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Nucleic Acids Research Pages 496-502  

Insertion of dGMP and dAMP during in vitro DNA synthesis opposite an oxidized form of 7,8-dihydro-8-oxoguanine
Introduction
Materials And Methods
   Materials
   DNA oxidation
   Primer extension
   Mass spectrometric analysis of oxidized 8-oxoguanine-containing oligonucleotides
Results
   Insertion of dNTPs opposite an oxidized form of 8-oxoG
   Insertion of dNTPs opposite an oxidized form of 8-oxoA
   Mass spectrometric analysis of oxidized 8-oxoG-containing oligonucleotides
Discussion
Acknowledgements
References

Insertion of dGMP and dAMP during in vitro DNA synthesis opposite an oxidized form of 7,8-dihydro-8-oxoguanine

Insertion of dGMP and dAMP during in vitro DNA synthesis opposite an oxidized form of 7,8-dihydro-8-oxoguanine

Victor Duarte, James G. Muller and Cynthia J. Burrows*

Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, UT 84112-0850, USA

Received October 1, 1998; Revised and Accepted November 18, 1998

ABSTRACT

Oxidative damage to DNA bases commonly resultsin the formation of oxidized purines, particularly 7,8-dihydro-8-oxoguanine (8-oxoG) and 7,8-dihydro-8-oxoadenine (8-oxoA), the former being a well-known mutagenic lesion. Since 8-oxoG is readily subject to further oxidation compared with normal bases, the insertion of a base during DNA synthesis opposite an oxidized form of 8-oxoG was investigated in vitro. A synthetic template containing a single 8-oxoG lesion was first treated with different one-electron oxidants or under singlet oxygen conditions and then subjected to primer extension catalyzed by Klenow fragment exo- (Kf exo-), calf thymus DNA polymerase [alpha] (pol [alpha]) or human DNA polymerase [beta] (pol [beta]). Consistent with previous reports, dAMP and dCMP are inserted selectively opposite 8-oxoG with all three DNA polymerases. Interestingly, oxidation of 8-oxoG was found to induce dAMP and dGMP insertion opposite the lesion by Kf exo- with transient inhibition of primer extension occurring at the site of the modified base. Furthermore, the lesion constitutes a block during DNA synthesis by pol [alpha] and pol [beta]. Experiments with an 8-oxoA-modified template oligonucleotide show that both 8-oxoA and an oxidized form of 8-oxoA direct insertion of dTMP by Kf exo-. Mass spectrometric analysis of 8-oxoG-containing oligonucleotides before and after oxidation with IrCl62- are consistent with oxidation of primarily the 8-oxoG site, resulting in formation of a guanidinohydantoin moiety as the major product. No evidence for formation of abasic sites was obtained. These results demonstrate that an oxidized form of 8-oxoG, possibly guanidinohydantoin, may direct misreading and misinsertion of dNTPs during DNA synthesis. If such a process occurred in vivo, it would represent a point mutagenic lesion leading to G->T and G->C transversions. However, the corresponding oxidized form of 8-oxoA primarily shows correct insertion of T during DNA synthesis with Kf exo-.

INTRODUCTION

Intracellular DNA damage caused by reactive oxygen species is known to be mutagenic (1-3) and is possibly involved in the aging process and many human diseases including cancer (4-6). The mutagenicity of the damaged DNA arises partly during its replication by a DNA polymerase (7). Thus, polymerase fidelity during DNA replication is essential to prevent mutagenesis and consequently carcinogenesis. Free radical forms of oxygen are probably the most important source of spontaneous DNA damage. Oxidative damage to DNA bases commonly results in the formation of oxidized purines, particularly 7,8-dihydro-8-oxoguanine (8-oxoG) and, to a lesser extent, 7,8-dihydro-8-oxoadenine (8-oxoA) (8,9). In vitro experiments concerning the mutagenic potential of 8-oxoG have shown that DNA polymerases can synthesize past the 8-oxoG lesion and incorporate selectively dAMP in addition to dCMP opposite the damage (2,10-12). Furthermore, the ratio between dAMP and dCMP insertion depends on the polymerase used. Replicative DNA polymerases insert preferentially dAMP, while the DNA polymerases associated with repair processes incorporate the correct dCMP (11).

