©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Formation of 2-Hydroxydeoxyadenosine Triphosphate, an Oxidatively Damaged Nucleotide, and Its Incorporation by DNA Polymerases
STEADY-STATE KINETICS OF THE INCORPORATION (*)

(Received for publication, February 8, 1995; and in revised form, May 19, 1995)

Hiroyuki Kamiya Hiroshi Kasai (§)

From the Department of Environmental Oncology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

We found that hydroxylation occurs at the C-2 position of adenine by oxygen radical treatment (Fe-EDTA) of dA, dATP, and single- and double-stranded DNA. This oxidatively damaged base, 2-hydroxyadenine, was produced 3-6-fold and 40-fold less than 8-hydroxyguanine when monomers and polynucleotides, respectively, were treated. To determine whether the damaged nucleotide, 2-hydroxydeoxyadenosine triphosphate (2-OH-dATP), is incorporated into a growing DNA, and to reveal the kinds of nucleotides opposite which 2-OH-dATP is incorporated, calf thymus DNA polymerase alpha and the Klenow fragment of Escherichia coli DNA polymerase I were used in in vitro DNA synthesis in the presence of 2-OH-dATP. DNA polymerase alpha incorporated the nucleotide opposite T and C in the DNA template. On the other hand, in an experiment using the Klenow fragment, incorporation of 2-OH-dATP was observed only opposite T. Steady-state kinetic studies indicated that incorporation of 2-OH-dATP by DNA polymerase alpha opposite T was favored over that opposite C by a factor of only 4.5. These results indicate that 2-OH-dATP, an oxidatively damaged nucleotide, is a substrate for DNA polymerases and is incorporated incorrectly by the replicative DNA polymerase.


INTRODUCTION

Oxygen radicals produced in cells damage DNA and nucleotides, and these processes are probably involved in mutagenesis and carcinogenesis. One of the oxidative DNA damage is 8-hydroxyguanine (8-OH-Gua). (^1)8-OH-Gua is produced by treatment of DNA with reagents that produce oxygen radicals(1) , and it induces mainly G to T mutations(2, 3, 4, 5, 6, 7) . The mutT mutant of Escherichia coli, in which AbulletT to CbulletG transversions are induced with high frequency(8) , lacks the activity that hydrolyzes 8-OH-dGTP(9) . Together with the fact that DNA polymerases incorporate 8-OH-dGTP as a substrate(7, 9) , it appears that 8-OH-Gua is generated by incorporation of the damaged nucleotide as well as by direct modification of G in DNA(1) .

A question that has arisen is whether other modified nucleotides that are produced by oxygen radicals are incorporated by DNA polymerases, and whether the incorporated nucleotides induce mutations. We report here that when dA, dATP, and DNA were treated with oxygen radicals (Fe-EDTA)(10) , hydroxylation at the C-2 position of adenine occurred to form 2-OH-Ade (2-hydroxyadenine, 1,2-dihydro-2-oxoadenine, or isoguanine) and that the hydroxylation in dATP and dA occurred more frequently than that in DNA. Although Switzer et al.(11, 12) observed that the Klenow fragment of E. coli DNA polymerase I (KF) and other DNA polymerases incorporate 2-OH-dATP (``d-iso-GTP'' in their reports) opposite T in a template DNA, they studied 2-OH-Ade as a component of additional base pairs in nucleic acids, and not as oxidative damage. They did not investigate the possibility of the incorporation opposite A and G (C was used as a control). Moreover, they did not use mammalian enzymes, which are important for studies of carcinogenesis. We describe the results of in vitro DNA synthesis reactions with mammalian DNA polymerase alpha (pol alpha) and KF in the presence of 2-OH-dATP.


EXPERIMENTAL PROCEDURES

Materials

T4 polynucleotide kinase was purchased from Toyobo Co. Calf thymus pol alpha was obtained from Molecular Biology Resources Inc. The Klenow fragment of DNA polymerase I was from Life Technologies, Inc. Calf thymus DNA, dA, dG, dATP, and dGTP, which were used in the Fe-EDTA treatment, were from Sigma. The FPLC-grade deoxynucleoside triphosphates used in the DNA polymerase reactions were from Pharmacia Biotech Inc. 2-OH-dA was prepared from 2,6-diaminopurine-9-deoxyriboside (Sigma) according to the method of Davoll(13) .

