Nucleic Acids Research, Vol 25, Issue 8 1570-1577, Copyright © 1997 by Oxford University Press
M Yoshida, K Makino, H Morita, H Terato, Y Ohyama and H Ide
5-Formyluracil (fU) is one of the thymine lesions produced by reactive
oxygen radicals in DNA and its constituents. In this work, 5-formyl-2'-
deoxyuridine 5'-triphosphate (fdUTP) was chemically synthesized and
extensively purified by HPLC. The electron withdrawing 5-formyl group
facilitated ionization of fU. Thus, p K a of the base unit of fdUTP was
8.6, significantly lower than that of parent thymine (p K a = 10.0 as
dTMP). fdUTP efficiently replaced dTTP during DNA replication catalyzed by
Escherichia coli DNA polymerase I (Klenow fragment), T7 DNA polymerase
(3'-5'exonuclease free) and Taq DNA polymerase. fU-specific cleavage of the
replication products by piperidine revealed that when incorporated as T,
incorporation of fU was virtually uniform, suggesting minor sequence
context effects on the incorporation frequency of fdUTP. fdUTP also
replaced dCTP, but with much lower efficiency than that for dTTP. The
substitution efficiency for dCTP increased with increasing pH from 7.2 to
9.0. The parallel correlation between ionization of the base unit of fdUTP
(p K a = 8.6) and the substitution efficiency for dCTP strongly suggests
that the base- ionized form of fdUTP is involved in mispairing with
template G. These data indicate that fU can be specifically introduced into
DNA as unique lesions by in vitro DNA polymerase reactions. In addition, fU
is potentially mutagenic since this lesion is much more prone to form
mispairing with G than parent thymine.
ARTICLES
Substrate and mispairing properties of 5-formyl-2'-deoxyuridine 5'- triphosphate assessed by in vitro DNA polymerase reactions
Department of Polymer Science and Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606, Japan.
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