Oxidative DNA damage may be repaired in cells by a variety of repair enzymes. In both bacteria and mammalian cells, repair enzymes have been discovered that possess multiple activities toward oxidative DNA damages. A repair system for the 8-oxoG lesion involving several enzymes has been elucidated in Escherichia coli by Michaels et al. (13,14). The MutM protein, also known as Fpg (15) or 8-oxoguanine glycosylase, removes the 8-oxoG lesion from the double-stranded DNA when paired to a cytosine. On the other hand, the 8-oxoG·A mismatch is repaired by a different DNA glycosylase, MutY. This protein removes the unmodified base adenine when it is paired with the 8-oxoG (13,14). Another protein, MutT, is an 8-oxo-dTPase that prevents incorportation of 8-oxo-dGTP into DNA (16). The mutagenic process resulting from an 8-oxoG-modified base and the repair system evolved to prevent these mutations have been extensively studied. However, the mutagenic potential of an oxidized 8-oxoguanine is unknown.

It has long been recognized that 8-oxoG is readily subjected to further oxidation since electrochemical detection is used for its analysis by HPLC (17). A redox potential in the range of 0.58-0.75 V versus normal hydrogen electrode (NHE) has been reported for 8-oxo-2[prime]-deoxyguanosine (18-20), significantly lower than that of 2[prime]-deoxyguanosine nucleoside (1.29 V versus NHE) (21). In vitro, the products of oxidation of 8-oxoguanosine have been analyzed under various reaction conditions, although products in oligonucleotides are unknown. Since the oxidation product profile of the parent heterocycle guanine differs in nucleosides versus duplex oligomers, caution must be exercised in extrapolating nucleoside products to those of the biopolymer(22). Electrochemical oxidation of 8-oxoguanosine was proposed to form the corresponding guanidinohydantoin ribonucleoside (Fig. 1), presumably via the 5-hydroxy-8-oxoG intermediate (23). It is noteworthy that this pathway is completely analogous to oxidative degradation of urate, which is converted both chemically and enzymatically to allantoin, the urea analog of guanidinohydantion, via 5-hydroxyurate (24). Photo-oxidation studies involving singlet oxygen generation led to different final products for the oxidation of 8-oxo-2[prime]-deoxyguanosine (25-29). Raoul and Cadet (25) found cyanuric acid as the major product of decomposition of 8-oxoG with singlet oxygen, but interestingly, imidazolone and oxazolone derivatives were also formed. These products have been observed in the photochemical one-electron oxidation of guanosine itself, in competition with formation of 8-oxoG (22,30,31). Sheu and Foote (29) reported the formation of cyanuric acid, parabanic acid and a seven-membered ring product for the oxidation of a silylated 8-oxoguanosine under conditions of singlet oxygen generation. Because 8-oxoG is easily oxidized, it is reasonable to assume that once 8-oxoG is formed, the products of further oxidative degradation of 8-oxoG may play a role in the mutagenic process associated with DNA damage.

The 8-oxoA-modified base has received less attention in the literature since the first reports on the mutagenic potential of this 8-oxopurine suggested that during in vitro DNA synthesis, dTMP was almost exclusively inserted opposite 8-oxoA (32). Wood et al. indicated that 8-oxoA was at least an order of magnitude less mutagenic than 8-oxoG in bacteria (33). However, Kamiya et al. (34) reported the insertion of dGMP, or dAMP plus dGMP, using DNA polymerases [alpha] or [beta], respectively. Little is known about the products of further oxidation of 8-oxoA since 8-oxoA is not as easily oxidized as 8-oxoG (0.92 and 0.58 V versus NHE, respectively) (18).

We recently reported the selective oxidation of 8-oxoG- and 8-oxoA-containing oligonucleotides by the one-electron oxidant IrCl62- (35). Moreover, the conversion of these 8-oxopurines into further oxidized products sensitive to piperidine allows their detection by gel electrophoresis. IrCl62- is a convenient, water-soluble, one-electron oxidant that does not exhibit any binding interactions with DNA because of its negative charge. Its redox potential, 0.90 V versus NHE (35), is convenient for oxidation of only oxopurines without significant reaction with normal DNA bases. A number of transition metal complexes also carry out one-electron oxidation of purines (36), including the carcinogenic metal nickel (37). Organonickel(II) complexes, including those of peptide ligands, are redox active in the presence of certain oxidants leading to oxidation of guanine and 8-oxoguanine in DNA (38). These species have been studied both as models for nickel carcinogenesis (39) and as probes of DNA (or RNA) structure due to their ability to oxidatively modify guanine residues (40).

