Oxidation of Thymine to 5-Formyluracil in DNA Promotes
Misincorporation of dGMP and Subsequent Elongation of a Mismatched
Primer Terminus by DNA Polymerase*
Aya
Masaoka,
Hiroaki
Terato,
Mutsumi
Kobayashi,
Yoshihiko
Ohyama, and
Hiroshi
Ide
From the Department of Mathematical and Life Sciences, Graduate
School of Science, Hiroshima University,
Higashi-Hiroshima 739-8526, Japan
5-Formyluracil (fU) is a major oxidative thymine
lesion generated by ionizing radiation and reactive oxygen species. In
the present study, we have assessed the influence of fU on DNA
replication to elucidate its genotoxic potential. Oligonucleotide
templates containing fU at defined sites were replicated in
vitro by Escherichia coli DNA polymerase I Klenow
fragment deficient in 3'-5'-exonuclease. Gel electrophoretic analysis
of the reaction products showed that fU constituted very weak
replication blocks to DNA synthesis, suggesting a weak to negligible
cytotoxic effect of this lesion. However, primer extension assays with
a single dNTP revealed that fU directed incorporation of not only
correct dAMP but also incorrect dGMP, although much less efficiently.
No incorporation of dCMP and dTMP was observed. When fU was substituted
for T in templates, the incorporation efficiency of dAMP
(fA = Vmax/Km) decreased to
1/4 to 1/2, depending on the nearest neighbor base pair, and that of dGMP (fG) increased 1.1-5.6-fold.
Thus, the increase in the replication error frequency
(fG/fA for fU
versus T) was 3.1-14.3-fold. The misincorporation rate of
dGMP opposite fU (pKa = 8.6) but not T
(pKa = 10.0) increased with pH (7.2-8.6) of the
reaction mixture, indicating the participation of the ionized (or
enolate) form of fU in the mispairing with G. The resulting mismatched
fU:G primer terminus was more efficiently extended than the T:G
terminus (8.2-11.3-fold). These results show that when T is oxidized
to fU in DNA, fU promotes both misincorporation of dGMP at this site
and subsequent elongation of the mismatched primer, hence potentially mutagenic.
*
This work was supported by grants-in-aid from the Ministry
of Education, Science, and Culture of Japan (to H. I.) and by JSPS Research Fellowships for Young Scientists (to A. M.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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