The competitive miscoding of O6-methylguanine and O6-ethylguanine and the possible importance of cellular deoxynucleoside 5'-triphosphate pool sizes in mutagenesis and carcinogenesis

Using initiated poly(dG,O6-RdG) and poly(dA,O6-RdG) polynucleotides as templates for DNA polymerase I in vitro the promutagenic potential of O6-MeG and O6-EtG has been confirmed, together with the possibility of minor miscoding pathways for O6-RG. These lead to the incorporation of dAMP and dGMP, which could give rise to some of the limited number of transversions that have been observed arising from the action of alkylating agents. The results are compatible with the current knowledge of oncogenes, explaining the changes in base sequence that have been observed. The competition for the miscoding of O6-RG which leads to the incorporation of dCMP in addition to the expected dTMP is also shown. The relative amounts of these two nucleotides incorporated depend upon the concentrations of the dCTP and dTTP in the assay. The mutagenic efficiency of O6-MeG is constant at approx. 0.4 over a wide range of dTTP and dCTP concentrations and only increases when the dCTP in the assay ceases to saturate the polymerizing enzyme, indicating that the DNA polymerase I plays a role in determining the mutagenic efficiency of a modified base. Although the mutagenic efficiency of both O6-MeG and O6-EtG depends upon the relative concentrations. of dTTP and dCTP in the assay, a reduction in the concentration of dCTP can be more effective at increasing the mutagenic efficiency than a corresponding increase in the concentration of dTTP. These results indicate the importance of cellular dNTP pools in determining the cellular response to agents.     

DNA-synthesis with methylated poly(dA-dT) templates: possible role of O4-methylthymine as a pro-mutagenic base.

The alternating copolymer poly(dA-dT) has been methylated with either dimethyl sulphate (DMS) or N-methyl-N-nitrosourea (MNU) and the levels of the various methylation products determined. In addition to the methylated adenines formed by both methylating agents, MNU resulted also in the formation of 3-methylthymine, O4-methylthymine and phosphotriesters. The methylated polymers have been ution of complementary and non-complementary nucleotides determined. With the DMS methylated template no wrong nucleotide incorporation was detectable, but with the MNU methylated polymer the incorporation of dGMP was observed. The amount of dGMP incorporated correlated with the level of O4-methylthymine in the template over the range of methylation studied. The results indicate that O4-methylthymine is capable of miscoding on a one-to-one basis while the products of DMS methylation (1-, 3- and 7-methyladenines), and also possibly the phosphotriesters, do not lead to any misincorporation.     

Transcriptional regulation of nuclear orphan receptor,NOR-1, by Ca(2+)/calmodulin-dependent protein kinase cascade

It has been proposed that the REV1 protein plays an important role in the induced-mutagenesis pathway. We show that purified REV1 protein inserts dCMP opposite template G, A, T and C, and dGMP and dTMP opposite template G in the presence of magnesium, while in the presence of manganese the specificity for dCMP was found to be relaxed and the REV1 protein acquired the ability to insert dCMP, dGMP, dAMP and dTMP opposite templates G, A, T, and C. Kinetic analysis provided evidence for high affinity for dCTP with template G, suggesting that the REV1 protein is specialized for dCTP and template G.     

Translesion synthesis past 2'-deoxyxanthosine, a nitric oxide-derived DNA adduct, by mammalian DNA polymerases

