N7 Position Of Guanine Research Articles

Etoposide metabolites enhance DNA topoisomerase II cleavage near leukemia-associated MLL translocation breakpoints.

Chromosomal breakage resulting from stabilization of DNA topoisomerase II covalent complexes by epipodophyllotoxins may play a role in the genesis of leukemia-associated MLL gene translocations. We investigated whether etoposide catechol and quinone metabolites can damage the MLL breakpoint cluster region in a DNA topoisomerase II-dependent manner like the parent drug and the nature of the damage. Cleavage of two DNA substrates containing the normal homologues of five MLL intron 6 translocation breakpoints was examined in vitro upon incubation with human DNA topoisomerase IIalpha, ATP, and either etoposide, etoposide catechol, or etoposide quinone. Many of the same cleavage sites were induced by etoposide and by its metabolites, but several unique sites were induced by the metabolites. There was a preference for G(-1) among the unique sites, which differs from the parent drug. Cleavage at most sites was greater and more heat-stable in the presence of the metabolites compared to etoposide. The MLL translocation breakpoints contained within the substrates were near strong and/or stable cleavage sites. The metabolites induced more cleavage than etoposide at the same sites within a 40 bp double-stranded oligonucleotide containing two of the translocation breakpoints, confirming the results at a subset of the sites. Cleavage assays using the same oligonucleotide substrate in which guanines at several positions were replaced with N7-deaza guanines indicated that the N7 position of guanine is important in metabolite-induced cleavage, possibly suggesting N7-guanine alkylation by etoposide quinone. Not only etoposide, but also its metabolites, enhance DNA topoisomerase II cleavage near MLL translocation breakpoints in in vitro assays. It is possible that etoposide metabolites may be relevant to translocations.

Formation and structural determinants of multi-stranded guanine-rich DNA complexes

We have investigated the complexes formed by oligonucleotides with the general sequence d(T 15G n ), where n=4–15. Two distinct classes of structures are formed, namely, the four-stranded tetraplex and frayed wires. Frayed wires differ from four-stranded tetraplexes in both strand association stoichiometry and the ability of dimethyl sulfate to methylate the N7 position of guanine. Thus, it appears that these two guanine-rich multistranded assemblies are stabilised by different guanine–guanine interactions. The number of contiguous guanine residues determines which of the complexes is favoured. Based on the stoichiometry of the associated species and the accessibility of the N7 position of guanine to methylation we have found that oligonucleotides with smaller number of contiguous guanines; n=5–8, form primarily four-stranded tetraplex. Oligonucleotides with larger numbers of contiguous guanines adapt primarily the frayed wire structure. The stability of the complexes formed by this series of oligonucleotides is determined by the number and arrangement of the guanines within the sequences. We propose that the formation of the two types of complex proceed by a parallel reaction pathways that may share common intermediates.

Butadiene diolepoxide- and diepoxybutane-derived DNA adducts at N7-guanine: a high occurrence of diolepoxide-derived adducts in mouse lung after 1,3-butadiene exposure.