In the present work, we used IrCl62- in addition to two nickel catalysts along with HSO5- or SO32-/O2 as one-electron oxidants as well as singlet oxygen to study the mutagenic potential of oxidized forms of 8-oxoG and 8-oxoA. First, we examined nucleotide insertion opposite an oxidized form of 8-oxoG during in vitro DNA synthesis catalyzed by Kf exo-, pol [alpha] or pol [beta], and the oxidation product of 8-oxoA was studied with Kf exo-. The present results demonstrate that an oxidized form of 8-oxoG induces insertion of dAMP and dGMP when DNA synthesis is performed with Kf exo-, suggesting that it may be a potent mutagenic lesion leading to G->T and G->C transversions. However, further oxidation of 8-oxoG represents a block during replication of the template by pol [alpha] and pol [beta]. Electrospray mass spectrometry was used to identify the major lesion produced from IrCl62- oxidation as guanidinohydantoin. Secondly, experiments conducted with 8-oxoA and an oxidized form of 8-oxoA show that this 8-oxopurine or a further oxidized product of 8-oxoA induces mainly dTMP insertion during DNA synthesis by Kf exo-.

MATERIALS AND METHODS

Materials

Reagents and enzymes were purchased from the following sources: Na2IrCl6 from Alfa Aesar, (Ward Hill, MA), Rose Bengal from Acros (Norcross, GA), potassium monoperoxysulfate (Oxone) and sodium sulfite were from Sigma and Fluka (Ronkonkoma, NY), respectively. 8-OxoG and 8-oxoA phosphor-amidites were from Glen Research (Sterling, VA), dNTPs from Pharmacia (Piscataway, NJ), [[gamma]-32P]ATP from Amersham(Arlington Heights, IL), T4 polynucleotide kinase and Klenow fragment (3[prime]->5[prime]exo-) were from New England Biolabs (Beverly, MA). Calf thymus pol [alpha] and human pol [beta] were purchased from Molecular Biology Resources (Milwaukee, WI). Oligodeoxynucleotides were synthesized with an Applied Biosystems synthesizer (ABI 392 B) using the manufacturer's protocols and incorporating 0.25 M into the final manual deprotection step of oligonucleotides containing 8-oxopurines (41). Purification was carried out by PAGE using 15 or 20% polyacrylamide/7 M urea. Oligonucleotides were 5[prime]-end-labeled using T4 polynucleotide kinase and [[gamma]-32P]ATP. Synthesis of the complex NiCR (42,43) and the peptide KGH-NH2 (44) were previously described. The NiKGH-NH2 complex was formed in situ by the addition of 1 equivalent of Ni(CH3CO2)2 to an aqueous solution of KGH-NH2 followed by the addition of two equivalents NaOH (38).

DNA oxidation

Oxidation reactions were carried out with 5 µM oligonucleotides containing 8-oxoG or 8-oxoA in a 10-µl solution of 10 mM NaPi buffer (pH 7) with 100 mM NaCl. For the oxidation with IrCl62-, the template was incubated for 1 h at room temperature in the presence of 100 µM IrCl62- (35). For the experiments with singlet oxygen, the reactions were performed with Rose Bengal (50 µM) for 30 min at 12°C under a 300 W tungsten lamp. The template oxidations with NiCR (42) or NiKGH-NH2 (38) were conducted as previously described. The DNA was incubated with 5 µM NiCR or 10 µM NiKGH-NH2 in the presence of 20 µM KHSO5 for 30 min at room temperature or 40 µM Na2SO3 for 1 h at room temperature, respectively.

Primer extension

Reactions catalyzed by Kf exo- were carried out in 10 µl solutions of 10 mM Tris-HCl (pH 7.5) plus 5 mM MgCl2 and 7.5 mM DTT containing the oligonucleotide template and the 5[prime]-end-labeled primer (Fig. 1) with a template/primer nanomolar ratio: 1/4 = 50/15; 2/4 and 3/4 = 90/15. The solution was heated at 90°C for 5 min then cooled to room temperature over a period of 2.5 h. DNA polymerization reactions were conducted with 100 µM of a single dNTP or 100 µM of a mixture of all four dNTPs and 0.02 U of Kf exo- (for duplex 1/4) or 0.1 U of Kf exo- (for duplexes 2/4 and 3/4) at 37°C for 20 min. Reactions were stopped by adding 5 µl of termination solution (95% formamide, 0.1% bromophenol blue and 0.1% xylene cyanol). The samples were then heated at 95°C for 3 min, applied to a 15% polyacrylamide gel in the presence of 7 M urea and then analyzed by phosphorimagery (Molecular Dynamics Storm 840) using ImageQuaNT software.