Cellular DNA is damaged by nitric oxide (NO), a multifunctional bioregulator and an environmental pollutant that has been implicated in diseases associated with cancer and chronic inflammation. 2'-Deoxyxanthosine (dX) is a major NO-derived DNA lesion. To explore the mutagenic potential of dX, a 38-mer oligodeoxynucleotide ((5')CATGCTGATGAATTCCTTCXCTTCTTTCCTCTCCCTTT) modified site-specifically with dX at the X position was prepared post-synthetically and used as a DNA template in primer extension reactions catalyzed by calf thymus DNA polymerase (pol) alpha and human DNA pol beta, eta, and kappa. Primer extension reactions catalyzed by pol alpha or beta in the presence of four dNTPs were retarded at the dX lesion while pol eta and kappa readily bypassed the lesion. The fully extended products were analyzed to quantify the miscoding specificity and frequency of dX using two-phase polyacrylamide gel electrophoresis (PAGE). With pol alpha, eta and kappa, incorrect dTMP was preferentially incorporated opposite the lesion, along with lesser amounts of dCMP, the correct base. When pol beta was used, direct incorporation of correct dCMP was primarily observed, accompanied by small amounts of misincorporation of dTMP, dAMP and dGMP. Steady-state kinetic analyses supported the results obtained from the two-phase PAGE assay. dX is a miscoding lesion capable of preferentially generating G-->A mutations. The miscoding frequency varied depending on DNA polymerase used.     

Mechanism of translesion synthesis past an equine estrogen-DNA adduct by Y-family DNA polymerases

4-Hydroxyequilenin (4-OHEN)-dC is a major, potentially mutagenic DNA adduct induced by equine estrogens used for hormone replacement therapy. To study the miscoding property of 4-OHEN-dC and the involvement of Y-family human DNA polymerases (pols) eta, kappa and iota in that process, we incorporated 4-OHEN-dC into oligodeoxynucleotides and used them as templates in primer extension reactions catalyzed by pol eta, kappa and iota. Pol eta inserted dAMP opposite 4-OHEN-dC, accompanied by lesser amounts of dCMP and dTMP incorporation and base deletion. Pol kappa promoted base deletions as well as direct incorporation of dAMP and dCMP. Pol iota worked in conjunction with pol kappa, but not with pol eta, at a replication fork stalled by the adduct, resulting in increased dTMP incorporation. Our results provide a direct evidence that Y-family DNA pols can switch with one another during synthesis past the lesion. No direct incorporation of dGMP, the correct base, was observed with Y-family enzymes. The miscoding potency of 4-OHEN-dC may be associated with the development of reproductive cancers observed in women receiving hormone replacement therapy.     

Miscoding properties of 6alpha- and 6beta-diastereoisomers of the N(2)-(estradiol-6-yl)-2'-deoxyguanosine DNA adduct by Y-family human DNA polymerases

Treatment with estrogen increases the risk of breast, ovary, and endometrial cancers in women. DNA damage induced by estrogen is thought to be involved in estrogen carcinogenesis. In fact, Y-family human DNA polymerases (pol) eta and kappa, which are highly expressed in the reproductive organs, miscode model estrogen-derived DNA adducts during DNA synthesis. Since the estrogen-DNA adducts are a mixture of 6alpha- and 6beta-diastereoisomers of dG-N(2)-6-estrogen or dA-N(6)-6-estrogen, the stereochemistry of each isomeric adduct on translesion synthesis catalyzed by DNA pols has not been investigated. We have recently established a phosphoramidite chemical procedure to insert 6alpha- or 6beta-isomeric N(2)-(estradiol-6-yl)-2'-deoxyguanosine (dG-N(2)-6-E(2)) into oligodeoxynucleotides. Using such site-specific modified oligomer as a template, the specificity and frequency of miscoding by dG-N(2)-6alpha-E(2) or dG-N(2)-6beta-E(2) were explored using pol eta and a truncated form of pol kappa (pol kappaDeltaC). Translesion synthesis catalyzed by pol eta bypassed both the 6alpha- and 6beta-isomers of dG-N(2)-6-E(2), with a weak blockage at the adduct site, while translesion synthesis catalyzed by pol kappaDeltaC readily bypassed both isomeric adducts. Quantitative analysis of base substitutions and deletions occurring at the adduct site showed that pol kappaDeltaC was more efficient than pol eta by incorporating dCMP opposite both 6alpha- and 6beta-isomeric dG-N(2)-6-E(2) adducts. The miscoding events occurred more frequently with pol eta, but not with pol kappaDeltaC. Pol eta promoted incorporation of dAMP and dTMP at both the 6alpha- and 6beta-isomeric adducts, generating G --> T transversions and G --> A transitions. One- and two-base deletions were also formed. The 6alpha-isomeric adduct promoted slightly lower frequency of dCMP incorporation and higher frequency of dTMP incorporation and one-base deletions, compared with the 6beta-isomeric adduct. These observations were supported by steady-state kinetic studies. Taken together, the miscoding property of the 6alpha-isomeric dG-N(2)-6-E(2) is likely to be similar to that of the 6beta-isomeric adduct.     

