The pyrimidine ring-opened derivative of 1,N6-ethenoadenine is excised from DNA by the Escherichia coli Fpg and Nth proteins.

It was previously shown that 1,N(6)-ethenoadenine (epsilonA) in DNA rearranges into a pyrimidine ring-opened derivative of 20-fold higher mutagenic potency in Escherichia coli (AB1157 lacDeltaU169) than the parental epsilonA (Basu, A. K., Wood, M. L., Niedernhofer, L. J., Ramos, L. A., and Essigmann, J. M. (1993) Biochemistry 32, 12793-12801). We have found that at pH 7.0, the stability of the N-glycosidic bond in epsilondA is 20-fold lower than in dA. In alkaline conditions, but also at neutrality, epsilondA depurinates or converts into products: epsilondA --> B --> C --> D. Compound B is a product of water molecule addition to the C(2)-N(3) bond, which is in equilibrium with a product of N(1)-C(2) bond rupture in epsilondA. Compound C is a deformylated derivative of ring-opened compound B, which further depurinates yielding compound D. Ethenoadenine degradation products are not recognized by human N-alkylpurine-DNA glycosylase, which repairs epsilonA. Product B is excised from oligodeoxynucleotides by E. coli formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III (Nth). Repair by the Fpg protein is as efficient as that of 7,8-dihydro-8-oxoguanine when the excised base is paired with dT and dC but is less favorable when paired with dG and dA. Ethenoadenine rearrangement products are formed in oligodeoxynucleotides also at neutral pH at the rate of about 2-3% per week at 37 degrees C, and therefore they may contribute to epsilonA mutations.

Endogenous and exogenous DNA lesions recognized by N-alkylpurine-DNA glycosylases.

The combined action of glycosylases and abasic site-specific endonucleases on damaged bases in DNA results in single strand breaks. In plasmid DNA, as a consequence, the covalently closed circular (ccc) form is converted to the open circular (oc) form, and this can be quantitated by agarose gel electrophoresis. We studied DNA lesions sensitive to E. coli 3-methyladenine-DNA glycosylase II (AlkA) and cloned human N-alkylpurine-DNA glycosylase (ANPG-40) which are known to excise alkylated bases and etheno adducts. pBR322 and pAlk10 plasmids not pretreated with mutagens were cleaved by both glycosylases in the presence of enzymes possessing endonucleolytic activity, which indicates that plasmids contain unknown, endogenously formed adducts. Plasmids pretreated with chloroacetaldehyde, a mutagen forming etheno adducts, exhibited enhanced sensitivity to both glycosylases. Adducts formed by acrolein and croton aldehyde were excised by AlkA, but not by ANPG-40, whereas malondialdehyde adducts were not excised by either glycosylase. Bulky p-benzochinone adducts were not excised by AlkA, however, the plasmid pretreated with this mutagen was incised by endonucleases, possibly without prior generation of an abasic site. These examples show that examination of conformational changes of plasmid DNA can be taken advantage of to study the specificity of N-alkylpurine-DNA-glycosylases.

Template-directed base pairing of 2-chloro-2'-deoxyadenosine catalyzed by AMV reverse transcriptase.

2-Chloro-2'-deoxyadenine (2CldA) is used for treatment of several lymphoid malignancies. Since this drug is incorporated into DNA, we have undertaken studies on base pairing of 2-chloroadenine (2ClA). 2CldA phosphoramidite was synthesized and used for preparation of 25-mer templates with 2ClA located at site 21 from the 3'-end. Kinetic parameters (Km and Vmax) for the incorporation of deoxynucleoside-5'-triphosphates by AMV reverse transcriptase opposite the 2ClA template, as well as for the extension of 2ClA.T pair, were determined. The efficiency (Vmax/Km) of incorporation of dGTP, dCTP, and dATP opposite 2ClA is at least one order of magnitude lower than opposite unmodified A. The efficiency of incorporation of dTTP opposite 2ClA is about 30-fold lower than opposite A and extension of 2ClA.T pair is 3-fold lower than of A.T pair. From the analysis of the parameters of dTTP incorporation we conclude that formation of 2ClA.T pair is thermodynamically, but not kinetically controlled. The difference in binding energy (deltadeltaG) between 2ClA.T and A.T pairs in the environment of the polymerase active site is 2 kcal/mol. Our results indicate that the presence of 2ClA in DNA slows down replication, but does not lead to base-substitution mutations.