Figure 1. Sequences of oligodeoxynucleotides.

For the experiments with pol [alpha] and pol [beta], the template and the 5[prime]-end-labeled primer (nanomolar ratio 1/4, 2/4, 3/4 = 50/15) were annealed as described above in 10 µl solutions of 50 mM Tris-HCl (pH 8) plus 10 mM MgCl2, 2 mM DTT and 0.5 µg/µl BSA. For DNA synthesis, the reaction mixtures containing the desired duplex, 100 µM of a single dNTP or 100 µM of a mixture of the four dNTPs and 0.05 U of pol [alpha] or pol [beta] were incubated at 25°C for 1 h. The reactions were stopped and the samples analyzed as described above.

Mass spectrometric analysis of oxidized 8-oxoguanine-containing oligonucleotides

Oxidations were carried out with 10-50 µl samples containing 12 µM of oligo 5 and 100 µM IrCl62- in 10 mM NaPi buffer (pH 7) and 100 mM NaCl for 60 min. Reactions were quenched by the addition of 2 µl of a 50 mM HEPES/250 mM EDTA (pH 7) solution. The reactions were combined and dialyzed (2 K Dalton MWCO) against water for 24 h and then against 10 mM ammonium acetate for an additional 24 h. The existing solution was lyophilized to dryness and dissolved in 50 µl of water and 50 µl of 10 M ammonium acetate. After 2 h, 150 µl of cold ethanol-isopropanol (1:1) mixture was added and the DNA was precipitated for 8 h at -80°C. After incubation, the sample was centrifuged (13 000 r.p.m.) for 30 min at 4°C and the supernatant liquid was carefully removed leaving the DNA as a pellet. After an additional DNA precipitation using the same procedure, the DNA pellet was lyophilized for 2 h prior to submission for electrospray mass spectrometric (ESI-MS) analysis. MS analysis was carried out on a Micromass Quattro I at the University of Illinois Mass Spectrometry Facility. A portion of the same sample used for MS was retained and radiolabeled with a 5[prime]-32P-phosphate, subjected to hot piperidine treatment (0.2 M, 60 min) and analyzed by PAGE. The procedure for this has already been described elsewhere (35).

RESULTS

Insertion of dNTPs opposite an oxidized form of 8-oxoG

To examine the effects of an oxidized form of 8-oxoG during in vitro DNA synthesis, a 40mer template 2, containing 8-oxoG at position 10 from the 5[prime] end (Fig. 1) was subjected to various oxidants and annealed to a 20mer primer, 4 (Fig. 1). The primer was then extended (37°C, 20 min) using three different DNA polymerases (0.1 U) in the presence of a single dNTP (100 µM) or a mixture of all four dNTPs (100 µM). An unmodified template 1, containing a guanine at position 10, and the unoxidized template 2 were used as controls for nucleotide insertion. In the case of the unmodified template 1, 0.02 U enzyme concentration was sufficient.


Figure 2. Nucleotide incorporation opposite G (template 1), 8-oxoG (template 2) or an oxidized form of 8-oxoG. Primer extension reactions were catalyzed by Kf exo- using a single dNTP (lanes A, C, G and T) or a mixture of the four dNTPs (lane Ex). Reaction conditions are described in the text.