Treatment of Nucleosides, Nucleotides, and DNA with Fe-EDTA

Single- and double-stranded DNA, dA, dG, dATP, or dGTP (1 mg) was treated with Fe-EDTA (5 mM FeSO(4), 5 mM EDTA) in 10 mM sodium phosphate (pH 7.4) in a total volume of 1.0 ml at 37 °C for 30 min. Samples were then analyzed directly (for monomers) or after enzymatic digestion (for DNA) by reverse-phase HPLC. Separation of 2-OH-dA was done using two Ultrasphere ODS 5 µ columns (4.6 250 mm, Beckman) connected in series, with an isocratic system consisting of 20 mM sodium phosphate (pH 7.0). A single Ultrasphere ODS 5-µm column (4.6 250 mm) was used for 8-OH-dG, with an isocratic system consisting of 10 mM NaH(2)PO(4) and 8.0% methanol. 8-OH-dGTP and 2-OH-dATP were separated by HPLC using a YMC-pack ODS-AM-303 column (4.6 250 mm, 5 µm), with an isocratic system consisting of 12.5 mM citric acid, 25 mM sodium acetate, 10 mM acetic acid, and 30 mM sodium hydroxide. Detection was performed with a Hewlett Packard 1040M HPLC Detection System.

Purification of 2-OH-dATP

dATP (5 mg), dissolved in 1 ml of 100 mM sodium phosphate (pH 7.4), was treated with 25 mM Fe-EDTA in an open 10-ml test tube by shaking at 37 °C for 120 min. The reaction mixture was directly injected into the HPLC column, and 2-OH-dATP was purified by reverse-phase HPLC, as described above.

In Vitro DNA Synthesis

Primers (10 pmol) were labeled at the 5`-ends by T4 polynucleotide kinase (10 units) in the presence of [-P]ATP (0.74 MBq, DuPont NEN) in a buffer solution of 50 mM Tris-HCl (pH 8.0), 10 mM MgCl(2), and 10 mM 2-mercaptoethanol in a total volume of 10 µl. After incubation at 37 °C for 60 min, the unincorporated ATP was removed with NENSORB® 20 (Du Pont NEN).

Reactions catalyzed by KF were carried out in a buffer solution containing oligonucleotide templates annealed with primers (0.05 µM), 50 mM Tris-HCl (pH 8.0), 8 mM MgCl(2), 5 mM 2-mercaptoethanol, various amounts of dNTP(s), and enzyme in a total volume of 10 µl at 20 °C. Experiments with pol alpha were conducted at 25 °C in a 10-µl reaction mixture containing oligonucleotide templates annealed with primers (0.05 µM), 20 mM Tris-HCl (pH 8.0), 5 mM MgCl(2), 3.3 mM 2-mercaptoethanol, bovine serum albumin (0.2 mg/ml), and various amounts of dNTP(s) and enzyme. Reactions were stopped by the addition of termination solution (95% formamide, 0.1% bromphenol blue, and 0.1% xylene cyanol). Samples were heated at 95 °C for 3 min and were then applied to a 7 M urea, 20% polyacrylamide gel. Autoradiograms were obtained with a Fujix BAS 2000 bioimage analyzer.

Steady-state Kinetics

Experiments with KF were done under the conditions described above using 0.05-50 µM dNTP and 0.002 units (one unit incorporates 10 nmol of deoxyribonucleotides into acid-precipitable material in 30 min at 37 °C) of the enzyme at 20 °C for 1-6 min. Reactions with pol alpha were measured under the conditions described above, using 0.05-200 µM dNTP and 0.14-0.7 units (one unit incorporates 1 nmol of deoxyribonucleotides in 1 h at 37 °C) of the enzyme at 25 °C for 3-30 min. The Michaelis constant (K) and the maximum velocity of the reaction (V(max)) were obtained from Lineweaver-Burk plots of the kinetic data(14) . Insertion frequencies (F) were determined relative to AbulletT or CbulletG according to the equations developed by Mendelman et al.(15) . F = (V(max)/K of wrong pair)/(V(max)/K of right pair), with the ``wrong pair'' defined as any base pair with 2-OH-Ade. All reaction rates were linear during the course of the reaction, in which less than 20% of the primer was extended(16) .