Activity of Escherichia coli DNA-glycosylases on DNA damaged by methylating and ethylating agents and influence of 3-substituted adenine derivatives.

Methylating and ethylating agents are used in the chemical industry and produced during tobacco smoking. They generate DNA base damage whose role in cancer induction has been documented. Alkylated bases are repaired by the base excision repair pathway. We have established the repair efficiency of methylated and ethylated bases by various Escherichia coli repair proteins, namely 3-methyladenine-DNA-glycosylase I (TagA protein), which excises 3-methyladenine and 3-methylguanine, 3-methyladenine-DNA-glycosylase II (AlkA protein), which has a broad substrate specificity including 3- and 7-alkylated purines and the formamidopyrimidine(Fapy)-DNA-glycosylase (Fpg protein) repairing imidazole ring-opened 7-methylguanine. The comparison of the Km values of these various enzymes showed that methylated bases were excised more efficiently than ethylated bases. Several 3-alkyladenine derivatives have been synthesized and examined for their ability to inhibit the activity of the various repair proteins. We have shown that 3-ethyl-, 3-propyl-, 3-butyl- and 3-benzyladenine were much more efficient inhibitors of TagA protein than 3-methyladenine. The inhibitory effect was increased with the increase of the size of alkyl-group and IC50 for 3-benzyladenine was 0.4 +/- 0.1 microM as compared to 1.5 +/- 0.3 mM for 3-methyladenine. These compounds inhibited neither the AlkA protein nor human 3-methyladenine-DNA-glycosylase (ANPG protein). Moreover, 3-hydroxyethyladenine did not affect the activity of any of these enzymes. Taken together, these results suggest that hydrophobic interactions are involved in the mechanism of inhibition and/or recognition and excision of alkylated purines by TagA protein.

Miscoding properties of isoguanine (2-oxoadenine) studied in an AMV reverse transcriptase in vitro system.

We have found that isoguanine (iG) can pair with thymine (iG.T) and the non-natural base, 5-methylisocytosine (iG.iCM) during template directed synthesis catalyzed by AMV reverse transcriptase. The ratio of these pairings is about 1:10, irrespectively which of the templates, poly(C,iG) or poly(I,iG) is used. This ratio corresponds to the ratio of 2-OH and 2-keto tautomers in monomer in aqueous solution and apparently it is not influenced by the template context. Our results indicate also that formation of the reverse transcriptase catalyzed base pairs between iG and A, G or C can occur only at a low frequency, comparable to the frequency, of mismatches of.(ABSTRACT TRUNCATED)

The induction of adaptive response to alkylating agents in Escherichia coli reduces the frequency of specific C-->T mutations in chloroacetaldehyde-treated M13 glyU phage.

The mutagenicity and repair of cytosine adducts formed in reactions of chloroacetaldehyde (CAA), a metabolite of the human carcinogen vinyl chloride, have been studied. The treatment of single-stranded DNA M13 JCM15472 (glyU313) phage with CAA and subsequent transfection of Escherichia coli K-12 JC15419 (trpA461) tester strain resulted in a dose-dependent increase of phage C-->T transitions and a decrease of phage survival. The induction of the adaptive response to alkylating agents in bacterial cells significantly decreased the frequency of examined C-->T transitions and increased phage survival. The results indicate that both CAA adducts to cytosine, the initially formed 3,N4-(N4-alpha-hydroxyethano)cytosine and the product of its dehydration, 3,N4-ethenocytosine, provoke C-->T transitions and are repaired in adapted bacteria. The role of 3-methyladenine-DNA glycosylase II, which is a part of the adaptive response system in E. coli, in excision of CAA adducts to cytosine, is discussed.

Chloroacetaldehyde-induced mutagenesis in Escherichia coli: specificity of mutations and modulation by induction of the adaptive response to alkylating agents.