Butadiene (BD) is a high production volume chemical and is known to be tumorigenic in rodents. BD is metabolized to butadiene monoepoxide (BMO), diepoxybutane (DEB) and butadiene diolepoxide (BDE). These epoxides are genotoxic and alkylate DNA both in vitro and in vivo, mainly at the N7 position of guanine. In this study, a 32P-post-labeling/thin-layer chromatography (TLC)/high-pressure liquid chromatography (HPLC) assay for BDE and DEB adducts at the N7 of guanine was developed and was used in determining the enantiomeric composition of the adducts and the organ dose of BD exposure in lung. Exposure of 2'-deoxyguanosine (dGuo), 2'-deoxyguanosine-5'-phosphate (5'-dGMP) and 2'-deoxyguanosine-3'-phosphate (3'-dGMP) to racemic BDE followed by neutral thermal hydrolysis gave two products (products 1 and 2) that were identified by MS and UV and NMR spectroscopy as a diastereomeric pair of N7-(2,3,4-trihydroxybutan-1-yl)-guanines. Exposure of dGuo nucleotides to RR/SS DEB (also referred to as dl DEB) followed by thermal depurination resulted in a single product coeluting with the BDE product 1. If the reaction mixture of BDE and 5'-dGMP was analyzed by HPLC before hydrolysis of the glycosidic bond, four major nucleotide alkylation products (A, B, C and D) with identical UV sepectra were detected. The products were isolated and hydrolyzed, after which A and C coeluted with product 1 and B and D coeluted with the product 2. The major adduct of DEB-exposed 5'-dGMP was N7-(2-hydroxy-3,4-epoxy-1-yl)-dGMP (product E). A 32P-post-labeling assay was used to detect BDE- and DEB-derived N7-dGMP adducts in DNA. Levels of adducts increased with a dose of BDE and DEB and exhibited a half life of 30 +/- 3 (r = 0.98) and 31 +/- 4 h (r = 0.95), respectively. Incubation of DEB-modified DNA at 37 degrees C at neutral pH for up to 142 h did not lead to an increase of N7-(2,3,4-trihydroxybutan-1-yl)-dGMP in the DNA. These observations led to the conclusion that the N7-(2,3, 4-trihydroxybutan-1-yl)-dGMP adducts in DNA can be used as a marker of BDE exposure and that N7-(2-hydroxy-3,4-epoxy-1-yl)-dGMP adducts are related to DEB exposure. Dose-related levels of BDE- and DEB-derived adducts were detected in lungs of mice inhaling butadiene. Most of the N7-dGMP adducts (73%; product D) were derived from the 2R-diol-3S-epoxide of 1,3-butadiene. The data presented in this paper indicate that in vivo, 98% of N7-dGMP alkylation after BD exposure is derived from BDE, and approximately 2% of the adducts were derived from DEB and BMO.

Quantitation and mapping of aflatoxin B1-induced DNA damage in genomic DNA using aflatoxin B1-8,9-epoxide and microsomal activation systems

Aflatoxin B1 (AFB1) is a mutagenic and carcinogenic mycotoxin which may play a role in the etiology of human liver cancer. In vitro studies have shown that AFB1 adducts form primarily at the N7 position of guanine. Using quantitative PCR (QPCR) and ligation-mediated PCR (LMPCR), we have mapped total AFB1 adducts in genomic DNA treated with AFB1-8,9-epoxide and in hepatocytes exposed to AFB1 activated by rat liver microsomes or human liver and enterocyte microsomal preparations. The p53 gene-specific adduct frequencies in DNA, modified in cells with 40–400 μM AFB1, were 0.07–0.74 adducts per kilobase (kb). In vitro modification with 0.1–4 ng AFB1-8,9-epoxide per microgram DNA produced 0.03–0.58 lesions per kb. The adduct patterns obtained with the epoxide and the different microsomal systems were virtually identical indicating that adducts form with a similar sequence-specificity in vitro and in vivo. The lesions were detected exclusively at guanines with a preference towards GpG and methylated CpG sequences. The methods utilizing QPCR and LMPCR thus provide means to assess gene-specific and sequence-specific AFB1 damage. The results also prove that microsomally-mediated damage is a suitable method for avoiding manipulations with very unstable DNA-reactive metabolites and that this damage can be detected by QPCR and LMPCR.

DNA adducts of heterocyclic amine food mutagens: implications for mutagenesis and carcinogenesis.

The heterocyclic amines (HCAs) are a family of mutagenic/carcinogenic compounds produced during the pyrolysis of creatine, amino acids and proteins. The major subclass of HCAs found in the human diet comprise the aminoimidazoazaarenes (AIAs) 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). All, except DiMeIQx, have been shown to be carcinogenic in animals. These compounds are present in cooked muscle meats at the p.p.b. level. Since the discovery of the HCAs in the late 1970s, many studies have examined the DNA adducts of these compounds. This review compiles the literature on AIA-DNA adducts including their identification and characterization, pathways of formation, mutagenesis in vitro and in vivo, and their association with carcinogenesis in animal models. It is now known that metabolic activation leading to the formation of DNA adducts is critical for mutagenicity and carcinogenicity of these compounds. All of the AIAs studied adduct to the guanine base, the major adduct being formed at the C8 position. Two AIAs, IQ and MeIQx, also form minor adducts at the N2 position of guanine. A growing body of literature has reported on the mutation spectra induced by AIA-guanine adducts. Studies of animal tumors induced by AIAs have begun to relate AIA-DNA adduct-induced mutagenic events with the mutations found in critical genes associated with oncogenesis. Several studies have demonstrated the feasibility of chemoprevention of AIA tumorigenesis. Only a few studies have reported on the detection of AIA-DNA adducts in human tissues; difficulties persist in the routine detection of AIA-DNA adducts in humans for the purpose of biomonitoring of exposure to AIAs. The AIAs are nevertheless regarded as possible human carcinogens, and future research on AIA-DNA adducts is likely to help address the role of AIAs in human cancer.

