Mechanism of action of nalidixic acid on Escherichia coli. Vi. Cell-free studies

The effects of nalidixic acid in vitro on deoxyribonucleic acid (DNA)- polymerase (deoxyribonucleosidetriphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.7), deoxyribonucleotide kinases (ATP: deoxymono- and diphosphate phosphotransferases), and deoxyribosyl transferase (nucleoside: purine deoxyribosyltransferase, EC 2.4.2.6) were examined employing partially purified and crude extracts of Escherichia coli ATCC 11229 and E. coli 15TAU. Nalidixic acid had no inhibitory effect on the DNA-polymerase of the wild-type strain E. coli ATCC 11229 at concentrations of 1.4 x 10(-3) to 2.8 x 10(-3)m. No inhibition of deoxyribonucleotide kinase activity was observed at concentrations of nalidixic acid ranging from 2 x 10(-3) to 8.6 x 10(-3)m. Nalidixic acid (0.43 x 10(-4) to 0.43 x 10(-3)m) had no inhibitory effect on the deoxyribosyl transferase activity of crude extracts obtained from E. coli ATCC 11229 or E. coli 15TAU. Analytical CsCl density gradient centrifugation demonstrated that the DNA obtained after treatment of E. coli 15TAU with nalidixic acid was not cross-linked. These results suggest that the prevention of DNA synthesis in vivo by nalidixic acid is not attributable to inhibition of DNA polymerase, deoxyribonucleotide kinase, deoxyribosyl transferase, or to cross-linking of the DNA of treated cells.     

Formation of 3-(2'-deoxyribofuranosyl) and 9-(2'-deoxyribofuranosyl) nucleosides of 8-substituted purines by nucleoside deoxyribosyltransferase

Earlier results suggested that although the N-deoxyribosyltransferase from lactobacilli is a convenient tool for the preparation of analogs of 2'-deoxyadenosine, 8-substituted purines do not act as substrates. However, eight of nine 8-substituted purines that were examined proved to be substrates for the transferase from Lactobacillus leichmannii, and deoxyribonucleosides of four of these bases have been prepared. The substituents at C-8 of the purine greatly affect the rate of deoxyribosyl transfer to the base, and in all cases the rate is slower than transfer to purines lacking an 8-substituent. The 8-substituent also affects the nature of the nucleoside formed. With the electron-donating methyl group at position 8 of adenine, the transferase forms the expected 8-methyl-9-(2'-deoxyribofuranosyl)adenine. However, when purines bearing an electron-withdrawing substituent at the 8-position are used as substrates, the deoxyribosyl moiety is preferentially transferred to N-3 of the base. In the case of 8-trifluoromethyladenine the 3-deoxyribonucleoside is the only product detectable. With 8-bromo or 8-chloroadenine as substrate the 3- and 9-deoxyribonucleosides can both be isolated from the enzymatic reaction mixture. Time course studies indicated that with thymidine and 8-bromoadenine as substrates the 3-deoxyribonucleoside is initially the major product, but that the 9-deoxyribonucleoside becomes the major product after long incubation periods. Negligible interconversion of these nucleosides occurs in the absence of transferase, but conversion in either direction occurs readily in the presence of the enzyme. Significant hydrolysis of pyrimidine and purine deoxyribonucleosides occurs in the presence of the transferase. This was more obvious during the course of reactions involving 8-substituted purines because the slowness of deoxyribosyl transfer required longer incubation periods and larger amounts of enzyme. The hydrolysis is proportional to enzyme concentration, little affected by the nature of the base and is attributed to hydrolysis of a deoxyribosyl derivative of the transferase which is an obligatory intermediate of deoxyribosyl transfer. 8-Trifluoromethyl-3-(2'-deoxyribofuranosyl)adenine, 8-methyl-9-(2'-deoxyribofuranosyl)adenine, and 8-bromo-9-(2'-deoxyribofuranosyl)adenine were tested for their ability to inhibit the growth of CCRF-CEM cells in culture. Unlike the potent 2-halogeno-2'-deoxyadenosine derivatives, these three nucleosides cause less than 50% inhibition at concentrations up to 100 microM.     