Results of primer extension reactions catalyzed by Kf exo- are shown in Figure 2. DNA synthesis on the unmodified template 1 (lanes 1-5) led to the expected insertion of dCMP opposite dG at position 10 (lane 3). In the presence of the four dNTPs (lane 1), extension of the oligomer to approximately full length was observed. When the 8-oxoG-modified oligonucleotide 2 was used as the template (lanes 6-10), dAMP and dCMP were preferentially inserted opposite the lesion (lanes 7 and 8), as expected. In the presence of all four dNTPs, we observed a transient inhibition opposite the 8-oxoG damage (lane 6) but essentially full length product was also obtained. The relative amounts of dAMP and dCMP insertion are dependent on the concentration of enzyme used in the reaction and on the polymerization time (data not shown). dAMP insertion opposite 8-oxoG, first reported by Shibutani et al. (11), is representative of the mutagenic potential of 8-oxoG during DNA replication. Interestingly, when the 8-oxoG-containing template 2 was selectively oxidized by IrCl62-, Kf exo- inserts exclusively dAMP and dGMP opposite the damage (lanes 12 and 14). When all the dNTPs were present in the reaction, the over-oxidized 8-oxopurine was a stop point for the enzyme and only a small amount of fully extended primer was observed (lane 11), although the integrity of this complement was not examined. Similar results were obtained when template 2 was oxidized in the presence of singlet oxygen. The oxidized form of 8-oxoG induces selective insertion of dAMP as well as dGMP (lanes 16 and 18) opposite the lesion, with strong inhibition during DNA synthesis when all dNTPs are present (lane 20).

Oxidation of 8-oxoG by nickel complexes that are models for nickel-mediated DNA damage and toxicity(45) was also studied, and the resulting template 2 was subjected to primer extension reaction catalyzed by Kf exo-. The results of nucleotide insertion by Kf exo- after treatment of 2 by NiCR/HSO5- or NiKGH-NH2/SO32-/O2 are presented in Figure 3. In both cases, oxidation of 8-oxoG by NiCR (lanes 1-5) and NiKGH-NH2 (lanes 6-10) in the presence of HSO5- and SO32-/O2, respectively, led to dAMP and dGMP insertion opposite the lesion (lanes 1 and 3, and 6 and 8). With the four dNTPs in the reaction mixture, DNA polymerization by Kf exo- is strongly inhibited, and no full or partial extension of the primer was obtained (lanes 5 and 10). Treatment of 2 by HSO5- or SO32-/O2 without nickel complexes induced insertion of dAMP and dCMP as expected opposite 8-oxoG (data not shown), indicating that there is little background reaction of the oxidants in the absence of nickel catalysts.


Figure 3. DNA synthesis opposite an oxidized form of 8-oxoG after treatment of template 2 by Ni complexes in the presence of KHSO5 or Na2SO3/O2. Primer extension reactions were catalyzed by Kf exo- using a single dNTP (lanes A, C, G and T) or a mixture of the four dNTPs (lane Ex). Reaction conditions are described in the text.

DNA synthesis catalyzed by pol [alpha] and pol [beta] were also conducted on template 2 after treatment by IrCl62-, and the results of primer extension are presented in Figure 4. With both enzymes, extension of the 8-oxoG containing template led to the expected insertion of dAMP and dCMP (lanes 1 and 2 for pol [alpha], lanes 11 and 12 for pol [beta]). Consistent with previous reports, pol [alpha] inserts predominantly dAMP compared with dCMP (11). The 8-oxoG lesion retarded the extension but some full length product was obtained (lanes 5 and 15). Interestingly when 8-oxoG is oxidized in the presence of IrCl62-, the resulting lesion inhibits the primer extension by pol [alpha] (lanes 6-10) and pol [beta] (lanes 16-20) beyond the damage. An oxidized form of 8-oxoG may induce a change in the conformation of the template opposite the damage not suitable for DNA synthesis by pol [alpha] and pol [beta].


Figure 4. Nucleotide incorporation opposite 8-oxoG (template 2) or an oxidized form of 8-oxoG. Primer extension reactions were catalyzed by calf thymus pol [alpha] (lanes 1-10) and by human pol [beta] (lanes 11-20) in the presence of a single dNTP (lanes A, C, G and T) or a mixture of the four dNTPs (lane Ex). Reaction conditions are described in the text.

Insertion of dNTPs opposite an oxidized form of 8-oxoA

The 8-oxoA-modified template 3, incubated in the presence of IrCl62- or NiCR/HSO5-, was used to examine the miscoding potential of an oxidized form of 8-oxoA. The relative insertion of dNTPs opposite of 8-oxoA, or a further oxidized form of 8-oxoA, catalyzed by Kf exo-, is shown in Figure 5. As previously reported in the literature (32), Kf exo- inserts predominantly dTMP opposite 8-oxoA (lane 5). In the presence of the four dNTPs, the 8-oxoA lesion is a stop point for the enzyme but some extended primer was also observed (lane 1). When 8-oxoA is subjected to further oxidation by IrCl62- (lanes 6-10) or NiCR/HSO5- (lanes 10-15) prior to primer extension, similar results were obtained. dTMP is mainly inserted opposite the damage; however, small amounts of dAMP were also detected (lane 12). Moreover, essentially fully extended primer was observed with transient inhibition opposite the lesion (lanes 6 and 11).