Human mRNA Capping Enzyme (RNGTT) and Cap Methyltransferase (RNMT) Map to 6q16 and 18p11.22–p11.23, Respectively

Eukaryotic cellular and viral mRNAs contain a 59-terminal mGpppN cap structure, which has important consequences at multiple levels of gene expression including RNA splicing, 39 end formation, transcript stability, nuclear export, and translation initiation (6, 12, 15, 16). Recent studies have demonstrated that capping enzyme selectively binds to the phosphorylated, elongating form of RNA polymerase II (pol II) (23). This finding accounts for the specific capping of pol II transcripts. It may also provide new insights into how HIV tat protein promotes pol II phosphorylation by tat-activated kinase (TAK) to enhance polymerase processivity (5). Caps are formed on nascent pre-mRNAs by the sequential action of RNA 59-triphosphatase, which removes the g-phosphate of the initiating nucleotide, RNA guanylyltransferase, which transfers GMP from GTP to the resulting diphosphate end, and RNA (guanine-7) methyltransferase, which methylates the guanine N7 position of the newly formed GpppN termini (4, 12, 15). These three enzymatic activities are present as separate polypeptides in yeasts, and strains null for the triphosphatase (18), guanylyltransferase (14), or methyltransferase (7) are nonviable. Metazoans including humans contain a separate cap methyltransferase (RNA guanine-7-methyltransferase, RNMT), but the RNA 59-triphosphatase and guanylyltransferase activities are contained as Nand C-terminal domains in a bifunctional capping enzyme (RNGTT) encoded by a single gene (23). Despite this difference in genomic organization between uniand multicellular organisms, capping enzyme is functionally conserved, and Saccharomyces cerevisiae strains null for guanylyltransferase or RNA 59-triphosphatase are complemented for growth by the mammalian counterpart (21, 23). In light of the importance of mRNA capping for gene expression, we determined the chromosomal location of the human capping enzyme gene (RNGTT) by PCR using as template the NIGMS human/rodent somatic cell hybrid panel 2, version 3 (Coriell Institute for Medical Research Cell Repositories, Camden, NJ). This panel consists of DNA isolated from 24 human/rodent somatic cell hybrid lines that each retained one intact human chromosome. Primers 1, 59-CAGTGTGTCCATTCTGGCAGTGGTTTTG-39, and 2, 59GTGGGAGAGCATTTTCTGGTGACAGC-39, from the 39-UTR of the human capping enzyme cDNA sequence (Accession No. AF025654) were designed to give a human-specific 318-bp PCR product. PCR results demonstrated that the human capping enzyme gene RNGTT is located on chromosome 6 (data not shown). Fine-mapping was performed by PCR analysis of the GeneBridge 4 radiation hybrid panel (Research Genetics, Inc., Huntsville, AL) with 25 ng template genomic DNA and primers 1 and 2 in a 12.5-ml reaction. After an initial denaturation step at 94°C for 5 min, 35 cycles of amplification consisting of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s were performed, followed by a final extension of 5 min at 72°C. Amplification products were analyzed by agarose gel electrophoresis. Data were submitted to the Whitehead Institute/MIT Center for Genome Research STS mapping server for statistical analysis with the RHMAPPER software package (http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl). The data vector for RNGTT was 00100 01101 02101 0011

Cloning and Characterization of Three Human cDNAs Encoding mRNA (Guanine-7-)-methyltransferase, an mRNA Cap Methylase