Deoxyribosyl transfer catalysis with trans-N-deoxyribosylase. Kinetic study of purine(pyrimidine) to pyrimidine(purine) trans-N-deoxyribosylase.

Kinetic studies were carried out in order to investigate the enzymic mechanism of a 215-fold-purified purine(pyrimidine) nucleoside: purine(pyrimidine) deoxyribosyl transferase fraction from Lactobacillus helveticus. A variety of natural deoxyribonucleosides and bases were used as substrates. Initial velocity, product inhibition and isotopic exchange studies are consistent with a ping-pong bi-bi mechanism. The kinetic parameters are used to show that this fraction is free from any contamination by a specific purine nucleoside: purine deoxyribosyl transferase also found in the same strain of L. helveticus.     

Translesion synthesis past equine estrogen-derived 2'-deoxyadenosine DNA adducts by human DNA polymerases eta and kappa

Hormone replacement therapy (HRT) increases the risk of developing breast, ovarian, and endometrial cancers. Equilin and equilenin are the major components of the widely prescribed drug used for HRT. 4-Hydroxyequilenin (4-OHEN), a major metabolite of equilin and equilenin, promotes 4-OHEN-modified dC, dA, and dG DNA adducts. These DNA adducts were detected in breast tumor and adjacent normal tissues of several patients receiving HRT. We have recently found that the 4-OHEN-dC DNA adduct is a highly miscoding lesion generating C --> T transitions and C --> G transversions. To explore the mutagenic potential of another major 4-OHEN-dA adduct, site-specifically modified oligodeoxynucleotides containing a single diastereoisomer of 4-OHEN-dA (Pk-1, Pk-2, and Pk-3) were prepared by a postsynthetic method and used as DNA templates for primer extension reactions catalyzed by human DNA polymerase (pol) eta and kappa that are highly expressed in the reproductive organs. Primer extension catalyzed by pol eta or pol kappa occurred rapidly on the unmodified template to form fully extended products. With the major 4-OHEN-dA-modified templates (Pk-2 and Pk-3), primer extension was retarded prior to the lesion and opposite the lesion; a fraction of the primers was extended past the lesion. Steady-state kinetic studies with pol eta and pol kappa indicated that dTMP, the correct base, was preferentially incorporated opposite the 4-OHEN-dA lesion. In addition, pol eta and pol kappa bypassed the lesion by incorporating dAMP and dCMP, respectively, opposite the lesion and extended past the lesion. The relative bypass frequency past the 4-OHEN-dA lesion with pol eta was at least 2 orders of magnitude higher than that observed with pol kappa. The bypass frequency past Pk-2 was more efficient than that past Pk-3. Thus, 4-OHEN-dA is a miscoding lesion generating A --> T transversions and A --> G transitions. The miscoding frequency and specificity of 4-OHEN-dA varied depending on the stereoisomer of the 4-OHEN-dA adduct and DNA polymerase used.     

Kinetic studies of thymidine phosphorylase from mouse liver

Initial velocity and product inhibition studies of thymidine phosphorylase from mouse liver revealed that the basic reaction mechanism of this enzyme is a rapid equilibrium random bi-bi mechanism with an enzyme-phosphate-thymine dead-end complex. Thymine displayed both substrate inhibition and nonlinear product inhibition, i.e., slope and intercept replots vs. 1/[thymine] were nonlinear, indicating that there is more than one binding site on the enzyme for thymine and that when thymine is bound to one of these sites, the enzyme is inhibited. Furthermore, both thymidine and phosphate showed "cooperative effects" in the presence of thymine at concentrations above 60 microM, suggesting that the enzyme may have multiple interacting allosteric and/or catalytic sites. The deoxyribosyl transferase reaction catalyzed by this enzyme is phosphate-dependent, requires nonstoichiometric amounts of phosphate, and can proceed by an "enzyme-bound" 2-deoxyribose 1-phosphate intermediate. These findings are in accord with the rapid equilibrium random bi-bi mechanism and demonstrate that deoxyribosyl transfer by this enzyme involves an indirect-transfer mechanism. These results strongly suggest that phosphorolysis and deoxyribosyl transfer are catalyzed by the same site on thymidine phosphorylase.     