RESULTS

Formation of 2-OH-Ade in Nucleosides, Nucleotides, and DNA by Fe-EDTA Treatment

When dATP and dA were treated with Fe-EDTA, formation of modified monomers, which were separated from starting materials by HPLC, was observed. Fig. 1shows the HPLC elution profile when the reaction mixture of dATP was injected. The peak, indicated as 2-OH-dATP by the arrow (retention time, 5.5 min), showed the UV spectrum characteristic of 2-OH-dA or 2-OH-Ade derivatives (17) (Fig. 1, inset). The modified monomer obtained by Fe-EDTA treatment of dA also showed similar UV spectrum (data not shown), suggesting that 2-OH-dA was formed. The structure of the modified dA was confirmed to be 2-OH-dA by comparing its chromatographic behavior with that of the synthetic sample(13) . Although we have no standard of 2-OH-dATP, the dephosphorylated product of the 5.5-min peak, which was obtained by Fe-EDTA treatment of dATP, was eluted at the same retention time as the synthetic 2-OH-dA (data not shown). Therefore, it was concluded that 2-OH-dA and 2-OH-dATP were produced by Fe-EDTA treatment of dA and dATP, respectively.


Figure 1: Separation of 2-OH-dATP by reverse-phase HPLC. Fe-EDTA treatment and separation by HPLC were done as described under ``Experimental Procedures.'' The material eluted at 5.4 min was identified as 2-OH-dATP by UV spectra (inset).



The yields of 2-OH-dA and 2-OH-dATP were 800/10^5 dA and 650/10^5 dA, respectively, and were 3.6- and 5.7-fold less than those of 8-OH-dG and 8-OH-dGTP (Table 1). On the other hand, the formation of 2-OH-Ade was less efficient, about 40-fold less than that of 8-OH-Gua, when double- and single-stranded DNA were treated (9.7 and 17.7/10^5 dA, Table 1). Therefore, 2-OH-Ade appears to be produced more efficiently in monomers than in polynucleotides.



Incorporation of 2-OH-dAMP by Klenow Fragment

Next, we analyzed the incorporation of 2-OH-dAMP during in vitro DNA synthesis in the presence of 2-OH-dATP. Template 1 (15-mer) annealed with a P-labeled primer 1 (10-mer) was used in the reactions (Table 2). The primer was elongated to an 11-mer when either dAMP or 2-OH-dAMP was incorporated by a DNA polymerase opposite the T residue in the template. Subsequent incorporations of dGMP and of dGMP and dCMP generated the 12-mer and the 13-mer, respectively. The incorporation of dGMP, dCMP, and dTMP after the insertion of dAMP or 2-OH-dAMP generated a 14-mer and/or 15-mer. When a nucleotide was inserted incorrectly, an extra band was observed in a denaturing polyacrylamide gel. Fig. 2shows the PAGE analysis of the primer extensions catalyzed by KF. In control experiments in which dATP was used, the primer was extended by one nucleotide by the addition of dATP (Fig. 2A, lane2). The primer was further extended, corresponding to the nucleotides added (Fig. 2A, lanes 3-5). A similar pattern was observed in the presence of 2-OH-dATP, and no extra elongations were detected (Fig. 2A, lanes6-9). These results indicate that KF incorporated 2-OH-dAMP only opposite T. This conclusion was confirmed by the polymerase reactions in the presence of the three dNTPs and 2-OH-dATP. Namely, the primed template was treated with KF in the presence of three of the four dNTPs. The full-length product was produced when the added 2-OH-dATP was used by the DNA polymerase instead of the missing dNTP. The fully elongated product was observed only when 2-OH-dATP was added to the reaction lacking A (Fig. 2B, lanes2 and 3). Again, it was concluded that KF incorporated 2-OH-dAMP only opposite T. Moreover, these results indicate that the incorporated 2-OH-dAMP did not terminate the reaction and was further elongated (in this case, KF incorporated dGMP next to the inserted 2-OH-dAMP). We obtained the same conclusion when another template (template 2, Table 2) was used (data not shown).