The mutational specificity of chloroacetaldehyde (CAA), one of the metabolites of the human carcinogen vinyl chloride (VC), has been determined through the examination of Arg+ revertants in Escherichia coli AB2497 (Arg-) and identification of their tRNA suppressors. The predominant mutations were GC-->AT transitions (65%) followed by AT-->TA transversions (12.5%). The observed mutational specificity of CAA is very similar to the reported specificity of the other VC metabolite, chloroethylene oxide. The induction of the adaptive response to alkylating agents significantly decreased the frequency of CAA-induced Rifr and Arg+ mutants in E. coli AB2497 and increased the cell survival. Likewise, the adaptation of bacterial cells decreased the frequency of GC-->AT transitions in CAA-treated M13glyU phage transformed to E. coli JC15419 and increased the phage survival. Experiments with strain MS23, which is an alkA mutant deficient in 3-methyladenine-DNA glycosylase II, and with MS23 harboring the pYN1000 plasmid carrying the alkA+ gene, have shown that induction of this repair enzyme is responsible for reduction of the level of CAA-induced mutations. The role of N2,3-ethenoguanine, among the other etheno-adducts, in CAA-induced mutagenesis and as a target for repair in 3-methyladenine-DNA glycosylase II proficient bacterial cells is discussed.

Development of monoclonal antibodies specific for 1,N2-ethenodeoxyguanosine and N2,3-ethenodeoxyguanosine and their use for quantitation of adducts in G12 cells exposed to chloroacetaldehyde.

Monoclonal antibodies specific for N2,3-ethenodeoxyguanosine (N2,3-epsilon dGuo) and 1,N2-ethenodeoxyguanosine (1,N2-epsilon dGuo) were developed. In a competitive ELISA, 50% inhibition of binding of the N2,3-epsilon dGuo specific antibody (ETH1) was achieved with 18 fmol of N2,3-epsilon dGuo. Fifty per cent inhibition of the 1,N2-epsilon dGuo-specific antibody (ETH2) required 11 pmol 1,N2-epsilon dGuo. Immunoassays for N2,3-epsilon dGuo and 1,N2-epsilon dGuo in single-stranded DNA were developed using these antibodies. The immunoassays could detect as little as 48 fmol of N2,3-epsilon dGuo or 340 fmol 1,N2-epsilon dGUO in 25 micrograms of single stranded DNA. These assays and previously developed immunoassays for 1,N6-ethenodeoxy-adenosine (1,N6-epsilon dAdo) and 3,N4-ethenodeoxycytidine (3,N4-epsilon dCyd) were used to measure etheno adduct levels in DNA of cells exposed to chloroacetaldehyde. The cells used were V79 cells with an inactivated hprt gene and a single copy of the bacterial gpt gene (G12 cells). The most abundant etheno adduct was 1,N6-epsilon dAdo, followed by 3,N4-epsilon dCyd and N2,3-epsilon dGuo. 1,N2-epsilon dGuo was not detected in chloro-acetaldehyde-treated G12 cells. Chloroacetaldehyde was also shown to be mutagenic in these same cells.

The effect of neighboring bases on miscoding properties of N2,3-ethenoguanine.

The miscoding potential of N2,3-ethenoguanine (epsilon G), one of the carcinogen vinyl chloride adducts to DNA bases, has been examined by copying of poly (A, epsilon G) templates with DNA-dependent RNA polymerase and reverse transcriptase. In contrast to the results previously obtained with poly (C, epsilon G) templates where epsilon G acts as G and A, in poly (A, epsilon G) templates epsilon G acts almost exclusively as A. These results suggest that mutagenic potential of epsilon G in vivo can depend on the nature of neighboring bases.

Both purified human 1,N6-ethenoadenine-binding protein and purified human 3-methyladenine-DNA glycosylase act on 1,N6-ethenoadenine and 3-methyladenine.

We previously described a protein, isolated from human tissues and cells, that bound to a defined double-stranded oligonucleotide containing a single site-specifically placed 1,N6-ethenoadenine. It was further demonstrated that this protein was a glycosylase and released 1,N6-ethenoadenine. We now find that this enzyme also releases 3-methyladenine from methylated DNA and that 3-methyladenine-DNA glycosylase behaves in the same manner, binding to the ethenoadenine-containing oligonucleotide and cleaving both ethenoadenine and 3-methyladenine from DNA containing these adducts. The rate and extent of glycosylase activities toward the two adducts are similar.