The mRNA cap structure is synthesized by a series of reactions catalyzed by capping enzyme and mRNA (guanine-7-)-methyltransferase. mRNA (guanine-7-)-methyltransferase catalyzes the methylation of GpppN- at the guanine N7 position, which is an essential step for gene expression in eukaryotic cells. Here we isolated three human cDNAs encoding mRNA (guanine-7-)-methyltransferase termedhCMT1a, hCMT1bandhCMT1c. hCMT1aandhCMT1bencode 476 and 504 amino acids, respectively, and differ only at the region coding for the C-terminal portion of the enzyme after amino acid residue 465. The third cDNAhCMT1cseems to encode the same polypeptide as hCMT1a, however, the 3′-noncoding region ofhCMT1ccontains sequences corresponding to part of the C-terminal coding and noncoding regions ofhCMT1bthus consisting of a mosaic ofhCMT1aandhCMT1b.RT-PCR showed that all 3 types of mRNAs were expressed in every tissue examined. Comparison of the deduced amino acid sequences with those of other viral and cellular enzymes showed the regions which are highly conserved among mRNA (guanine-7-)-methyltransferases. The recombinant hCMT1a expressed inE. coliexhibited mRNA (guanine-7-)-methyltransferase activity. On the other hand, neither mRNA (guanine-7-)-methyltransferase nor mRNA (nucleoside-2′-O-)-methyltransferase activity was detected with the recombinant hCMT1b protein. Although the biological significance of the expression of these three mRNA (guanine-7-)-methyltransferase mRNA species remains unknown at present, the nucleotide sequences suggest that they are produced by alternative RNA splicing.

Styrene oxide DNA adducts: in vitro reaction and sensitive detection of modified oligonucleotides using capillary zone electrophoresis interfaced to electrospray mass spectrometry.

Styrene is one of the most important synthetic chemicals in the world and is subject to investigations concerning carcinogenicity and mutagenicity due to the active metabolite, styrene-7,8-oxide. This epoxide shows a tendency to react, among others, with DNA and DNA constituents. The in vitro reaction of styrene oxide with DNA was investigated by cleaving incubated calf thymus DNA with two different enzymes, namely Benzonase and alkaline phosphatase, to obtain oligonucleotides of the type n-nucleotide-(n-1)-phosphate with chain length from 2 to 8 bases. Alkylated and nonalkylated nucleotides were separated in groups according to their chain length using capillary zone electrophoresis and were detected with electrospray mass spectrometry. This improvement in sensitivity made it possible to obtain new information about the reaction of styrene oxide with DNA, especially to detect unknown reaction products. The results indicate that primarily purine bases were alkylated by styrene oxide before pyrimidine bases, which react with higher concentrations of styrene oxide. This means that in addition to the already reported adducts in DNA at the N-7-, O6- and N2-position of guanine also adducts at the nucleophilic sites of adenine can be found using mass spectrometry. We anticipate for the future this procedure will allow us to investigate base sequence specific reactions as well as interactions from xenobiotics and cytostatic drugs, since reaction products would directly be detectable.

32P-postlabelling/HPLC assay reveals an enantioselective adduct formation in N7 guanine residues in vivo after 1,3-butadiene inhalation exposure

We have established a protocol that allows qualitative and quantitative determination of butadiene monoepoxide-DNA adducts formed as a result of inhalation exposure to 1,3-butadiene. We observed that in this particular case in vivo samples required extensive sample purification to facilitate a low background. Sample preparation included a solid phase extraction carried out with a strong anion exchange column and one-dimensional ion exchange TLC. The ultimate analysis is based on reverse phase HPLC with on-line radioactivity and UV detectors. The qualitative identification and quantitation is based on characterized markers, which are used as external and internal standards. Modified 3'-dGMP markers were used to control labelling efficiency, which varies, and modified 5'-dGMP markers were used as an optical standard to qualitatively assign the products and to determine recovery of the sample preparation. Using this method we were able to demonstrate, for the first time, specific enantio- and regioisomeric adduct formation at the N7 position of guanine residues in liver DNA of rats inhalation-exposed to 1,3-butadiene. The major adduct formed was the C-2 isomer derived from the R enantiomer of butadiene monoepoxide, contributing 47% of all adducts formed at the N7 position of guanine. The relative proportions of the remaining three other adducts detected were 22 (R C-1), 18 (S C-2) and 14% (S C-1) respectively. Inhalation exposure to 200 p.p.m. for 5 days resulted in an alkylation level of 7.2 fmol/10 microg DNA or 2.4 adducts/10(-7) normal nucleotides.

