Substrate and Mispairing Properties of 5-Formyl-2'-Deoxyuridine 5'-Triphosphate Assessed by in vitro DNA Polymerase Reactions

Abstract

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.

Voltammetric determination of all DNA nucleotides

“…For cytosine, for pH < 4.5, a value of À88 mV for dE p /dpH was obtained and this can be associated to the first pK a of cytosine that is equal to 4.6 [1,20].…”

“…This indicates that the proton/electron ratio involved in the cytosine oxidation changed to $2, and the change in slope can be associated to the pK a of cytosine that is equal to 10.2 [1,20].…”

Enzymatic Repair of 5-Formyluracil

“…Preparation of 5-formyl-2Ј-deoxyuridine 5Ј-triphosphate (fdUTP) was reported previously (29). 5-Hydroxymethyl-2Ј-deoxyuridine 5Ј-triphosphate (hdUTP) was synthesized by NaBH 4 reduction of fdUTP and extensively purified by HPLC as described for fdUTP (29). The structure and purity of hdUTP were confirmed by alkaline phosphatase digestion of the triphosphate to the corresponding nucleoside followed by reversed phase HPLC analysis (data not shown).…”

“…Four normal dNTPs were obtained from Amersham Pharmacia Biotech, and N-methyl-NЈ-nitro-N-nitrosoguanidine (MNNG) was from Wako. Preparation of 5-formyl-2Ј-deoxyuridine 5Ј-triphosphate (fdUTP) was reported previously (29). 5-Hydroxymethyl-2Ј-deoxyuridine 5Ј-triphosphate (hdUTP) was synthesized by NaBH 4 reduction of fdUTP and extensively purified by HPLC as described for fdUTP (29).…”

“…We have recently shown that fU belonging to group IV products forms a mispair with guanine in addition to a correct base pair with adenine during DNA replication (29). Ionization of fU promoted by the electron-withdrawing formyl group is involved in the mispairing between fU and guanine.…”