1,N2-ethenodeoxyguanosine: properties and formation in chloroacetaldehyde-treated polynucleotides and DNA.

1,N2-Etheno-2'-deoxyguanosine (1,N2-epsilon dGuo), not previously reported as a product of chloroacetaldehyde (CAA) reaction, has been synthesized and characterized. Reaction of deoxyguanosine with CAA in dimethylformamide in the presence of K2CO3 led to preparation of pure 1,N2-epsilon dGuo with 55% yield. pKa values are 2.2 and 9.2. The anionic form of the compound exhibits weak but defined fluorescence; the intensity is similar to that of N2,3-etheno-2'-deoxyguanosine (N2,3-epsilon dGuo) at neutrality. The stability of the glycosyl bond of 1,N2-epsilon dGuo (t1/2 = 2.3 h at 37 degrees C, pH 1) is 10-fold greater than of unmodified deoxyguanosine and at least one thousand-fold greater than of isomeric N2,3-epsilon dGuo. Reaction of CAA with model polynucleotides indicates that hydrogen bonding of guanine residues in the double-stranded structures is, as expected, an important factor in the formation of 1,N2-ethenoguanine. In contrast, the formation of isomeric N2,3-ethenoguanine is relatively independent of whether the DNA is single- or double-stranded. In salmon sperm DNA, reacted with CAA at neutrality, the formation of 1,N2-ethenoguanine could be demonstrated. However, we find the efficiency of formation of this adduct in double-stranded DNA to be lower than that of all other etheno derivatives.

Miscoding potential of N2,3-ethenoguanine studied in an Escherichia coli DNA-dependent RNA polymerase in vitro system and possible role of this adduct in vinyl chloride-induced mutagenesis.

The miscoding potential of N2,3-ethenoguanine (epsilon G), one of the carcinogen vinyl chloride adducts to DNA bases, has been evaluated in an Escherichia coli DNA-dependent RNA polymerase in vitro system. Epsilon G present in poly(C) templates causes incorporation of cytosine (C), uridine (U) and adenosine (A) under competitive and non-competitive conditions, and in the presence of either Mn2+ and Mg2+ cations, indicating that this modified base still retains the coding properties of unmodified G and can also act as A or U. The formation of hydrogen bonded pairs between different tautomeric forms of epsilon G and C, U and A is proposed. The possible role of epsilon G, along with a role of other vinyl chloride adducts in causing of GC----AT transitions, the most frequent mutation induced by a vinyl chloride metabolite, is discussed.

Evidence for the mutagenic potential of the vinyl chloride induced adduct, N2, 3-etheno-deoxyguanosine, using a site-directed kinetic assay.

N2,3-Ethenoguanine (epsilon G) is a product of vinyl chloride reaction with DNA in vivo and of its ultimate metabolite, chloroacetaldehyde, in vitro. The synthesis of the very labile 5'-triphosphate of N2,3-etheno-deoxyguanosine (epsilon dGuo) has made it possible to study the base pairing properties of this derivative placed opposite a defined normal base in a 25-base oligonucleotide template. The kinetic parameters, Km and Vmax were determined from elongation of a [32P]5'-end labeled primer annealed one base prior to the designated template base, epsilon G.T pairs, which would be mutagenic, were formed with a frequency 2- to 4-fold greater than the analogous wobble pair, G.T. The non-mutagenic pairing, epsilon G.C, occurs with a lower frequency than G.C but neither epsilon G.T or epsilon G.C constitute a significant block to replication. The frequency of epsilon G.T formation was similar with all polymerases tested: Escherichia coli DNA polymerase I (Klenow fragment), exonuclease-free Klenow, Drosophila melanogaster polymerase alpha-primase complex and human immunodeficient virus-I reverse transcriptase (HIV-RT). It is concluded that these prokaryotic and eukaryotic replicating enzymes apparently recognize the same structural features, and on replication G----A transitions would occur, which in turn, could initiate malignant transformation. In contrast to the G.T mismatch which is known to have a specific repair system, etheno derivatives are apparently not repaired in vivo.

A new one-step method for the preparation of 3',5'-bisphosphates of acid-labile deoxynucleosides.