Reduction of the Toxicity and Mutagenicity of Aziridine in Mammalian Cells Harboring the Escherichia Coli fpg Gene

Aziridine (ethyleneimine) reacts with DNA in vitro, mainly at the N7 position of guanine and N3 of adenine, then imidazole ring opening of the modified guanine results in formation of formamidopyrimidine (FaPy) residues. The Escherichia coli fpg gene encodes a DNA glycosylase that removes FaPy residues from DNA. To determine whether aziridine produces FaPy lesions in mammalian cells we have expressed the E.coli fpg gene in CHO cells. The transfected cells, expressing high levels of the bacterial protein, are more resistant to the toxic and mutagenic effects of aziridine than the control population. Less DNA damage was measured by quantitative PCR analysis in transfected than in control cells treated with equimolar concentrations of aziridine. The results suggest that aziridine produces in vivo FaPy residues that could account for the deleterious effects of this compound.

Improved strategies for postoligomerization synthesis of oligodeoxynucleotides bearing structurally defined adducts at the N2 position of deoxyguanosine.

Improved methodology has been developed for preparation of oligodeoxynucleotides bearing adducts on the N2 position of guanine in which the adduction reaction is carried out in homogeneous solution rather than while the oligonucleotide is immobilized on a solid matrix. The methodology utilizes a new synthon, 2-fluoro-O6-(trimethylsilylethyl)-2'-deoxyinosine (3). Nucleoside 3 is stable to the conditions of oligonucleotide synthesis, but the O6 protection is eliminated under very mild conditions following displacement of the 2-fluoro group by amine nucleophiles. Oligonucleotides containing 3 could be removed from the solid support by treatment with 0.1 M NaOH (8 h, rt) without disruption of 3. Reaction of the crude, partially deprotected oligonucleotide with (R)-2-amino-2-phenylethanol in homogeneous solution, followed by removal of the remaining protective groups with NH4OH (60 degrees C, 8 h) and then 0.1% acetic acid, gave the adducted oligonucleotide in good purity and yield. Alternatively, fully deprotected oligonucleotide containing 3 could be prepared by use of labile phenoxyacetyl-type protecting groups on the exocyclic amino groups.

S-adenosyl methionine alters the DNA contacts of the EcoKI methyltransferase.

The EcoKI methyltransferase methylates two adenines on opposite strands of its bipartite DNA recognition sequence AAC(N6)GTGC. The enzyme has a strong preference for hemimethylated DNA substrates, but the methylation state of the DNA does not influence its binding affinity. Methylation interference was used to compare the contacts made by the EcoKI methyltransferase with unmodified, hemimethylated or fully modified DNAs. Contacts were seen at or near the N7 position of guanine, in the major groove, for all of the guanines in the EcoKI recognition sequence, and at two guanines on the edge of the intervening spacer sequence. The presence of the cofactor and methyl donor S-adenosyl methionine had a striking effect on the interference pattern for unmodified DNA which could not be mimicked by the presence of the cofactor analogue S-adenosyl homocysteine. In contrast, S-adenosyl methionine had no effect on the interference patterns for either kind of hemimethylated DNA, or for fully modified DNA. Differences between the interference patterns for the unmodified DNA and any of the three forms of methylated DNA provide evidence that methylation of the target sequence influences the conformation of the protein-DNA interface, and illustrate the importance of S-adenosyl methionine in the distinction between unmodified and methylated DNA by the methyltransferase.