Role of oxygen radicals in DNA damage and cancer incidence

The development of cancer in humans and animals is a multistep process. The complex series of cellular and molecular changes participating in cancer development are mediated by a diversity of endogenous and exogenous stimuli. One type of endogenous damage is that arising from intermediates of oxygen (dioxygen) reduction - oxygen-free radicals (OFR), which attacks not only the bases but also the deoxyribosyl backbone of DNA. Thanks to improvements in analytical techniques, a major achievement in the understanding of carcinogenesis in the past two decades has been the identification and quantification of various adducts of OFR with DNA. OFR are also known to attack other cellular components such as lipids, leaving behind reactive species that in turn can couple to DNA bases. Endogenous DNA lesions are genotoxic and induce mutations. The most extensively studied lesion is the formation of 8-OH-dG. This lesion is important because it is relatively easily formed and is mutagenic and therefore is a potential biomarker of carcinogenesis. Mutations that may arise from formation of 8-OH-dG involve GC --> TA transversions. In view of these findings, OFR are considered as an important class of carcinogens. The effect of OFR is balanced by the antioxidant action of non-enzymatic antioxidants as well as antioxidant enzymes. Non-enzymatic antioxidants involve vitamin C, vitamin E, carotenoids (CAR), selenium and others. However, under certain conditions, some antioxidants can also exhibit a pro-oxidant mechanism of action. For example, beta-carotene at high concentration and with increased partial pressure of dioxygen is known to behave as a pro-oxidant. Some concerns have also been raised over the potentially deleterious transition metal ion-mediated (iron, copper) pro-oxidant effect of vitamin C. Clinical studies mapping the effect of preventive antioxidants have shown surprisingly little or no effect on cancer incidence. The epidemiological trials together with in vitro experiments suggest that the optimal approach is to reduce endogenous and exogenous sources of oxidative stress, rather than increase intake of anti-oxidants. In this review, we highlight some major achievements in the study of DNA damage caused by OFR and the role in carcinogenesis played by oxidatively damaged DNA. The protective effect of antioxidants against free radicals is also discussed.     

The purine-2-deoxyribonucleosidase from Crithidia luciliae. Purification and trans-N-deoxyribosylase activity

Crude extracts of Crithidia luciliae catalysed a deoxyribosyl transfer from purine deoxynucleosides to free purine bases. Fractionation of a 0-80% (NH4)2SO4 fraction from C. luciliae on DEAE-cellulose resulted in the separation of three nucleosidase activities. Two of these were ribonucleosidases, one specific for inosine, uridine and xanthosine and the other for inosine and guanosine, whereas the third activity was specific for purine deoxyribonucleosides. This pattern is similar to that found in Leishmania donovani. Significant deoxyribosyltransferase activity was, however, associated with the purine-2'-deoxyribonucleosidase from C. luciliae. The purine-2'-deoxyribonucleosidase was purified to homogeneity by a six-step procedure involving (NH4)2SO4 fractionation and chromatography on DEAE-cellulose, hydroxyapatite, Sephadex G-75, and a chromatofocusing resin. The purified enzyme migrated as a single band of 17 kDa on SDS/polyacrylamide gel electrophoresis. The enzyme catalysed the hydrolysis of deoxyinosine, deoxyguanosine and deoxyadenosine with Km values of 80 +/- 10.5 microM, 20.7 +/- 3.2 microM and 17.3 +/- 5.3 microM, respectively, and V values for these substrates in the ratio 1:0.5:0.39. The pH optimum for deoxyribosyl transfer from deoxyinosine to guanine was at pH 7.7, while deoxyinosine hydrolysis in the presence of guanine was optimal in the range pH 6-7. During the synthesis of deoxyinosine from hypoxanthine and deoxyadenosine two products were formed. One of these coeluted with deoxyinosine on HPLC, while the second was tentatively identified as the positional isomer, 7-(beta-D-2'-deoxyribofuranosyl)hypoxanthine.     