A method is described for preparing 3',5'-bisphosphates of labile deoxynucleosides. Under strictly anhydrous conditions, pyrophosphoryl tetrakistriazole apparently forms a ring structure bridging the 3'- and 5'-hydroxyl groups of deoxynucleosides, since upon the addition of water the ring opens and the 3',5'-bisphosphate is formed. Due to the presence of triethylamine no acid is generated at any time so that the entire procedure is in neutral solution. The bisphosphates of N2,3-ethenodeoxyguanosine, O2-ethyldeoxythymidine, and O4-methyldeoxythymidine, all of which are acid-labile, were prepared in good yield without degradation. Other modified bisphosphates prepared include O6-benzyldeoxyguanosine and 1,N6-ethenodeoxyadenosine, as well as those of unmodified deoxyguanosine and thymidine. Characterization was by 31P NMR and UV spectroscopy. Both 5'p(dT)p3' and 5'p(dG)p3' were substrates for RNA ligase, further proving the structure of the phosphorylated compounds.

Synthesis of N2,3-ethenodeoxyguanosine, N2,3-ethenodeoxyguanosine 5'-phosphate, and N2,3-ethenodeoxyguanosine 5'-triphosphate. Stability of the glycosyl bond in the monomer and in poly(dG,epsilon dG-dC).

The synthesis of a new modified etheno-2'-deoxyguanosine is reported. N2,3-Ethenodeoxy-guanosine (epsilon dGuo) is a product in double-stranded DNA treated with the carcinogen vinyl chloride in vivo or its metabolite chloroacetaldehyde in vitro. The lability of its glycosyl bond has, however, interfered with its isolation from DNA. The synthesis, starting with O6-benzyl-2'-deoxyguanosine 5'-phosphate, reacted with bromoacetaldehyde, could only be accomplished in slightly alkaline media, which prevented significant loss of the sugar. The 5'-phosphate also decreased the lability of the glycosyl bond. The resulting compound, when deprotected, was converted to N2,3-ethenodeoxyguanosine 5'-triphosphate, as well as the corresponding nucleoside. Fluorescence, UV, and 1H NMR data were consistent with the assigned structures and almost identical with those of the previously synthesized much more stable ribo analogues [Kusmierek et al. (1987) J. Org. Chem. 52, 2374-2378]. A systematic study of the pH-dependent glycosyl bond cleavage gave a t1/2, 37 degrees C, pH 6, of approximately 3.5 h for the nucleoside and 7-10 h for the nucleotides. Comparison, under the same conditions, of stability of the glycosyl bond in poly(dG,epsilon dG-dC) showed an increased stability of 2 orders of magnitude, t1/2 = approximately 600 h. The rate of sugar loss was, in all cases, greatly decreased at higher pH's, over the range of pH 5-9. These stability data indicate that when slightly alkaline conditions can be used, studies on incorporation of epsilon dGuo into polymers for in vitro mutagenesis studies are possible.

Comparison of polymerase insertion and extension kinetics of a series of O2-alkyldeoxythymidine triphosphates and O4-methyldeoxythymidine triphosphate.

The effect of alkyl group size on ability to act as deoxythymidine triphosphate (dTTP) has been studied for the carcinogen products O2-methyl-, O2-ethyl-, and O2-isopropyl-dTTP by using three types of nucleic acids as template and DNA polymerase I (Pol I) or Klenow fragment as the polymerizing enzymes. Apparent Km and relative Vmax values were determined in primer extension on M13 DNA at a single defined site, in poly[d(A-T)], and in nicked DNA. These data are the basis for calculation of the relative rate of insertion opposite A, relative to dTTP. The insertion rate for any O2-alkyl-dTTP is much higher than for a mismatch between unmodified dNTPs. Unexpectedly, O2-isopropyl-dTTP is more efficiently utilized than O2-methyl-dTTP or O2-ethyl-dTTP on each of the templates. O2-isopropyl-dTTP also substitutes for dTTP over extended times of DNA synthesis at a rate only slightly lower than that of dTTP. Parallel experiments using O4-methyl-dTTP under the same conditions show that it is incorporated opposite A more frequently than is O2-methyl-dTTP. Therefore, both the ring position and the size of the alkyl group influence polymerase recognition. Once formed, all O2-alkyl-T.A termini permit elongation, as does O4-methyl-T.A. In contrast to the relative difficulty of incorporating the O-alkyl-dTTPs, formation of the following normal base pair (C.G) occurs rapidly when dGTP is present. This indicates that a single O-alkyl-T.A pair does not confer significant structural distortion recognized by Pol I.