Structural, steric and energetic requirements for induction of base substitutional mutations by methylated guanines and thymines

The products of methylation at the N3-, O6- and N7-positions of guanine and at the O2- and O4- positions of thymine are subjected to various possibilities for pairing with DNA bases, using calculations at the semiempirical PM3 SCF-MO level. It is predicted that the presence of the Watson–Crick protons in the modified bases would lead to non-mutagenic base-pairing schemes, while their absence facilitates promutagenic pairing schemes, modified guanines behaving like adenine and modified thymines like cytosine. Some degree of competition with non-mutagenic base-pairing schemes is also anticipated. Only the conformers of the O-methylated bases with the O-methyl group anti to the hydrogen bonding side furnish feasible base-mispairing schemes in the double-helical configuration. The syn conformers do not pair in the double-helical configuration. Correlation of these results with experimental and theoretically predicted Watson–Crick proton acidities for the nucleoside systems leads to the prediction that N3- and O6-methylguanines and O2 and O4-methylthymines would be promutagenic bases at biological pH, while N1-methylguanine would behave without miscoding properties. These predictions are largely confirmed by the reported experimental template properties of these modified DNA bases and are also corroborated by NMR, UV and crystallography studies on some of the modified bases considered here.

DNA sequence-specific adenine alkylation by the novel antitumor drug tallimustine (FCE 24517), a benzoyl nitrogen mustard derivative of distamycin.

FCE 24517, a novel distamycin derivative possessing potent antitumor activity, is under initial clinical investigation in Europe. In spite of the presence of a benzoyl nitrogen mustard group this compound fails to alkylate the N7 position of guanine, the major site of alkylation by conventional nitrogen mustards. Characterisation of DNA-drug adducts revealed only a very low level of adenine adduct formation. Using a modified Maxam-Gilbert sequencing method the consensus sequence for FCE 24517-adenine adduct formation was found to be 5'-TTTTGA-3'. A single base modification in the hexamer completely abolishes the alkylation of adenine. Using a Taq polymerase stop assay alkylations were confirmed at the A present in the hexamer TTTTGA and, in addition, in one out of three TTTTAA sequences present in the plasmid utilized. The sequence specificity of alkylation by FCE 24517 is therefore the most striking yet observed for an alkylating agent of small molecular weight.

Mechanisms of aflatoxin carcinogenesis.

Much progress has been made in elucidating the biochemical and molecular mechanisms that underlie aflatoxin carcinogenesis. In humans, biotransformation of AFB1 to the putative carcinogenic intermediate. AFB-8,9-exo-epoxide, occurs predominantly by cytochromes P450 1A2 and 3A4, with the relative importance of each dependent upon the relative magnitude of expression of the respective enzymes in liver. Genetic variability in the expression of these and other cytochromes P450 may result in substantial interindividual differences in susceptibility to the carcinogenic effects of aflatoxins. Detoxification of AFB-8,9-epoxide by a specific alpha class glutathione S-transferase is an important protective mechanism in mice, and it accounts for the resistance of this species to the carcinogenic effects of AFB. This particular form of GST is expressed constitutively only at low levels in rats, but it is inducible by antioxidants such as ethoxyquin, and it accounts for much of the chemoprotective effects of a variety of substances, including natural dietary components that putatively act via an "antioxidant response element" (ARE). In humans, the constitutively expressed GSTs have very little activity toward AFB1-8,9-exo-epoxide, suggesting that--on a biochemical basis--humans should be quite sensitive to the genotoxic effects of aflatoxins. If a gene encoding a high aflatoxin-active form of GST is present in the human genome, but is not constitutively expressed, and is inducible by dietary antioxidants (as occurs in rats), then chemo- and/or dietary intervention measures aimed at inducing this enzyme could be highly effective. However, as it is possible that human CYP 1A2 may also be inducible by these same chemicals (because of the possible presence of an ARE in this gene), the ultimate consequence of dietary treatment with chemicals that induce biotransformation enzymes via an ARE is uncertain. The balance of the rate of activation (exo-epoxide production) to inactivation (GST conjugation plus other P450-mediated non-epoxide oxidations) may be a strong indicator of individual and species susceptibility to aflatoxin carcinogenesis, if the experimental conditions are reflective of true dietary exposures. There is strong evidence that AFB-8,9-exo-epoxide binds to G:C rich regions of DNA, forming an adduct at the N7-position of guanine. Substantial evidence demonstrates that AFB1-8,9-epoxide can induce activating mutations in the ras oncogene in experimental animals, primarily at codon 12.(ABSTRACT TRUNCATED AT 400 WORDS)

Site-specific benzo[a]pyrene diol epoxide-DNA adducts inhibit transcription elongation by bacteriophage T7 RNA polymerase.

Benzo[a]pyrene, an extremely potent procarcinogen and mutagen, is metabolized to a variety of products, including the ultimate carcinogen 7,8-dihydroxy-9,10-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene. This product of biotransformation reacts with DNA, forming a series of adducts principally at the N2 position of guanine that differ in their stereochemistry and exhibit unique biological properties. In order to gain a better understanding of the effects on RNA synthesis of these adducts, we used purified bacteriophage T7 RNA polymerase to transcribe a series of templates containing one of four stereoisomerically pure BPDE-guanine lesions--(+)-trans-,(-)-trans-,(+)-cis-anti-N2-BPDE-guanine--or no damaged bases. To construct suitable double-stranded oligodeoxynucleotides for these studies, we annealed an 11-mer containing a site-specific stereoisomerically pure N2-BPDE-guanine adduct, a 37-mer, and a 10-mer to a complementary 58-base sequence of single-stranded DNA. The oligomers were ligated, purified, and reannealed. The resulting DNA template contained the promoter for T7 RNA polymerase and a BPDE adduct at position +16 following the transcription initiation site. The results of the transcription assays clearly demonstrate that each of the adducts inhibits elongation by T7 RNA polymerase, but they do so to significantly different extents, depending on the stereochemical characteristics of the BPDE-modified guanine. The order of inhibition is (+)-trans > (-)-trans > (+)-cis > (-)-cis, when the amount of full-length transcript for each is compared to that obtained for an unmodified template. Furthermore, premature termination of RNA synthesis occurs at or near the site of the BPDE lesion as evidenced by the formation of discrete, truncated transcripts. These results might be related to the fact that the pyrenyl moiety of the trans-BPDE adducts is situated in the minor groove of double-stranded DNA, but is quasi-intercalated into the double helix in the case of the cis stereoisomers.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequence-selective alkylation and cross-linking induced by mitomycin C upon activation by DT-diaphorase

Aerobic reduction of MMC by DTD, an obligate two-electron reductase, or chemical reduction by sodium borohydride results predominantly in monoalkylation of DNA at the guanine N7 position within 5'-GG-3' and 5'-GTC-3' sequences. The level of guanine N7 alkylation after DTD reduction increased as the pH was decreased from 7.8 and was optimal at pH 6.6. A similar profile of alkylation was obtained when the major metabolite of DTD-mediated MMC metabolism, 2,7-diaminomitosene, was further reduced by DTD. The sequence preference for DNA interstrand cross-linking (ISC) was also determined using singly end-labeled oligonucleotide duplexes. Reduction of MMC by DTD induced DNA cross-links which were resistant to piperidine cleavage. Exposure of cross-linked DNA to dimethyl sulfate or formic acid and subsequent piperidine cleavage displayed a discontinuity in band pattern which suggested a 5'-CG-3' preference for DNA ISC. Major groove alkylation is proposed to occur via generation, and subsequent metabolism by DTD, of 2,7-diaminomitosene. Cross-linking of DNA, at 5'-CG-3' sequences, is proposed to require the formation of either the protonated leucomitomycin C or the leucoaziridinomitosene during DTD-mediated metabolism of MMC.

Determining N7-alkylguanine adducts by immunochemical methods and HPLC with electrochemical detection: applications in animal studies and in monitoring human exposure to alkylating agents.

Many xenobiotics exert their toxic effects through interaction with DNA in the cells of the exposed organism. This interaction may lead to the formation DNA adducts. Some of these may give rise to mutations that initiate cell transformation and, ultimately, the formation of tumors. Sensitive methods for determining DNA adducts are indispensable for the study of chemical mutagenesis and carcinogenesis and for biomonitoring human exposure to genotoxic agents. Alkylating agents form an important class of genotoxic compounds. They react preferentially at the N7-position of guanine. Under neutral or acidic conditions, the adducts can be readily released from the DNA backbone as the free base N7-alkylguanine (N7-AlkGua). The imidazole ring of N7-alkyldeoxyguanosine (N7-AlkdGuo) can be opened under alkaline conditions, which results in formation of a more stable adduct in DNA. To develop immunochemical methods for the detection of N7-alkylations, we immunized mice with various alkylguanosines in the ring-opened form (RON7-AlkdGuo). Antibodies were selected to detect adducts in isolated DNA by competitive ELISA and in single cells by immunofluorescence microscopy (IFM). Various monoclonal antibodies were characterized in detail with respect to specificity and sensitivity toward methylated, ethylated, and hydroxyethylated DNAs. The antibodies showed extensive cross-reactivity toward N7-(m)ethyl- and N7-(2-hydroxyethyl)guanine modifications in the ring-opened form. The limits of detection in the direct and competitive ELISA were 5-10 and 1-2 adducts per 10(6) nucleotides, respectively. The detection limit of the IFM method was about 20 adducts per 10(6) nucleotides(ABSTRACT TRUNCATED AT 250 WORDS)

Determining N7-Alkylguanine Adducts by Immunochemical Methods and HPLC with Electrochemical Detection: Applications in Animal Studies and in Monitoring Human Exposure to Alkylating Agents

Many xenobiotics exert their toxic effects through interaction with DNA in the cells of the exposed organism. This interaction may lead to the formation DNA adducts. Some of these may give rise to mutations that initiate cell transformation and, ultimately, the formation of tumors. Sensitive methods for determining DNA adducts are indispensable for the study of chemical mutagenesis and carcinogenesis and for biomonitoring human exposure to genotoxic agents. Alkylating agents form an important class of genotoxic compounds. They react preferentially at the N7-position of guanine. Under neutral or acidic conditions, the adducts can be readily released from the DNA backbone as the free base N7-alkylguanine (N7-AlkGua). The imidazole ring of N7-alkyldeoxyguanosine (N7-AlkdGuo) can be opened under alkaline conditions, which results in formation of a more stable adduct in DNA. To develop immunochemical methods for the detection of N7-alkylations, we immunized mice with various alkylguanosines in the ring-opened form (RON7-AlkdGuo). Antibodies were selected to detect adducts in isolated DNA by competitive ELISA and in single cells by immunofluorescence microscopy (IFM). Various monoclonal antibodies were characterized in detail with respect to specificity and sensitivity toward methylated, ethylated, and hydroxyethylated DNAs. The antibodies showed extensive cross-reactivity toward N7-(m)ethyl- and N7-(2-hydroxyethyl)guanine modifications in the ring-opened form. The limits of detection in the direct and competitive ELISA were 5-10 and 1-2 adducts per 10(6) nucleotides, respectively. The detection limit of the IFM method was about 20 adducts per 10(6) nucleotides(ABSTRACT TRUNCATED AT 250 WORDS)

Use of [8- 3H]guanine-labeled deoxyribonucleic acid tostudy alkylating agent reaction kinetics and stability

Alkylation at the N7 position of guanine in DNA renders the C8-hydrogen acidic. This serves as the basis for an assay of guanine N7 alkylation using [8- 3H]-guanine-labeled DNA. I modified the assay by preparing a high specific activity substrate in vitro and by replacing the distillation step with charcoal adsorption of substrate. Using the appearance of noncharcoal-adsorbable label as a measure of guanine-N7 alkylation I examined the reaction of DNA with dimethyl sulfate and mechlorethamine. The rate of reaction of dimethyl sulfate with the N7 position of guanine in DNA was constant over time, i.e., loss of label from DNA proceeded linearly with time. On the other hand, the rate of reaction of mechlorethamine with DNA increased with time, consistent with the initial formation of the reactive aziridinium ion. The assay can also be used to compare the reaction rates of various alkylating agents with DNA. Thus, the acridine mustards ICR-170 and quinacrine mustard were far more potent alkylating agents than mechlorethamine. Furthermore the assay may be used to determine the alkylating potency and stability of various alkylating agent preparations: while frozen solutions of acridine mustards in organic solvents retained alkylating activity for several months, different commercial preparations of quinacrine mustard had little or no alkylating activity.