Human and bacterial DNA polymerases discriminate against 8-oxo-2'-deoxyadenosine- 5'-triphosphate

Aim. 8-Oxoadenine is an abundant DNA lesion associated with cancer and neurodegeneration. It may appear through direct oxidation of adenine in DNA or by incorporation from the oxidized dNTP pool. Methods. We developed an efficient method of synthesizing 8-oxo-2'-deoxyadenosine-5'-triphosphate and studied its incorporation by various DNA polymerases. Results. oA was weakly misincorporated opposite guanine by the DNA polymerase I Klenow fragment. Limited incorporation of oA was observed opposite guanine and adenine with DNA polymerase a, and opposite adenine, thymine and guanine with DNA polymerase b. Conclusions. Adenine oxidation in DNA may outweigh damage to dATP as a source of genomic oA.

Introduction.8-Oxoadenine (oA) is a major product of adenine damage by ionizing radiation and metabolically generated free radicals [1].The levels of oA in DNA are similar to those of other ubiquitous lesion, 8-oxoguanine [2], and increase in tumors [3].When present in DNA, oA is weakly mutagenic [4].Intriguingly, human cells possess two enzymes, OGG1 and NEIL1, that remove oA from oA:C pairs but not from oA:T [5].
oA can appear in DNA in two ways.A in DNA may be directly oxidized, producing oA:T pairs.Alternatively, A in dATP may be damaged, and the resulting odATP could be used by DNA polymerases to incorporate oA opposite T or another base.Human cells express specific odATPases [6], underscoring the importance of this damaged dNTP.Whereas a wealth of data exists for 8-oxoguanine and its dNTP [7], little is known about the utilization of odATP by DNA polymerases.It has been reported that, with T in the template, odATP is ~800-fold less efficient than dATP as a substrate for the exonuclease-deficient Klenow fragment (KF exo -) [8].We describe an efficient synthesis of odATP and an analysis of its use by DNA polymerases.
Materials and methods.Synthesis of odATP.8oxo-2'-deoxyadenosine (Fig. 1, 2) was synthesized from 8-bromo-2'-deoxyadenosine 1 («ChemGenes», USA) by treatment with 3 M equiv. of 2-mercaptoethanol and 10 M equiv. of triethylamine in water [9].The product (90 % yield) was purified by chromatography on a C 18 silica gel column in water-acetonitrile.Compound 2 was identified by comparison of 1 H NMR and MS spectra with the literature data [9].5'-Triphosphate of 2 was synthesized by the Ludwig method [10] with modifications.The solution of 2 (100 mg, 0.374 mmol) in anhydrous trimethyl phosphate and tributylamine (267 µl, 1.122 mmol) was chilled on ice, and freshly distilled phosphorous oxychloride (77 µl, 0.823 mmol) was added.The mixture was stirred for 20 min and mixed into 0.5 M bis(tetra-n-butylammonium) pyrophosphate in acetonitrile (2.24 ml, 1.122 mmol) and tributylamine (0.267 ml, 1 mmol) with vigorous stirring.After stirring for 30 min at room temperature, 30 ml of 1 M triethylammonium bicarbonate (pH 7.5) was added.After 2 h, the reaction mixture Results and discussion.The yield of odATP was 33 mg (45 % of the starting material).odATP was used for primer extension by KF exo -in a binary primertemplate system (Fig. 2, A).As shown in Fig. 2, B, little if any incorporation was observed opposite T, whereas the primer was quickly extended when dATP was present.This observation confirms the previous report of poor substrate properties of odATP for KF exo - [8].No incorporation of oA occurred opposite A or C.However, with G in the template, the primer was elongated by one and two nucleotides after 30 min.Incorporation of two oA residues is consistent with the presence of ano- ther G in the + 2 position (Fig. 2, A).The oA(syn): :G(anti) Hoogsteen pair is stable [11], permitting oA incorporation opposite G.
KF is a member of DNA polymerase Family A, whereas most human DNA polymerases belong to other families.To assess the mutagenic potential of odATP in human cells, we performed the reaction using the highfidelity Pol a (Family B), and two Family X enzymes, Pol b and Pol l , normally participating in DNA repair [12].With the binary primer-template, all three enzymes efficiently incorporated A opposite T (Fig. 2, C).Binary primer-template is suboptimal for Pol b and Pol l, which prefer substrates with a short gap [13].Therefore, we studied the behavior of Pol a , Pol b , and Pol l when presented with odATP and a gapped substrate consisting of a template, a primer, and a downstream strand (Fig. 3, A).All polymerases efficiently incorporated A opposite T (Fig. 3, B-D).Pol a misincorporated A opposite A and, less efficiently, opposite C. In contrast, no incorporation of oA opposite C or T was observed.When A or G were in the template, oA was weakly incorporated with some extension to the +2 position, similar to that observed with KF and the binary primer-template.Pol b misincorporated A opposite A, C, and G, whereas oA was incorporated much worse, with the order of the template preference T > G > A. Pol l catalyzed only normal incorporation of A opposite T, did not form mismatches with A and did not incorporate oA.
Finally, we inquired whether odATP is used by DNA polymerases present in mammalian cell extracts.Whole-cell mouse embryonic fibroblast extracts efficiently incorporated A opposite T (Fig. 4).However, no incorporation of oA was evident.It is still possible that such incorporation is not observed due to the primer degradation by nucleases.
It is instructive to compare the situation when oA is present in dNTP and in DNA.KF bypasses template oA  in an error-free manner when all four dNTPs are available [4].On the contrary, when this lesion is present as odATP, it is poorly incorporated by KF and tends to be misincorporated opposite G. Small amounts of G or A are incorporated opposite oA by KF, Pol a and Pol b when only dGTP or dATP is present [4].In our experiments, a small degree of misincorporation of oA by Pol a and Pol b is also observed opposite A and G, suggesting that the acceptance of oA may require Hoogsteentype pairing.
Conclusions.Our data hint that oxidative damage of the dATP pool and incorporation of oA may be less important than direct oxidation of A in DNA as a source of genomic oA.However, this does not mean that odATP has no effect in vivo.The proposed synthetic pathway to oxodATP will permit a more detailed investigation of its properties.

Fig. 1 .Fig. 2 .
Fig. 1.Scheme of odATP synthesis: i -2-mercaptoethanol, TEA, H 2 O; ii -POCl 3 , n-Bu 3 N, (MeO) 3 PO; iii -(Bu 4 N) 2 H 2 P 2 O 7 However, only Pol b incorporated oA opposite T to any extent.We observed no incorporation of oA opposite C by Pol a , Pol b , or Pol l .Interestingly, taken in a large excess, Pol b and Pol l incorporated A opposite C, consistent with their lower fidelity in comparison with Pol a [12].

Fig. 4 .
Fig. 4. Extension of the primer-template substrate by mouse embryonic fibroblast extracts