Preparative electrochemical reduction of 2-amino-6-chloropurine and synthesis of 6-deoxyacyclovir, a fluorescent substrate of xanthine oxidase and a prodrug of acyclovir.

D.c. polarography of 2-amino-6-chloropurine in aqueous medium over a broad pH range revealed two diffusion waves, the first of which corresponds to reduction of the C(6)-Cl bond, leading to formation of 2-aminopurine in high yield. Condensation of the sodium salt of 2-aminopurine with (2-acetoxyethoxy)methyl chloride led to the two isomeric 9- and 7-(2-hydroxyethoxymethyl)-2-aminopurines. The 9- isomer, 6-deoxyacyclovir, a prodrug of acyclovir previously synthesized by another route, was readily converted to the latter by xanthine oxidase; the 7-isomer was not a substrate. The intense fluorescence of 6-deoxyacyclovir makes it a convenient fluorescent substrate for xanthine oxidase, although less sensitive than xanthine; it is shown that 2-aminopurine would be a very sensitive fluorescent substrate. The polarographic behaviour of the riboside of 2-amino-6-chloropurine was virtually identical with that of the parent purine, leading to a simple procedure for conversion of 2-amino-6-chloropurine nucleosides and acyclonucleosides to the corresponding 2-aminopurine congeners.

The vinyl chloride-derived nucleoside, N2,3-ethenoguanosine, is a highly efficient mutagen in transcription.

N2,3-Ethenoguanine (N2,3-epsilon G) was recently identified in the liver of vinyl chloride-exposed rats. We have now synthesized the nucleoside and the 5'-diphosphate which was copolymerized with CDP. The deoxypolynucleotide complement, synthesized by AMV reverse transcriptase contained, in addition to dG, dC and dT. The total pyrimidine content was approximately equivalent to the N2,3-epsilon G content of the template. Incorporation of dC is neither lethal nor mutagenic, while dT incorporation represents a mutagenic event, occurring with approximately 20% frequency. N2,3-epsilon G X dT base pairs can have two hydrogen bonds with minimal helical distortion, as is also the case for N2,3-epsilon G X C base pairs. N2,3-epsilon G is the only derivative formed in vivo by the human carcinogen, vinyl chloride, that can be shown to have a high probability of causing transitions which could initiate malignant transformation.

Pyrimidine homoribonucleosides: synthesis, solution conformation, and some biological properties.

Conversion of uridine and cytidine to their 5'-O-tosyl derivatives, followed by cyanation with tetraethylammonium cyanide, reduction and deamination, led to isolation of the hitherto unknown homouridine (1-(5'-deoxy-beta-D-allofuranosyl)uracil) and homocytidine (1-(5'-deoxy-beta-D-allofuranosyl)cytosine), analogues of uridine and cytidine in which the exocyclic 5'-CH2OH chain is extended by one carbon to CH2CH2OH. Homocytidine was also phosphorylated to its 6'-phosphate and 6'-pyrophosphate analogues. In addition, it was converted, via its 2,2'-anhydro derivative, to arahomocytidine, an analogue of the chemotherapeutically active araC. The structures of all the foregoing were established by various criteria, including 1H and 13C NMR spectroscopy, both of which were also applied to analyses of the solution conformations of the various compounds, particularly as regards the conformations of the exocyclic chains. The behaviour of the homo analogues was examined in several enzymatic systems. Homocytidine was a feeble substrate, without inhibitory properties, of E. coli cytidine deaminase. Homocytidine was an excellent substrate for wheat shoot nucleoside phosphotransferase; while homouridine was a good substrate for E. coli uridine phosphorylase. Although homoCMP was neither a substrate, nor an inhibitor, of snake venom 5'-nucleotidase, homoCDP was a potent inhibitor of this enzyme (Ki approximately 6 microM). HomoCDP was not a substrate for M. luteus polynucleotide phosphorylase. None of the compounds exhibited significant activity vs herpes simplex virus type 1, or cytotoxic activity in several mammalian cell lines.