Nucleic Acids Research, Vol 25, Issue 8 1548-1552, Copyright © 1997 by Oxford University Press
K Negishi, DM Williams, Y Inoue, K Moriyama, DM Brown and H Hayatsu
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.
ARTICLES
The mechanism of mutation induction by a hydrogen bond ambivalent, bicyclic N4-oxy-2'-deoxycytidine in Escherichia coli
Gene Research Center, Okayama University, Tsushima, Okayama 700, Japan. knegishi@grrsews1.okayama-u.ac.jp
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