Figure 5. Nucleotide incorporation opposite 8-oxoA (template 3) or an oxidized form of 8-oxoA. Primer extension reactions were catalyzed by Kf exo- using a single dNTP (lanes A, C, G and T) or a mixture of the four dNTPs (lane Ex). Reaction conditions are described in the text.

Mass spectrometric analysis of oxidized 8-oxoG-containing oligonucleotides

In order to examine the possible products of 8-oxoG oxidation that might be responsible for misinsertion of dGMP and dAMP, we carried out ESI-MS analysis of synthetic oligodeoxynucleotides. An oligomer consisting of the first 18 residues of template 1 was initially studied (data not shown), but ~20% of the products corresponded to imidazolone and oxazolone residues (although the major products were as discussed below). Since these are common one-electron oxidation products of G, and possible oxidation products of 8-oxoG, it was not clear whether these minor oxidation products arose from reaction at the single 8-oxoG site or from the five other Gs present in the 18mer. As a consequence, we chose to study an alternative oligomer with a single 8-oxoG site and no competing G oxidation sites, namely oligo 5 (Fig. 1).

Both the starting material, oligo 5, and its IrCl62- oxidized product were purified and subjected to ESI-MS analysis. As shown in Figure 6, none of the starting material remained after treatment with 100 µM IrCl62- for 1 h. The largest peak in the spectrum represents an overall loss of 10 Da from the mass of the starting material (5515 versus 5525 Da). This major product is best explained as the guanidinohydantoin derivative, as shown in Figure 7. Guanidinohydantoin was proposed to be the two-electron, electrochemical oxidation product of 8-oxoguanosine (23), and the pathway is entirely analogous to the well-known oxidation of uric acid to form allantoin (20,24). The second largest peak in the product spectrum occurred at a mass of 5541 (M+16) which is consistent with the addition of an oxygen atom to 8-oxoG. This nicely matches with the proposed intermediate, 5-hydroxy-8-oxoguanine, which is thought to be on the pathway to guanidinohydantoin, just as 5-hydroxyurate is an intermediate in allantoin formation (20,24). Interestingly, no evidence for formation of an abasic site was obtained by mass spectral analysis of the oxidized 8-oxoG containing oligomer, in contrast to a recent mechanistic proposal concerning [gamma]-irradiated 8-oxoG lesions (46).


Figure 6. ESI-MS spectra of oligo 5 (dashed line) and of its IrCl62- oxidation product (solid line). Arrows `a' and `b' indicate expected positions for imidazolone and oxazolone products, respectively.

Since the two oligonucleotide products observed in the present study are likely to give similar responses by ESI-MS, one can estimate the relative amounts of various oxidation products formed. Thus, the major products, guanidinohydantoin and 5-hydroxy-8-oxoG, are formed in an ~70:30 ratio and constitute >90% of the total products. Interestingly, only trace amounts, if any, of the oxazolone (or more likely the ring-opened form, guanidinooxalamide, M-37) and its precursor, imidazolone (M-55), are observed. When compared with the initial study of a G-rich 18mer where these products were significant, we now conclude that they are not generally formed from the one-electron oxidation of 8-oxoG, but rather from one-electron oxidation of the parent residue, G.

Alternative interpretations of the mass spectral data are possible, given the error limits in ESI-MS of approximately ±1 a.m.u. For example, our assignment of guanidinohydantoin for the M-10 peak in the mass spectrum is based on the observation of a logical intermediate, 5-hydroxy-8-oxoG (M+16), its observation in nucleoside oxidation studies,(23) and the strong analogy to urate oxidation (24). Alternatively, a seven-membered ring product that has been observed in singlet oxygen chemistry with 8-oxoG would also lead to an M-10 peak, but a pathway for its formation by one-electron oxidation is not evident. Thus, the most consistent explanation of the mass spectral data is that one-electron oxidation of 8-oxoG leads initially to 5-hydroxy-8-oxoguanine followed by hydrolysis to the major product, guanidinohydantoin (Fig. 7, top).