Figure 2: Incorporation of 2-OH-dATP by Klenow fragment. A, template 1 was annealed with P-labeled primer 1. The template-primer complex (0.05 µM) was treated with KF (0.1 units) in the presence of 50 µM of each of the dNTP(s) in a total volume of 10 µl under the conditions described under ``Experimental Procedures.'' The reaction mixtures were incubated at 20 °C for 60 min and were processed as described under ``Experimental Procedures.'' Lane1, untreated primer 1; lane2, in the presence of dATP; lane3, in the presence of dATP and dGTP; lane4, in the presence of dATP, dGTP, and dCTP; lane5, in the presence of dATP, dGTP, dCTP, and dTTP; lane6, in the presence of 2-OH-dATP; lane7, in the presence of 2-OH-dATP and dGTP; lane8, in the presence of 2-OH-dATP, dGTP, and dCTP; lane9, in the presence of 2-OH-dATP, dGTP, dCTP, and dTTP. B, the template-primer complex (0.05 µM) was treated with KF (0.1 units) in the presence of 50 µM of each of the three dNTPs, with or without 50 µM 2-OH-dATP, in a total volume of 10 µl under the conditions described under ``Experimental Procedures.'' The reaction mixtures were incubated at 20 °C for 30 min and were processed as described under ``Experimental Procedures.'' Lanes1 and 10, untreated primer 1; lane2, in the presence of dGTP, dCTP, and dTTP; lane3, in the presence of 2-OH-dATP, dGTP, dCTP, and dTTP; lane4, in the presence of dATP, dCTP, and dTTP; lane5, in the presence of 2-OH-dATP, dATP, dCTP, and dTTP; lane6, in the presence of dATP, dGTP, and dTTP; lane7, in the presence of 2-OH-dATP, dATP, dGTP, and dTTP; lane8, in the presence of dATP, dGTP, and dCTP; lane9, in the presence of 2-OH-dATP, dATP, dGTP, and dCTP.



Incorporation of 2-OH-dAMP Opposite T and C by DNA Polymerase alpha

In experiments using primed template 1 and 2-OH-dATP, the primer was elongated by pol alpha to an 11-mer by the addition of the modified nucleotide (Fig. 3A, lane2). Furthermore, a small amount of 12-mer was detected in the presence of a single 2-OH-dATP (Fig. 3A, lane2). This indicated that 2-OH-dAMP was incorporated opposite C in the template, in addition to T. No extra bands were observed in the other lanes (Fig. 3A, lanes3 and 4), indicating that 2-OH-dATP was not inserted opposite G and A in the template. Similar results were obtained when template 2 was used in the reactions. In this case, the base at position 11 in the template is C, and T is located at position 13 (Table 2). As shown in Fig. 3B, 2-OH-dAMP was incorporated opposite C (lane2). The extra 13-mer in the reaction containing 2-OH-dATP and dCTP was possibly produced by the insertion of 2-OH-dAMP opposite T (Fig. 3B, lane3). These results indicate that pol alpha inserted 2-OH-dAMP opposite C in addition to T.


Figure 3: Incorporation of 2-OH-dATP by DNA polymerase alpha. A, template 1 was annealed with P-labeled primer 1. The template-primer complex (0.05 µM) was treated with pol alpha (1.4 units) in the presence of 50 µM of each of the dNTP(s) in a total volume of 10 µl, under the conditions described under ``Experimental Procedures.'' The reaction mixtures were incubated at 25 °C for 120 min and were processed as described under ``Experimental Procedures.'' Lane1, untreated primer 1; lane2, in the presence of 2-OH-dATP; lane3, in the presence of 2-OH-dATP and dGTP; lane4, in the presence of 2-OH-dATP, dGTP, and dCTP; lane5, in the presence of 2-OH-dATP, dGTP, dCTP, and dTTP. B, reaction conditions were the same as described above, except that template 2 was used instead of template 1. Lane1, untreated primer 1; lane2, in the presence of 2-OH-dATP; lane3, in the presence of 2-OH-dATP and dCTP; lane4, in the presence of 2-OH-dATP, dCTP, and dATP; lane5, in the presence of 2-OH-dATP, dCTP, dATP, and dTTP.



