Oxidation of Thymine to 5-Formyluracil in DNA Promotes Misincorporation of dGMP and Subsequent Elongation of a Mismatched Primer Terminus by DNA Polymerase
| Autor: | Hiroaki Terato, Mutsumi Kobayashi, Aya Masaoka, Yoshihiko Ohyama, Hiroshi Ide |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
DNA Replication
DNA Repair Base Pair Mismatch DNA polymerase Base pair Biochemistry chemistry.chemical_compound Escherichia coli Uracil Molecular Biology Klenow fragment Base Sequence biology DNA replication Deoxyguanine Nucleotides DNA Templates Genetic Cell Biology DNA Polymerase I Molecular biology Thymine Kinetics Oligodeoxyribonucleotides chemistry biology.protein Primer (molecular biology) DNA polymerase I Oxidation-Reduction |
| Zdroj: | Journal of Biological Chemistry. 276:16501-16510 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m008598200 |
| Popis: | 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 (f(A) = V(max)/K(m)) decreased to (1/4) to (1/2), depending on the nearest neighbor base pair, and that of dGMP (f(G)) increased 1.1-5.6-fold. Thus, the increase in the replication error frequency (f(G)/f(A) for fU versus T) was 3.1-14.3-fold. The misincorporation rate of dGMP opposite fU (pK(a) = 8.6) but not T (pK(a) = 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. |
| Databáze: | OpenAIRE |
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