In order to correlate the mass spectrometric results with gel electrophoretic analysis of 8-oxoG oxidation, a portion of the sample used in MS analysis was radiolabeled, piperidine treated, and electrophoresed on a polyacrylamide gel. After 60 min of piperidine treatment at 90°C, the band corresponding to cleavage at the oxidized 8-oxoG site represented 72% of the total intensity in the lane, and no other cleavage sites were observed. This nicely correlates with the ~70:30 product ratio of guanidinohydantoin:5-hydroxy-8-oxoG and suggests that guanidinohydantoin is a piperidine-labile lesion while 5-hydroxy-8-oxoG, like 8-oxoG, is not.


Figure 7. Scheme depicting products of 8-oxoG oxidation and their relative masses. Electrochemical oxidation (23) and one-electron oxidation (this work) lead to 5-hydroxy-8-oxoG and guanidinohydantoin. Singlet oxygen leads to a complex array of products (25-29).

DISCUSSION

In this study, the polymerase insertion of nucleotides opposite oxidized forms of 8-oxoG and 8-oxoA has been investigated using modified templates containing these two 8-oxopurine lesions. Singlet oxygen or one-electron oxidation of 8-oxoG- and 8-oxoA-containing templates, 2 and 3 respectively, were used as substrates for primer extension catalyzed by Kf exo-, pol [alpha] and pol [beta]. In vitro DNA synthesis opposite the lesion and full extension of the primer past the damage were studied using a single dNTP and a mixture of all four dNTPs, respectively. Replication of the template containing 8-oxoG after oxidation by IrCl62-, singlet oxygen and NiCR/HSO5- or NiKGH-NH2/SO32-/O2 induces insertion of both dAMP and dGMP opposite the damage by Kf exo- (Figs 2 and 3). In contrast to the behavior of 8-oxoG (2,10-12), dCMP is no longer inserted after further treatment with an oxidant. This result also supports the proposal that treatment with 100 µM IrCl62- leads to complete conversion of 8-oxoG to new products, since any unoxidized 8-oxoG would be expected to show at least partial insertion of dCMP. Complete oxidation is also supported by mass spectro-metric results showing the absence of starting material (Fig. 6) as well as related gel electrophoretic studies with oligonucleotides (35).

Many reports focused on the oxidation of 8-oxoG suggested that this modified base is easily oxidized, and an array of products has already been identified for the degradation of the 8-oxoG nucleoside under various oxidative conditions (23,25-27,29). Until now, little was known about the hydrogen bonding, the base-pairing properties and the potent mutagenicity of these compounds. Our findings suggest that at least one of the products of oxidation of 8-oxoG is chemically competent to lead to G->T and G->C transversions, although further extension of oligomers containing oxidized 8-oxopurines is much less efficient than 8-oxopurine-containing substrates. Similar nucleotide insertion was observed when the 8-oxoG-containing template 2 was oxidized by NiCR and NiKGH-NH2 in the presence of HSO5- or SO32-/O2, respectively. Nickel complexes such as NiCR and NiKGH-NH2 mediate oxidative DNA damage, predominantly at guanine sites (38). Mechanistic studies have shown that the reaction with guanines does not involve freely diffusible radicals but a Ni(II) complex with a bound radical, such as a sulfate radical (38). Furthermore, we have recently observed that the 8-oxoG-modified base present in an oligonucleotide is the preferential site of reaction with these nickel complexes in the presence of HSO5- or SO32-/O2 (unpublished results). The lower redox potential of 8-oxoG compared with guanine (0.58 and 1.29 V versus NHE, respectively) explains the higher reactivity of the 8-oxoG compared with the unmodified guanine. The reactivity of the nickel-tripeptide complex associated with SO32-/O2 toward 8-oxoG, leading to insertion of purines rather than dCMP provides some evidence for the toxicity of sulfite mediated by nickel (45,47).