Miscoding events during DNA synthesis past the nitration-damaged base 8-nitroguanine

8-Nitro-2'-deoxyguanosine (8-NO(2)-dG) DNA adducts are induced by the reactive nitrogen species and may be associated with the development of cancer in inflammatory tissues. To explore the miscoding potential of 8-NO(2)-dG adduct, an oligodeoxynucleotide containing a single 8-NO(2)-dG adduct was prepared by photochemical synthesis and used as a template in primer extension reactions catalyzed by mammalian DNA polymerases (pol). Primer extension reactions catalyzed by pol alpha or beta were strongly retarded at the 8-NO(2)-dG lesion; a fraction of primers was extended past the lesion by incorporating preferentially dCMP, the correct base, opposite the lesion, accompanied by lesser amounts of dAMP and dGMP incorporation. In contrast, primer extension reactions catalyzed by pol eta or a truncated form of pol kappa (pol kappaDeltaC) readily extended past the 8-NO(2)-dG lesion. Pol eta and kappaDeltaC showed more broad miscoding spectra; direct incorporations of dCMP and dAMP were observed, along with lesser amounts of dGMP and dTMP incorporations and deletions. The miscoding frequencies induced by pol eta and kappaDeltaC were at least 8 times higher than that of pol alpha or beta. Miscoding frequency and specificity of 8-NO(2)-dG varied depending on the DNA polymerases used. These observations were supported by steady-state kinetic studies. 8-NO(2)-dG adduct may play an important role in initiating inflammation driven carcinogenesis.     

The miscoding potential of 5-hydroxycytosine arises due to template instability in the replicative polymerase active site

5-Hydroxycytosine (5-OHC) is a stable oxidation product of cytosine associated with an increased frequency of C → T transition mutations. When this lesion escapes recognition by the base excision repair pathway and persists to serve as a templating base during DNA synthesis, replicative DNA polymerases often misincorporate dAMP at the primer terminus, which can lead to fixation of mutations and subsequent disease. To characterize the dynamics of DNA synthesis opposite 5-OHC, we initiated a comparison of unmodified dCMP to 5-OHC, 5-fluorocytosine (5-FC), and 5-methylcytosine (5-MEC) in which these bases act as templates in the active site of RB69 gp43, a high-fidelity DNA polymerase sharing homology with human replicative DNA polymerases. This study presents the first crystal structure of any DNA polymerase binding this physiologically important premutagenic DNA lesion, showing that while dGMP is stabilized by 5-OHC through normal Watson-Crick base pairing, incorporation of dAMP leads to unstacking and instability in the template. Furthermore, the electronegativity of the C5 substituent appears to be important in the miscoding potential of these cytosine-like templates. While dAMP is incorporated opposite 5-OHC ~5 times more efficiently than opposite unmodified dCMP, an elevated level of incorporation is also observed opposite 5-FC but not 5-MEC. Taken together, these data imply that the nonuniform templating by 5-OHC is due to weakened stacking capabilities, which allows dAMP incorporation to proceed in a manner similar to that observed opposite abasic sites.     

Aphidicolin inhibits DNA synthesis by DNA polymerase alpha and isolated nuclei by a similar mechanism.

Aphidicolin is a selective inhibitor of DNA polymerase alpha. In contrast to earlier reports, the drug was found to inhibit DNA synthesis catalyzed by DNA polymerase alpha and isolated HeLa cell nuclei by a similar mechanism. For both systems aphidicolin primarily competed with dCTP incorporation. However, the apparent Vmax for dCTP incorporation was reduced by 50-60% at relatively low concentrations of aphidicolin, thus the mechanism of inhibition is complex. Furthermore, a 2-5 fold increase in apparent Km for dTTP was observed in the presence of aphidicolin, but the apparent Km values for dATP and dGTP were essentially unaltered. This, together with additional evidence, suggested that the mechanism of action of aphidicolin involves a strong competition with dCMP incorporation, a weaker competition with dTMP incorporation and very little, if any, competition with dGMP and dAMP incorporation.     

Mechanisms of dCMP transferase reactions catalyzed by mouse Rev1 protein.

The Rev1 protein, a member of a large family of translesion DNA polymerases, catalyzes a dCMP transfer reaction. Recombinant mouse Rev1 protein was found to insert a dCMP residue opposite guanine, adenine, thymine, cytosine, uracil, and an apurinic/apyrimidinic site and to have weak ability for transfer to a mismatched terminus. The mismatch-extension ability was strongly enhanced by a guanine residue on the template near the mismatched terminus; this was not the case with an apurinic/apyrimidinic site and the other template nucleotides. Kinetic analysis of the dCMP transferase reaction provided evidence for high affinity for dCTP with template G but not the other templates, whereas the template nucleotide did not much affect the V(max) value. Furthermore, it could be established that the mouse Rev1 protein inserts dGMP and dTMP residues opposite template guanine at a V(max) similar to that for dCMP.     

Translesional synthesis past acetylaminofluorene-derived DNA adducts catalyzed by human DNA polymerase kappa and Escherichia coli DNA polymerase IV.

Human DNA polymerase kappa (pol kappa) has a sequence significantly homologous with that of Escherichia coli DNA polymerase IV (pol IV). We used a truncated form of human pol kappa (pol kappaDeltaC) and full-length pol IV to explore the miscoding properties of these enzymes. Oligodeoxynucleotides, modified site-specifically with N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-AF), were used as DNA templates in primer extension reactions that included all four dNTPs. Reactions catalyzed by pol kappaDeltaC were partially blocked one base prior to dG-AAF or dG-AF, and also opposite both lesions. At higher enzyme concentrations, a significant fraction of primer was extended. Analysis of the fully extended reaction product revealed incorporation of dTMP opposite dG-AAF, accompanied by much smaller amounts of dCMP, dAMP, and dGMP and some one- and two-base deletions. The product terminating 3' to the adduct site contained AMP misincorporated opposite dC. On templates containing dG-AF, dAMP, dTMP, and dCMP were incorporated opposite the lesion in approximately equal amounts, together with some one-base and two-base deletions. Steady-state kinetics analysis confirmed the results obtained from primer extension reactions catalyzed by pol kappa. In contract, primer extension reactions catalyzed by pol IV were blocked effectively by dG-AAF and dG-AF. At high concentrations of pol IV, full-length products were formed containing primarily one- or two-base deletions with dCMP, the correct base, incorporated opposite dG-AF. The miscoding properties of pol kappa observed in this study are consistent with mutational spectra observed when plasmid vectors containing dG-AAF or dG-AF are introduced into simian kidney cells [Shibutani, S., et al. (2001) Biochemistry 40, 3717-3722], supporting a model in which pol kappa plays a role in translesion synthesis past acetylaminofluorene-derived lesions in mammalian cells.     

The Gly-952 residue of Saccharomyces cerevisiae DNA polymerase alpha is important in discriminating correct deoxyribonucleotides from incorrect ones

