Japan. knegishi@grrsews1.okayama-u.ac.jp
Williams
D M
DM
Inoue
Y
Y
Moriyama
K
K
Brown
D M
DM
Hayatsu
H
H
D001483:Base Sequence; D002876:Chromosomes, Bacterial; D017931:DNA Primers; D004269:DNA, Bacterial / Q000096:biosynthesis* / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D007700:Kinetics; D015394:Molecular Structure; D016296:Mutagenesis; D009153:Mutagens / Q000494:pharmacology*; D019914:Taq Polymerase; D013698:Templates, Genetic
D017931:DNA Primers; D004269:DNA, Bacterial; D009153:Mutagens; D003841:Deoxycytidine; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase