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J T Kuśmierek

Publications and source records attributed to J T Kuśmierek.

At least 19 recordsLinked to original sources
PubMed1998

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.

DNA Adducts↗
PubMed1998

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.

Animals↗
PubMed1996

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)

5-Methylcytosine↗
PubMed1994

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.

Acetaldehyde↗
PubMed1993

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.

Acetaldehyde↗
PubMed1992

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.

Adenine↗
PubMed1991

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.

Base Composition↗
PubMed1991

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.

DNA Damage↗
PubMed1989

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.

Alkylation↗
PubMed1987

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.

Base Composition↗
PubMed1987

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.

5'-Nucleotidase↗
PubMed1986

O4-Methyl, -ethyl, or -isopropyl substituents on thymidine in poly(dA-dT) all lead to transitions upon replication.

In a previous paper, we reported that O4-methyl dTTP can be incorporated into poly(dA-dT) in place of thymidine without distortion of the helical structure, but on replication it could behave as deoxycytidine and misincorporate dGTP. Only weak interactions are possible for any O4-modified T X A pair. While O4-alkyl T X G pairing should be favored, experiments to detect the ability of Escherichia coli DNA polymerase I (pol I) to utilize the triphosphate as dCTP were ambiguous. dTTPs with larger alkyl groups (ethyl, isopropyl) have now been synthesized and tested for their recognition as dTTP by pol I. Enhanced steric hindrance could be expected, particularly for O4-isopropyl dTTP, which has a three-carbon branched chain. However, both compounds behaved qualitatively like O4-methyl dTTP, being incorporated into poly(dA-dT) and then directing deoxyguanosine misincorporation by pol I. Quantitative comparisons of mutagenicity were not possible because of the finding that, unlike polymers made with O4-methyl dTTP, those made with ethyl or isopropyl dTTP were resistant to hydrolysis by using a variety of nucleases. The frequent misincorporations of dGTP would be expected to produce transitions in vivo. O4-ethyldeoxythymidine is very poorly repaired in vivo, which would also be expected for repair of O4-isopropyldeoxythymidine. Therefore, under suitable conditions, these particular carcinogen products are likely to be initiators of carcinogenesis.

DNA Polymerase I↗
PubMed1986

Neutral reactions of haloacetaldehydes with polynucleotides: mechanisms, monomer and polymer products.

The generally accepted mechanism for the formation of etheno derivatives upon reaction of adenosine or cytidine with haloacetaldehydes involves two intermediates. The first, a primary addition to the exocyclic amino group, has not been experimentally verified. The second, a cyclic form of the first intermediate, has been described in monomers but presumed to be too unstable to exist in polynucleotides since such derivatives would be readily dehydrated to other derivatives at pHs below neutrality. We have found that the cyclic intermediates of adenosine and cytidine are the predominant products in polynucleotides, even upon extensive reaction with chloroacetaldehyde at neutrality. The hydrated compounds have half-lives at pH 7, 37 degrees C, of 1.4 h and 13 h for adenosine and cytidine, respectively. Two types of evidence are presented for the existence of the first intermediate, a (1-hydroxy-2-chloroethyl)-substituted exocyclic amino group. Firstly, poly d[A-T] cannot form etheno derivatives (except when denatured) and the observed cross-linking is therefore attributed to alkylation by the chlorinated sidechain of the adenine residue (A), acting on the N6 of A on the opposite strand. Secondly, our results show that blocking of the acceptor nitrogen, needed for cyclization, leads to the formation of relatively stable derivatives of adenosine and cytidine. Guanosine, as a monomer, is modified extensively, but in synthetic polymers no reaction was detected, possibly due to secondary structure.

Acetaldehyde↗
PubMed1983

Escherichia coli polymerase I can use O2-methyldeoxythymidine or O4-methyldeoxythymidine in place of deoxythymidine in primed poly(dA-dT).poly(dA-dT) synthesis.

O2-and O4-alkyldeoxythymidine are among the four O-alkyl base-modified derivatives produced by the reaction of N-nitroso alkylating agents with nucleic acids in vitro and in vivo. We find that both O2- and O4-methyl-dTTP can substitute for dTTP in alternating poly(dA-dT)-primed DNA synthesis. Up to 22% of the pyrimidines in the newly synthesized polymer were found by HPLC analysis to be O-methyldeoxythymidine. Little polymer synthesis was observed in the absence of dTTP. However, the O-methyl-dTTPs did not inhibit polymerization of dATP and dTTP. Polymers containing O2- or O4-methyldeoxythymidine were obtained in good yield, retaining the secondary structure of alternating poly(dA-dT). This was shown by the data for thermal transition under different conditions. In contrast, poly(dA-dT).poly(dA-dT) methylated or ethylated to less than 4% total modification by alkylnitrosoureas had a distinctly less stable structure. Neither O2- nor O4-methyldeoxythymidine can form more than one hydrogen bond with adenosine. The unchanged secondary structure of polymers containing these modified thymidines indicates that stacking interactions must play a major role in helix stabilization. O-Alkyldeoxythymidine may be formed by N-nitroso carcinogens that react intracellularly. We have shown that the triphosphates can be utilized by Escherichia coli DNA polymerase I as dTTP. The incorporated O4-methyl-dT causes misincorporation of G, both in transcription and synthesis. When O2-methyl-dT is present, less, but definite, misincorporation results.

DNA Polymerase I↗
PubMed1983

In vitro discrimination of replicases acting on carcinogen-modified polynucleotide templates.

Three different poly(dC)s with modifications that block the N-3 of deoxycytidine were used as templates for polymer synthesis by Escherichia coli DNA polymerase I (EC 2.7.7.7). In contrast to previously reported results with transcriptases, the hydrated form of 3,N(4)-ethenodeoxycytidine (epsilondC.H(2)O) did not mispair. Both 3,N(4)-ethenodeoxycytidine (epsilondC) and 3-methyldeoxycytidine (m(3)dC) led to dTMP misincorporation: 1/20 epsilondC and 1/80 m(3)dC. No other misincorporations appeared to be significant in amount. Thus, both qualitatively and quantitatively, replication errors resulting from carcinogen-modified bases are less frequent than errors in transcription of the same deoxypolynucleotides. Replication of comparable ribopolynucleotide templates by cucumber RNA-dependent RNA polymerase (EC 2.7.7.48) was strongly inhibited by epsilonrC.H(2)O and epsilonrC, so that the fidelity of this enzyme could not be assessed. However, both poly(dC) and poly(rC) containing dU or rU led to incorporation of rA. The presence of even small amounts of purines in poly(rC) greatly depressed synthesis, but the complementary base was incorporated. The finding that an RNA replicase can utilize a deoxypolynucleotide template is a further indication that, at least in vitro, the specificity of the relationship of enzymes and their natural templates is not absolute.

Carcinogens↗
PubMed1982

Chloroacetaldehyde-treated ribo- and deoxyribopolynucleotides. 1. Reaction products.

The in vitro reaction of the vinyl chloride metabolite chloroacetaldehyde (CAA) with cytosine and adenine residues in ribo- and deoxyribopolynucleotides leads to the formation of the relatively stable hydrated etheno derivatives 3,N4-(N4-alpha-hydroxyethene)adenine (epsilon C . H2O) and 1,N6-(N6-alpha-hydroxyethene)cytosine (epsilon A . H2O). Under physiological conditions the hydrates are slowly converted to 3,N4-ethenocytosine (epsilon C) and 1,N6-ethenoadenine (epsilon A). The half-life at pH 7.25 of epsilon C . H2O in poly(rC) is 4.9 h at 50 degrees C and of epsilon A . H2O in poly(rA) is 1.4 h at 37 degrees C. These dehydration rates in polymers are similar to those for hydrates in monomers. The reactivity of A and C residues is greatly suppressed in double-stranded polymers. Adenine residues are about 10 times less reactive in poly(rA) . poly(rU) than A in single-stranded polymers. Under similar reaction conditions no reaction of C residues in poly(rC) . poly(rG) was detected. In vinyl chloride exposed cells, where CAA is formed, the cyclic etheno derivatives of A and C are likely to occur preferentially in single-stranded regions of nucleic acids, with the hydrate forming a major proportion of the modification.

Acetaldehyde↗
PubMed1982

Chloroacetaldehyde-treated ribo- and deoxyribopolynucleotides. 2. Errors in transcription by different polymerases resulting from ethenocytosine and its hydrated intermediate.

Chloroacetaldehyde-modified poly(rC) or poly(dC) was prepared containing either 8-36% 3,N4-ethenocytidine (epsilon C) or 8-36% of a mixture of epsilon C and the hydrated epsilon C (epsilon C . H2O), with the hydrate greatly predominating (greater than 90%). These ribo- and deoxyribonucleotide templates were transcribed with DNA-dependent RNA polymerases from Escherichia coli and calf thymus, in the presence of either Mn2+ or Mg2+ and all four ribonucleoside triphosphates. All the polymers tested were transcribed with either cation present. In an earlier report from this laboratory [Spengler, S., & Singer, B. (1981) Nucleic Acids Res. 9. 365], transcriptional ambiguities resulting from epsilon C residues in enzymatically synthesized poly(rC, epsilon rC) were studied with E. coli DNA-dependent RNA polymerase in the presence of Mn2+. The misincorporations there reported were confirmed when poly(rC, epsilon rC) and poly(dC, epsilon dC), prepared by reaction of poly(rC) and poly(dC) with CAA, were transcribed in the presence of either Mn2+ or Mg2+. We now report that the presence of hydrated epsilon C in polymers also leads to misincorporations but with reproducible differences from those found with epsilon C alone. Nearest-neighbor analysis of the transcription products showed that the hydrate caused misincorporation of A greater than U much greater than C while epsilon C caused misincorporation of U greater than A much greater than C. The extent of misincorporation in transcription was less with Mg2+ than with Mn2+, but the pattern of ambiguity was the same with both cations and with both ribo- and deoxyribocytidylate polymers. Calf thymus DNA-dependent RNA polymerase IIB was also used to transcribe deoxyribocytidine polymers with Mn2+ as the cation. epsilon C and epsilon C . H2O both caused a high level of misincorporation of U , A, and C, but the preferred misincorporations differed slightly from those found with E. coli DNA-dependent RNA polymerase. For both prokaryotic and eukaryotic enzymes, the type of misincorporation resulting from the loss of hydrogen bonding by modification of the N-3 of C not only differed between epsilon C and the hydrated intermediate but also both differed from the transcriptional errors resulting from the presence of 3-methylcytidine in poly(dC) or poly(rC). We conclude that the errors made by these polymerases during transcription do not result primarily from the conditions used (cation, ribo- or deoxyribotemplate) but must be at least in part attributed to the enzyme recognizing some facet of the modified base other than the lack of normal hydrogen bonding.

Acetaldehyde↗