Gly-952 is a conserved residue in Saccharomyces cerevisiae DNA polymerase alpha (pol alpha) that is strictly required for catalytic activity and for genetic complementation of a pol alpha-deficient yeast strain. This study analyzes the role of Gly-952 by characterizing the biochemical properties of Gly-952 mutants. Analysis of the nucleotide incorporation specificity of pol alpha G952A showed that this mutant incorporates nucleotides with extraordinarily low fidelity. In a steady-state kinetic assay to measure nucleotide misincorporation, pol alpha G952A incorporated incorrect nucleotides more efficiently than correct nucleotides opposite template C, G, and T. The fidelity of the G952A mutant polymerase was highest at template A, where the ratio of incorporation of dCMP to dTMP was as high as 0.37. Correct nucleotide insertion was 500- to 3500-fold lower for G952A than for wild type pol alpha, with up to 22-fold increase in pyrimidine misincorporation. The Km for G952A pol alpha bound to mismatched termini T:T, T:C, C:A, and A:C was 71- to 460-fold lower than to a matched terminus. Furthermore, pol alpha G952A preferentially incorporated pyrimidine instead of dAMP opposite an abasic site, cis-syn cyclobutane di-thymine, or (6-4) di-thymine photoproduct. These data demonstrate that Gly-952 is a critical residue for catalytic efficiency and error prevention in S. cerevisiae pol alpha.     

Avian myeloblastosis virus DNA polymerase. Kinetic studies on the incorporation of noncomplementary nucleotides.

The high error rate characteristic of DNA polymerases from RNA tumor viruses has permitted measurements on the simultaneous incorporation of complementary and noncomplementary nucleotides during DNA synthesis. For example, avian myeloblastosis virus DNA polymerase incorporates 1 molecule of dCMP for approximately 500 molecules of dTMP polymerized using polyriboadenylic acid as a template. The parallel incorporation of complementary and noncomplementary nucleotides afer gel filtration of avian myeloblastosis virus DNA polymerase indicates that the observed fidelity is catalyzed by the polymerase itself. Nearest neighbor analysis of the product indicates that noncomplementary nucleotides are incorporated as single base substitutions. The incorporation of the noncomplementary dCMP is not reduced by a 20-fold greater amount of the complementary nucleotide, dTTP. Conversely, the concentration of the noncomplementary nucleotides does not effect the rate of incorporation of the complementary nucleotide. A similar lack of competition between complementary dGTP and noncomplementary dATP is exhibited using poly(rC)-oligo(dG) as a template-primer. Furthermore, there was no detectable competition between the different noncomplementary nucleotides. Possible explanations for this lack of competition are considered.     

Separation of the SOS-dependent and SOS-independent components of alkylating-agent mutagenesis.

Escherichia coli plasmids containing the rpsL+ gene (Strs phenotype) as the target for mutation were treated in vitro with N-methyl-N-nitrosourea. Following fixation of mutations in E. coli MM294A cells (recA+ Strs), an unselected population of mutant and wild-type plasmids was isolated and transferred into a second host, E. coli 6451 (recA Strr). Strains carrying plasmid-encoded forward mutations were then selected as Strr isolates, while rpsL+ plasmids conferred the dominant Strs phenotype in the second host. Mutation induction and reduced survival of N-methyl-N-nitrosourea-treated plasmids were shown to be dose dependent. Because this system permitted analysis and manipulation of the levels of certain methylated bases produced in vitro by N-methyl-N-nitrosourea, it afforded the opportunity to assess directly the relative roles of these bases and of SOS functions in mutagenesis. The methylated plasmid DNA gave a mutation frequency of 6 X 10(-5) (a 40-fold increase over background) in physiologically normal cells. When the same methylated plasmid was repaired in vitro by using purified O6-methylguanine DNA methyltransferase (to correct O6-methylguanine and O4-methylthymine), no mutations were detected above background levels. In contrast, when the methylated plasmid DNA was introduced into host cells induced by UV light for the SOS functions, rpsL mutagenesis was enhanced eightfold over the level seen without SOS induction. This enhancement of mutagenesis by SOS was unaffected by prior treatment of the DNA with O6-methylguanine DNA methyltransferase. These results demonstrate a predominant mutagenic role for alkylation lesions other than O6-methylguanine or O4-methylthymine when SOS functions are induced. The mutation spectrum of N-methyl-N-nitrosourea under conditions of induced SOS functions revealed a majority of mutagenic events at A . T base pairs.     