Synthesis and DNA binding properties of a new benzo[f]imidazo[1,5b]-isoquinoline-butan-1,2-diol derivative

A benzo[f]imidazo[1,5b]-isoquinoline derivative 4 with a 1,2-butandiol linker was prepared by reaction of a trimethylsilylated 5-naphthylidenehydantoin 3 with a 2,3-dideoxy-D-glycero-pentafuranoside 2 in 22% yield. After deprotection, the resulting compound 5 was converted to a DMT protected phosphoramidite building block 7 for standard DNA synthesis. DNA/DNA, DNA/RNA duplexes with 5 inserted as bulges were destabilized, except when the new amidite was used for the synthesis of a zipping duplex.     

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.     

HNO induces DNA deletions in the yeast S. cerevisiae

HNO is genotoxic but its mechanism is not well understood. There are many possible mechanisms by which HNO can attack DNA. Since HNO is electrophilic, it may react with exocyclic amine groups on DNA bases and through a series of subsequent reactions form a deaminated product. Alternatively, HNO may induce radical chemistry through O(2)-dependent (or possibly O(2)-independent) chemistry. In cell free systems, experiments have shown that HNO does react with DNA, resulting in base oxidation and strand cleavage. In this study, we used a whole-cell system in the yeast Saccharomyces cerevisiae to study the mechanism of HNO induced DNA damage with Angeli's salt as HNO donor. The yeast DEL assay provided a measure of intrachromosomal recombination leading to DNA deletions. We also examined interchromosomal recombination leading to genomic rearrangements and used the canavanine (CAN) assay to study induction of forward point mutations. HNO was a potent inducer of DNA deletions and recombination but it was negative for induction of point mutations. This suggests that HNO causes DNA strand breaks rather than base damage. Genotoxicity was observed under aerobic and anaerobic conditions and NAC protected against HNO induced DNA deletions. Since HNO is genotoxic under anaerobic conditions, NAC probably protected against radicals generated by HNO independent of oxygen.     

Manganese-dependent ribonucleotide reductase from Propionibacterium freudenreichii subsp. Shermanii: partial purification, characteristics and role in DNA biosynthesis

Like Lactobacillus leichmanii, Rhizobium meliloti, and Euglena gracilis, P. freudenreichii implicates cobalamin in DNA anabolism via adenosylcobalamin-dependent ribonucleotide reductase. However, in the absence of corrinoids, P. freudenreichii is able to synthesize DNA with the involvement of an alternative ribonucleotide reductase, which is independent of adenosylcobalamin. This enzyme is localized in both the cytoplasm (80% of activity) and the cytoplasmic membrane (20% of activity), being loosely bound to the latter. Experiments with crude ribonucleotide reductase isolated from extracts of corrinoid-deficient cells showed that manganese specifically stimulates this enzyme and that it is composed of two protein subunits, a feature that is typical of all metal-containing reductases activated by molecular oxygen. Low concentrations of manganese ions enhanced DNA synthesis in corrinoid-deficient manganese-limited cells. This effect was prevented by the addition of 80 mM hydroxyurea, a specific inhibitor of metal-containing aerobic ribonucleotide reductases. It was concluded that, in adenosylcobalamin-deficient P. freudenreichii cells, DNA synthesis is provided with deoxyribosyl precursors through the functioning of manganese-dependent aerobic ribonucleotide reductase composed of two subunits.     