Primer extension reactions catalyzed by pol [alpha] and pol [beta] on template 2 in the presence of IrCl62- led to different results in comparison with Kf exo-. Both enzymes were unable to insert a nucleotide opposite the lesion, leading to a stop at the site of damage. This result might suggest a change in the template conformation such that the modified base is no longer a substrate for pol [alpha] and pol [beta]. Alternatively, small amounts of an incorrectly inserted nucleotide may have been removed by exonuclease activity of these polymerases, but this remains to be tested. Nucleotide insertion opposite an oxidized form of 8-oxoG, seems to be dependent on the DNA polymerase used; in the case of 8-oxoG-containing oligonucleotides, many studies revealed differences among various polymerases on nucleotide insertion opposite the damage (11,12,48). Shibutani et al. (11) found that dAMP was inserted opposite 8-oxoG by pol [alpha] and pol [delta], while pol [beta] and Klenow fragment favor insertion of dCMP. Furthermore, Feig and Loeb (7) pointed out that the mutation spectrum induced by DNA damage generated by oxygen radicals is highly dependent on the DNA polymerase.

In conclusion, dAMP and dGMP insertion by Kf exo- provides a molecular basis for misincorporations opposite an oxidized form of 8-oxoG during DNA synthesis. Mass spectrometric analysis is consistent with high-yield conversion of 8-oxoG to guanidinohydantoin following treatment with IrCl62-, and this is the first report of this lesion being observed in an oligomer. While it is tempting to propose that this heterocycle is responsible for the subsequent incorrect insertions observed, in principle, any or all of the products in Figure 7 could be active mutagenic lesions. Based on related work, it is likely that both of the nickel-catalyzed reactions of 8-oxoG lead to the same guanidinohydantoin product; however, 1O2 chemistry clearly yields a different product distribution based on extensive literature surrounding this reaction. It is therefore quite interesting that the entire group of oxidation products (Fig. 7) gives the same dAMP + dGMP incorporation. What these products have in common is that they are all heterocycles of roughly the same dimensions as a pyrimidine, and they (or their tautomers) present a variety of H-bonding opportunities. This may indicate that purine insertion is favored for Kf exo- on the basis of size and shape as opposed to highly specific and complementary hydrogen bonding, as already suggested by Moran et al. for non-hydrogen-bonding nucleoside analogs (49,50). That very stable base pairs are not formed may account for the lack of efficient extension beyond the initial misinsertion. Thus, a tentative conclusion is that oxidative degradation of purines to one-ring heterocycles generally leads to purine insertion with Kf exo-.

Indeed, G->C and G->T transversions resulting from dAMP and dGMP incorporation have been reported during replication of DNA treated with oxygen radicals. McBride et al. (51) found that Fe2+/oxygen damage to DNA is a non-random mechanism, leading to single base substitution with the most frequent being G->C transversions followed by C->T and G->T. McBride et al. (52) also found, using singlet oxygen mediated DNA damage, that among the transversions observed after DNA replication, G->C transversion was predominant with G->T transversions occurring at lower frequency. G->C transversions produced by singlet oxygen have also been detected in a mutation assay in mammalian cells (53). More recently, Valentine et al. (54) studied the DNA damage generated by peroxyl radicals. The spectrum of mutations induced in Escherichia coli upon transfection of DNA exposed to ROO· indicates predominantly G->C and G->T transversions. The authors reported that the oxidative lesions at G were sensitive to digestion by MutM, for which 8-oxoG, but not imidazolone or oxazolone products (22,55), are substrates. Efforts to detect 8-oxoG by HPLC/electrochemical analysis suggest that 8-oxoG was not present and that the G->T transversion was not caused by this 8-oxopurine. Taken together these results suggest that in oxygen-mediated DNA damage, 8-oxoguanine is not responsible for G->C transversions, and may not induce all of the G->T transversions observed during DNA replication. Until now, oxidized products of 8-oxoG have not been reported to be implicated in DNA mutagenesis. Our findings suggest that an oxidized form of 8-oxoG may account for some of the G->C and G->T transversions observed under oxidative conditions.

Given the high susceptibility of 8-oxoG to further oxidation, and the findings reported herein that the oxidized 8-oxoG lesion, possibly the major product guanidinohydantoin, leads to insertion of dGMP and dAMP during in vitro DNA synthesis, it will now be important to learn how these lesions might be recognized and excised by the DNA repair enzymes. Such studies are currently in progress.

ACKNOWLEDGEMENTS

We thank Prof. T. Widlanski (Indiana University) and Prof. S. Rokita (University of Maryland) for helpful discussions. Support of this work by a research grant from the National Science Foundation (CHE-9521216) and in part by a travel grant from NSF (INT-9613313) is gratefully acknowledged.

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