Structural basis for proficient incorporation of dTTP opposite O6-methylguanine by human DNA polymerase iota

O(6)-methylguanine (O(6)-methylG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, even physiological ones (e.g. S-adenosylmethionine). The efficiency of a truncated, catalytic DNA polymerase ι core enzyme was determined for nucleoside triphosphate incorporation opposite O(6)-methylG, using steady-state kinetic analyses. The results presented here corroborate previous work from this laboratory using full-length pol ι, which showed that dTTP incorporation occurs with high efficiency opposite O(6)-methylG. Misincorporation of dTTP opposite O(6)-methylG occurred with ～6-fold higher efficiency than incorporation of dCTP. Crystal structures of the truncated form of pol ι with O(6)-methylG as the template base and incoming dCTP or dTTP were solved and showed that O(6)-methylG is rotated into the syn conformation in the pol ι active site and that dTTP misincorporation by pol ι is the result of Hoogsteen base pairing with the adduct. Both dCTP and dTTP base paired with the Hoogsteen edge of O(6)-methylG. A single, short hydrogen bond formed between the N3 atom of dTTP and the N7 atom of O(6)-methylG. Protonation of the N3 atom of dCTP and bifurcation of the N3 hydrogen between the N7 and O(6) atoms of O(6)-methylG allow base pairing of the lesion with dCTP. We conclude that differences in the Hoogsteen hydrogen bonding between nucleotides is the main factor in the preferential selectivity of dTTP opposite O(6)-methylG by human pol ι, in contrast to the mispairing modes observed previously for O(6)-methylG in the structures of the model DNA polymerases Sulfolobus solfataricus Dpo4 and Bacillus stearothermophilus DNA polymerase I.     

Epigenetically modified N6-methyladenine inhibits DNA replication by human DNA polymerase η

N⁶-methyladenine (6mA), as a newly reported epigenetic marker, plays significant roles in regulation of various biological processes in eukaryotes. However, the effect of 6mA on human DNA replication remain elusive. In this work, we used Y-family human DNA polymerase η as a model to investigate the kinetics of bypass of 6mA by hPol η. We found 6mA and its intermediate hypoxanthine (I) on template partially inhibited DNA replication by hPol η. dTMP incorporation opposite 6mA and dCMP incorporation opposite I can be considered as correct incorporation. However, both 6mA and I reduced correct incorporation efficiency, next-base extension efficiency, and the priority in extension beyond correct base pair. Both dTMP incorporation opposite 6mA and dCTP opposite I showed fast burst phases. However, 6mA and I reduced the burst incorporation rates (k_pol) and increased the dissociation constant (K_d,dNTP), compared with that of dTMP incorporation opposite unmodified A. Biophysical binding assays revealed that both 6mA and I on template reduced the binding affinity of hPol η to DNA in binary or ternary complex compared with unmodified A. All the results explain the inhibition effects of 6mA and I on DNA replication by hPol η, providing new insight in the effects of epigenetically modified 6mA on human DNA replication.     

Misincorporation of dAMP opposite 2-hydroxyadenine, an oxidative form of adenine.

Nucleotide incorporation opposite an oxidative form of adenine, 2-hydroxyadenine (2-OH-Ade) was investigated. When a primed template with 2-OH-Ade was treated with an exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I (KFexo-), recombinant rat DNA polymerase beta (pol beta) or calf thymus DNA polymerase alpha (pol alpha), incorporation of dTMP and dAMP was observed. In addition, KFexo- inserted dGMP as well. A steady-state kinetic study indicated that the insertion of dAMP and dTMP opposite the DNA lesion occurred with similar frequency with KFexo- and pol beta. Insertion of dTMP opposite 2-OH-Ade was favored to that of dAMP by pol alpha. Chain extension from the A.2-OH-Ade pair is less favored than that from the T.2-OH-Ade pair by all three DNA polymerase. Analysis of full-length products of in vitro DNA synthesis showed that dTMP and dAMP were incorporated by DNA polymerases and that exonuclease-proficient and -deficient Klenow fragments also inserted dGMP opposite 2-OH-Ade. These results suggest that formation of 2-OH-Ade from A in DNA will induce A-->T and A-->C transversions in cells.     