We recently found that DNA polymerases (DNA polymerases alpha and beta and KF) misincorporated dAMP opposite 2-OH-Ade in a template DNA(18) . However, we did not detect the incorporation of 2-OH-dATP opposite A in template DNAs, as shown in Fig. 2and Fig. 3. Since the nucleotide sequence used in the previous study (5`-dGCTA*ATATTCCGTCAT-3`, where A* represents 2-OH-Ade) was different from those used in this study, we tested whether 2-OH-dATP was inserted opposite A in a template with the same flanking sequences as used in the previous study (Table 2, template 3). However, pol alpha did not insert 2-OH-dATP opposite A, whereas the enzyme incorporated the nucleotide opposite T and C (data not shown). The modified nucleotide was incorporated opposite T and C, but not opposite A, even in the sequence where dAMP is inserted opposite 2-OH-Ade.

Steady-state Kinetics

We measured the kinetic parameters during in vitro DNA synthesis. The incorporation of 2-OH-dATP opposite T and C, using templates 1 and 2, respectively, was studied. Table 3shows the parameters of the reactions. In reactions catalyzed by KF, the Kvalue of 2-OH-dATP was 96 times higher than that of dATP. The V(max) value of 2-OH-dATP was 8 times lower than that of dATP. The relative V(max)/K (the F) of 2-OH-dATP was 1.3 10, which means that the insertion of 2-OH-dAMP opposite T was disfavored by a factor of 745 over that of dAMP. With pol alpha, the K value of 2-OH-dATP was 445 times higher than that of dATP. On the other hand, the V(max) value of 2-OH-dATP was higher than that of dATP. The relative V(max)/K (the F) of 2-OH-dATP was 3.4 10, which means that the insertion of 2-OH-dAMP opposite T was disfavored by a factor of 288 over that of dAMP. In reactions containing pol alpha and template 2, the K value of 2-OH-dATP was 88 times higher than that of dGTP. The V(max) value of 2-OH-dATP was 14 times lower than that of dGTP. The F of 2-OH-dATP was 7.5 10, which means that the insertion of 2-OH-dAMP opposite C was disfavored by a factor of 1232 over that of dGMP. Thus, the incorporation of 2-OH-dAMP opposite T was favored only 4.5 times over that opposite C. These results suggest that the formation of 2-OH-dATP will be mutagenic because it is incorporated incorrectly by pol alpha.




DISCUSSION

In this study we found that 2-OH-dATP is efficient produced from dATP by treatment with oxygen radicals and that it is incorporated into DNA incorrectly. It is interesting to compare the efficiency of incorporation of 2-OH-dATP into DNA with that of 8-OH-dGTP. The relative V(max)/K values of 8-OH-dGTP opposite C and A are 4.4 10 and 7.2 10, respectively, with exonuclease-free KF(19) . The obtained relative V(max)/Kvalues (the F) of 2-OH-dATP opposite T and C (Table 3) were comparable to those of 8-OH-dGTP reported with exonuclease-free KF(19) . Therefore, 2-OH-dATP appears to be incorporated as effectively as 8-OH-dGTP by DNA polymerases.

In aqueous solution, the major tautomers of the 9-methyl and ribosyl derivatives of 2-OH-Ade are in the N(1)H, 2-keto form(17) . The keto and enol forms are calculated to be about 90% and about 10%, respectively, of the total in water(17) . The postulated base pairs of 2-OH-Ade with T and C are shown in Fig. 4(A and B). With T, the enol tautomer of 2-OH-Ade can form a pair in a Watson-Crick manner, although the keto tautomer can also pair in a wobble alignment. In this putative Watson-Crick pair, the two bases form three hydrogen bonds as GbulletC. With C, both tautomers of 2-OH-Ade can form wobble pairs. The kinetic data obtained in this study indicated that the V(max) value of Tbullet2-OH-Ade with pol alpha was larger than that of TbulletA, although the K value of Tbullet2-OH-Ade was larger (Table 3). This fact may be explained by the small ratio of the enol tautomer in the solution used in this study, and by rapid phosphodiester bond formation due to the more stable hydrogen bonding of Tbullet2-OH-Ade (enol) than TbulletA.