A large "footprint" at the boundary of the human beta-globin locus control region hypersensitive site-2

The 5'-boundary region of the human beta-globin locus control region hypersensitive site-2 (HS-2) was examined for protein-DNA interactions. The HS-2 is an erythroid specific DNase I hypersensitive site that extends for approximately 600 bp. Erythroid K562 cells and non-erythroid HeLa cells were damaged by bleomycin and hedamycin--these agents are able to "footprint" nucleosome cores and proteins bound to DNA. The fragments generated by DNA damage were amplified by the ligation-mediated polymerase chain reaction with primers specific for the 5'-boundary region of HS-2 and examined at base pair resolution on DNA sequencing gels. The intensity of damage in intact cells was compared with that in purified DNA. The comparison between intact cells and purified DNA revealed a protected region of 226 bp with bleomycin and 182 bp with hedamycin in K562 cells. The length of the protected region was consistent with the presence of a nucleosome core. We postulate that an erythroid-specific protein binds next to the positioned nucleosome at the boundary of HS-2 to prevent sliding of the nucleosome into the hypersensitive site--this would also account for the large size of the protected region. HeLa cells (lacking a hypersensitive site in the beta-globin cluster) did not have an area of protection in this region.     

Error-free bypass of 2-hydroxyadenine by human DNA polymerase lambda with Proliferating Cell Nuclear Antigen and Replication Protein A in different sequence contexts

1,2-dihydro-2-oxoadenine (2-OH-A), a common DNA lesion produced by reactive oxygen species, is a strong replicative block for several DNA polymerases (DNA pols). We have previously shown that various bases can be misincorporated opposite the 2-OH-A lesion and the type of mispairs varies with either the sequence context or the type of DNA pol tested. Here, we have analysed the ability of the human pol family X member DNA pol λ, to bypass the 2-OH-A lesion. DNA pol λ can perform error-free bypass of 2-OH-A when this lesion is located in a random sequence, whereas in a repeated sequence context, even though bypass was also largely error-free, misincorporation of dGMP could be observed. The fidelity of translesion synthesis of 2-OH-A in a repeated sequence by DNA pol λ was enhanced by the auxiliary proteins Proliferating Cell Nuclear Antigen (PCNA) and Replication Protein A (RP-A). We also found that the DNA pol λ active site residue tyrosine 505 determined the nucleotide selectivity opposite 2-OH-A. Our data show, for the first time, that the 2-OH-A lesion can be efficiently and faithfully bypassed by a human DNA pol λ in combination with PCNA and RP-A.     

Chemical synthesis of 2''-deoxyoligonucleotides containing 5-fluoro-2''-deoxycytidine.

2'-Deoxyoligonucleotides with 5-fluorocytosine residues incorporated at specific positions of the nucleotide sequence are tools of great potential in the study of the catalytic mechanism by which DNA cytosine methyltransferases methylate the 5-position of DNA cytosine residues in specific sequence contexts. Chemical synthesis of such oligonucleotides is described. Two alternative approaches have been developed, one of which proceeds via a fully protected phosphoramidite of 5-fluoro-4-methylmercapto-2'-deoxyuridine 2, the other via a fully protected phosphoramidite of 5-fluoro-2'-deoxycytidine 3. Either building block can be used in automated oligonucleotide synthesis applying standard elongation cycles and deprotection procedures exclusively. The methylmercapto function of 2 is replaced by an amino group in the final ammonia treatment used for cleavage from support and base deprotection.     

