Oxidation of Thymine to 5-Formyluracil in DNA Promotes Misincorporation of dGMP and Subsequent Elongation of a Mismatched Primer Terminus by DNA Polymerase

Abstract

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.Faithful replication of DNA is essential for maintaining genetic integrity of living organisms. High fidelity of DNA replication is achieved by two cellular functions that involve discrimination of correct versus incorrect nucleotides by DNA polymerases (1, 2) and postreplication mismatch repair (3). The overall error frequency of DNA replication is one in 10 8 to 10 10 base pairs when they function properly. Fidelity of DNA replication also relies on the structural integrity of DNA itself that serves as a template for the newly synthesized strand. A number of endogenous and exogenous agents have been identified to induce structural deterioration of DNA (4). Among them, reactive oxygen species generate a very complicated spectrum of DNA damage (5, 6). These lesions are mostly restored by the base excision repair pathway both in prokaryotic and eukaryotic cells, but if left unrepaired, they arrest DNA synthesis or direct misincorporation of nucleotides during DNA replication, hence exerting deleterious effects on cells (7,8). Replication blocks and nucleotide misincorporation have been related to lethality and mutation of cells, respectively, until recently. However, this concept is now challenged by the discovery of numerous error-prone and error-free DNA polymerases that can bypass the blocking lesions (9). Although the past several years have witnessed the discovery...

Derivatization with Girard Reagent T Combined with LC−MS/MS for the Sensitive Detection of 5-Formyl-2‘-deoxyuridine in Cellular DNA

“…Hela-S3 cells were from the National Cell Culture Center (Minneapolis, MN). [1,[3][4][5][6][7][8][9][10][11][12][13][14][15] N 2 , 2′-D]-5-formyl-2′-deoxyuridine was prepared according to previously described procedures 8 .…”

Fluorescence Properties of 5‐(5,6‐Dimethoxybenzothiazol‐2‐yl)‐2′‐deoxyuridine (dbtU) and Oligodeoxyribonucleotides Containing dbtU

“…[5] DNA polymerization across fo Ura in vitro with the E. coli Pol I Klenow fragment introduces guanine (Gua) as well as adenine (Ade) in DNA. [6] Zhang et al reported that dCTP as well as dATP are frequently incorporated but only a small amount of dGTP is incorporated into the site opposite to fo Ura in DNA by the Klenow fragment (exo-). [7] These misincorporations induce mutation (from A:T to G:C and A:T to C:G) by mispairing between an ionized form of fo Ura and Gua or between fo Ura and Cyt during DNA replication.…”

Differential Specificity of Human and Escherichia coli Endonuclease III and VIII Homologues for Oxidative Base Lesions

“…30UR containing a urea residue was prepared by mild alkaline treatment of 30TG5R and 30TG5S (18,41). 25FU, 25HMU, and 25OG containing 5-formyluracil (fU), 5-hydroxymethyluracil (hmU), and 7,8-dihydro-8-oxoguanine (8-oxoG), respectively, were chemically synthesized (42)(43)(44). 25HOU, 25HOC, 34FP, and 25OXA containing 5-hydroxyuracil (hoU), 5-hydroxycytosine (hoC), 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (mFapyG), and oxanine (Oxa), respectively, were prepared by DNA polymerase reactions with modified 2Ј-deoxynucleoside 5Ј-triphosphates as reported previously (19,(43)(44)(45).…”