Figure 4: Postulated base pairs involving 2-OH-Ade. A, 2-OH-AdebulletT pair; B, 2-OH-AdebulletC pair; C: 2-OH-AdebulletA pair.



We recently found that KF and pol alpha inserted dAMP as well as dTMP opposite 2-OH-Ade in in vitro DNA synthesis(18) . On the other hand, the two DNA polymerases did not insert 2-OH-dATP opposite A in template DNAs, and pol alpha incorporated 2-OH-dATP opposite C (Figs. 2 and 3). This discrepancy may depend on the different sequence contexts used in both studies (see ``Results''). However, we did not observe the incorporation of 2-OH-dATP opposite A and G in the template that has the same flanking sequences near the target position (Table 2). The reasons for the different tendencies remain to be resolved. One possibility is that 2-OH-Ade can form a base pair with A when the modified base is in the enol form (Fig. 4C). Sepiol et al. reported that the equilibrium between the keto and enol forms of 2-OH-Ade shifts in the direction of the enol form with a decrease in the solvent polarity(17) . It is known that a base moiety in DNA is surrounded by neighboring bases that are stabilized by stacking, and is in a more hydrophobic environment than the monomer. Therefore, the structure of 2-OH-Ade in DNA may shift more to the 2-enol form than in the monomer. Furthermore, the DNA molecule is surrounded by a DNA polymerase when DNA synthesis occurs. The polarity of the active site of a DNA polymerase may be less polar than water. Thus, the structure of 2-OH-Ade in a template DNA to which a DNA polymerase binds may significantly shift to the enol form, as suggested by Switzer et al.(11) , and may pair with A (Fig. 4C). On the other hand, the ratio of the enol form in 2-OH-dATP is probably similar to that in the 9-methyl and ribosyl derivatives of 2-OH-Ade. If the equilibrium shifts from the keto to the enol form slowly, then 2-OH-dATP exists almost in the keto form, even near the active site of a DNA polymerase, and could dissociate from the position opposite A in a template. In contrast, the facts that 2-OH-dAMP was incorporated opposite C (Fig. 3), and that dCTP was not inserted opposite 2-OH-Ade in the template(18) , may be explained if C pairs with the keto form of 2-OH-Ade (Fig. 4B).

In the case of 8-OH-dGTP, the MutT protein and its homologues prevent the mutagenic nucleotide from being incorporated into DNA, by hydrolyzing 8-OH-dGTP to 8-OH-dGMP(9, 20) . A MutT-like activity that destroys 2-OH-dATP may be present in bacterial and mammalian cells.

In this report, we have shown that 2-OH-Ade is formed by oxygen radicals, and that KF and a replicative polymerase, pol alpha, incorporated 2-OH-dATP. The latter enzyme inserted the nucleotide opposite C, in addition to T, in template DNAs.


FOOTNOTES

*
This work was supported by grants-in-aid from the Ministry of Health and Welfare of Japan and from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-93-691-7468; Fax: 81-93-601-2199.

(^1)
The abbreviations used are: 8-OH-Gua, 8-hydroxyguanine; 2-OH-Ade, 2-hydroxyadenine; 2-OH-dA, 2-hydroxy-2`-deoxyadenosine; 2-OH-dATP, 2-hydroxy-2`-deoxyadenosine 5`-triphosphate; 2-OH-dAMP, 2-hydroxy-2`-deoxyadenosine 5`-monophosphate; 8-OH-dG, 8-hydroxy-2`-deoxyguanosine; 8-OH-dGTP, 8-hydroxy-2`-deoxyguanosine 5`-triphosphate; 8-OH-dGMP, 8-hydroxy-2`-deoxyguanosine 5`-monophosphate; KF, Klenow fragment of E. coli DNA polymerase I; pol alpha, DNA polymerase alpha; F, frequency of insertion; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography.


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