Mutational specificities of brominated DNA adducts catalyzed by human DNA polymerases

Chronic inflammation is known to lead to an increased risk for the development of cancer. Under inflammatory condition, cellular DNA is damaged by hypobromous acid, which is generated by myeloperoxidase and eosinophil peroxidase. The reactive brominating species induced brominated DNA adducts such as 8-bromo-2′-deoxyguanosine (8-Br-dG), 8-bromo-2′-deoxyadenosine (8-Br-dA), and 5-bromo-2′-deoxycytidine (5-Br-dC). These DNA lesions may be implicated in carcinogenesis. In this study, we analyzed the miscoding properties of the brominated DNA adducts generated by human DNA polymerases (pols). Site-specifically modified oligodeoxynucleotides containing a single 8-Br-dG, 8-Br-dA, or 5-Br-dC were used as a template in primer extension reactions catalyzed by human pols α, κ, and η. When 8-Br-dG-modified template was used, pol α primarily incorporated dCMP, the correct base, opposite the lesion, along with a small amount of one-base deletion (4.8%). Pol κ also promoted one-base deletion (14.2%), accompanied by misincorporation of dGMP (9.5%), dAMP (8.0%), and dTMP (6.1%) opposite the lesion. Pol η, on the other hand, readily bypassed the 8-Br-dG lesion in an error-free manner. As for 8-Br-dA and 5-Br-dC, all the pols bypassed the lesions and no miscoding events were observed. These results indicate that only 8-Br-dG, and not 5-Br-dC and 8-Br-dA, is a mutagenic lesion; the miscoding frequency and specificity vary depending on the DNA pol used. Thus, hypobromous acid-induced 8-Br-dG adduct may increase mutagenic potential at the site of inflammation.     

Mechanism of protection by the flavonoids, quercetin and rutin, against tert-butylhydroperoxide- and menadione-induced DNA single strand breaks in Caco-2 cells

Protection by the flavonoids, quercetin and rutin, against tert-butylhydroperoxide (tert-BOOH)- and menadione-induced DNA single strand breaks was investigated in Caco-2 cells. Both tert-BOOH and menadione induced DNA single strand breaks in a concentration-dependent manner. Pre-incubation of Caco-2 cells with either quercetin or rutin for 24 h significantly decreased the formation of DNA single strand breaks evoked by tert-BOOH (P <.05). Iron chelators, 1,10-phenanthroline (o-Phen) and deferoxamine mesylate (DFO), also protected against tert-BOOH-induced DNA damage, whereas butylated hydroxytoluene (BHT) had no effect. Quercetin, and not rutin, decreased the extent of menadione-induced DNA single strand breaks. DFO and BHT, and not o-Phen, protected against menadione-induced DNA strand break formation (P <.05). From the results of this study, iron ions were involved in tert-BOOH-induced DNA single strand break formation in Caco-2 cells, whereas DNA damage evoked by menadione was far more complex. We demonstrated that the flavonoids, quercetin and rutin, protected against tert-BOOH-induced DNA strand breaks by way of their metal ion chelating mechanism. However, quercetin, and not rutin, protected against menadione-induced DNA single strand breaks by acting as both a metal chelator and radical scavenger.     

Characterization of DNA structures by laser Raman spectroscopy

Oriented fibers drawn from aqueous gels of calf-thymus DNA were maintained at constant relative humidites of 75 and 92% to yield canonical A-DNA and B-DNA structures, respectively. Raman spectra of the two forms of DNA were recorded over the spectral range 300–4000 cm^?1. The authenticated DNA fibers were deuterated in hygrostatic cells containing D₂O at appropriate relative humidities, and the corresponding spectra of deuterated DNAs were also obtained. The spectra reveal all of the Raman scattering frequencies and intensities characteristic of A- and B-DNA structures in both nondeuterated and deuterated froms, as well as the frequencies and intensities of adsorbed solvent molecules from which the hydration content of DNA fibers can be calculated. Numerous conformation-sensitive vibrational modes of DNA bases and phosphate groups have been identified throughout the 300–1700-cm^?1 interval. Evidence has also been obtained for conformation sensitivity of deoxyribosyl CH stretching modes in the 2800–3000-cm^?1 region. Raman lines of both the backbone and the bases are proposed as convenient indicators of A- and B-DNA structures. The results are extended to Z-DNA models investigated previously. Some implications of these findings for the determination of DNA or RNA structure from Raman spectra of nucleoproteins and viruses are considered.     

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.