Studies on the inhibition of highly purified calf thymus 8S and 7.3S DNA polymerase alpha by aphidicolin.

On activated DNA aphidicolin competitively inhibits the incorporation of dCMP by both calf thymus DNA polymerase alpha A2 and C enzymes and inhibits the incorporation of the other three deoxynucleoside monophosphates apparently non-competitively. However, aphidicolin does not inhibit the incorporation of dAMP into poly(dT) . oligo(A)10 nor does it inhibit the incorporation of dGMP into poly(dC) . oligo(dG)10, but, it does competitively inhibit the incorporation of dTMP into poly(dA) . oligo(dT)10.     

Miscoding potential of the N2-ethyl-2'-deoxyguanosine DNA adduct by the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I

Acetaldehyde, a major metabolite of ethanol, reacts with dG residues in DNA, resulting in the formation of the N(2)-ethyl-2'-deoxyguanosine (N(2)-Et-dG) adduct. This adduct has been detected in lymphocyte DNA of alcohol abusers. To explore the miscoding property of the N(2)-Et-dG DNA adduct, phosphoramidite chemical synthesis was used to prepare site-specifically modified oligodeoxynucleotides containing a single N(2)-Et-dG. These N(2)-Et-dG-modified oligodeoxynucleotides were used as templates for primer extension reactions catalyzed by the 3' --> 5' exonuclease-free (exo(-)) Klenow fragment of Escherichia coli DNA polymerase I. The primer extension was retarded one base prior to the N(2)-Et-dG lesion and opposite the lesion; however, when the enzyme was incubated for a longer time or with increased amounts of this enzyme, full extension occurred. Quantitative analysis of the fully extended products showed the preferential incorporation of dGMP and dCMP opposite the N(2)-Et-dG lesion, accompanied by a small amounts of dAMP and dTMP incorporation and one- and two-base deletions. Steady-state kinetic studies were also performed to determine the frequency of nucleotide insertion opposite the N(2)-Et-dG lesion and chain extension from the 3' terminus from the dN.N(2)-Et-dG (N is C, A, G, or T) pairs. These results indicate that the N(2)-Et-dG DNA adduct may generate G --> C transversions in living cells. Such a mutational spectrum has not been detected with other methylated dG adducts, including 8-methyl-2'-deoxyguanosine, O(6)-methyl-2'-deoxyguanosine, and N(2)-methyl-2'-deoxyguanosine. In addition, N(2)-ethyl-2'-deoxyguanosine triphosphate (N(2)-Et-dGTP) was efficiently incorporated opposite a template dC during DNA synthesis catalyzed by the exo(-) Klenow fragment. The utilization of N(2)-Et-dGTP was also determined by steady-state kinetic studies. N(2)-Et-dG DNA adducts are also formed by the incorporation of N(2)-Et-dGTP into DNA and may cause mutations, leading to the development of alcohol- and acetaldehyde-induced human cancers.     

DNA damage reduces Taq DNA polymerase fidelity and PCR amplification efficiency

DNA damage blocks DNA polymerase progression and increases miscoding. In this study, we assessed the effects of specific lesions on Taq DNA polymerase fidelity and amplification efficiency. In the presence of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), Taq DNA polymerase inserted dCMP and to a lesser extent dAMP. 8-Oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA) instructed the incorporation of dTMP and caused a pronounced n-1 deletion not observed in other systems. The presence of an abasic lesion led to dAMP incorporation and n-1 deletions. In addition, we introduce the mean modified efficiency (MME) as a more precise method for determining PCR amplification efficiency of damaged templates. Using this method, we were able to quantify reductions in amplification efficiency of templates containing 8-oxodG (single or multiple), 8-oxodA, or abasic sites. Because the MME method can detect small reductions in amplification efficiency, it may be useful in comparing the extent of damage in environmentally degraded or archival DNA specimens.