Putative three-stranded DNA pairing intermediate in recA protein-mediated DNA strand exchange: no role for guanine N-7.

As an early step in DNA strand exchange reactions, the recA protein aligns homologous sequences within two DNA molecules to form a putative triple-stranded intermediate. In virtually all models for three-stranded DNA proposed to date, hydrogen bonds involving the N-7 position of guanine have played a prominent structural role. To determine whether the N-7 position of guanine is required for triple helix and heteroduplex formation in the recA protein-mediated DNA pairing reaction, guanine was completely replaced by the base analog 7-deazaguanine in both strands of the duplex DNA substrate using polymerase chain reaction. This modified double-strand DNA was reacted with unmodified single-strand DNA in vitro. The 7-deazaguanine-substituted DNA functioned as well as the unsubstituted DNA in recA protein-mediated DNA three-strand exchange reactions. Strand exchange reactions involving four strands also proceeded normally when three of the four strands contained 7-deazaguanine rather than guanine. In fact, the rate of strand exchange improved somewhat when the modified DNA substrates were used. This indicates either that the N-7 position of guanine is not essential for the formation of the putative triple-stranded DNA pairing intermediate, or that a three-stranded (or four-stranded) structure is not an obligate intermediate in recA protein-mediated DNA strand exchange.     

Sites of methylation of DNA bases by the action of organophosphorus insecticides in vitro

Methylation in vitro of DNA by three methyl-14C-labelled organophosphorus insecticides has been studied. The ability of methylbromphenvinphos, methylparathion and malathion to methylate N-7 of guanine in DNA can be expressed as 100:40:15. Among the methylation products, no O6-methylguanine, a known mutagen, was found. Both in the reaction with dsDNA and with ssDNA 7-methyl-guanine was the main methylation product. However, all methyl derivatives of adenine (3-methyladenine, 1-methyladenine and 7-methyladenine) constituted about 40% and 50% of all methylation products in the case of dsDNA and ssDNA, respectively. The only methyl derivative of pyrimidine we have identified was 3-methylcytosine. In the case of dsDNA 3-methylcytosine appeared in small amounts but in the alkylated ssDNA 3-methylcytosine C constituted about 20% of all alkylation products.     

Characterization of a Trypanosoma brucei RNA cap (guanine N-7) methyltransferase

The m7GpppN cap structure of eukaryotic mRNA is formed by the sequential action of RNA triphosphatase, guanylyltransferase, and (guanine N-7) methyltransferase. In trypanosomatid protozoa, the m7GpppN is further modified by seven methylation steps within the first four transcribed nucleosides to form the cap 4 structure. The RNA triphosphatase and guanylyltransferase components have been characterized in Trypanosoma brucei. Here we describe the identification and characterization of a T. brucei (guanine N-7) methyltransferase (TbCmt1). Sequence alignment of the 324-amino acid TbCmt1 with the corresponding enzymes from human (Hcm1), fungal (Abd1), and microsporidian (Ecm1) revealed the presence of conserved residues known to be essential for methyltransferase activity. Purified recombinant TbCmt1 catalyzes the transfer of a methyl group from S-adenosylmethionine to the N-7 position of the cap guanine in GpppN-terminated RNA to form the m7GpppN cap. TbCmt1 also methylates GpppG and GpppA but not GTP or dGTP. Mutational analysis of individual residues of TbCmt1 that were predicted-on the basis of the crystal structure of Ecm1--to be located at or near the active site identified six conserved residues in the putative AdoMet- or cap-binding pocket that caused significant reductions in TbCmt1 methyltransferase activity. We also report the identification of a second T. brucei RNA (guanine N-7) cap methyltransferase (named TbCgm1). The 1050-amino acid TbCgm1 consists of a C-terminal (guanine N-7) methyltransferase domain, which is homologous with TbCmt1, and an N-terminal guanylyltransferase domain, which contains signature motifs found in the nucleotidyl transferase superfamily.     

Topography of the interaction of recA protein with single-stranded deoxyoligonucleotides

recA protein, in the presence of adenosine 5'-(gamma-thio)triphosphate, formed stable complexes with single-stranded deoxyoligonucleotides between 9 and 20 residues in length but not with those 8-residues long. The binding of recA protein to a 15-mer and 20-mer completely protected the sugar-phosphate backbone of the nucleic acid from digestion by pancreatic deoxyribonuclease I and protected the 5'-terminal phosphate from cleavage by calf intestinal alkaline phosphatase. Ethylation of the phosphate backbone at any position by ethylnitrosourea blocked the binding of recA protein to the 15-mer but not to the 20-mer. Ethylation of phosphates near the ends of the 15-mer interfered less, suggesting a minimum binding site requirement. In contrast to the protection of the nucleic acid backbone, recA protein did not protect the N-7 position of guanine or the N-3 position of adenine from methylation by dimethyl sulfate, but rather enhanced the methylation of guanine. These results indicate that recA protein binds primarily to the phosphate backbone of single-stranded DNA, leaving the bases free for homologous pairing. We present a model for the organization of the presynaptic filament.     

Methylation of DNA in various organs of C57B1 mice by a carcinogenic dose of N-methyl-N-nitrosourea and stabiltty of some methylation products up to 18 hours.

Female 6–8-week-old C57B1 mice were injected i.p. with N-methyl-N-nitrosourea (MNUA) (¹⁴C or ³H-methyl-labelled) in saline (80 mg/kg) and DNA was isolated from bone marrow, small bowel, kidneys, liver, lungs, spleen and thymus at various times thereafter up to 18 h. Methylation of DNA was found in all organs examined, and by analyses using column or paper chromatography of DNA hydrolysates, the extent of methylation of DNA purines was determined.

Methylated guanine residues (at N-3, N-7 and O-6 positions) were stable in DNA up to 18 h, but methylated adenines (at N-3 or N-7) were removed from DNA of all organs examined; the overall half-life of methyladenines was about 3 h, but removal appeared to occur in a biphasic manner, with a proportion of methyladenine remaining relatively stable. This relative stability was somewhat more marked in bone marrow than in other organs.     

West Nile virus 5'-cap structure is formed by sequential guanine N-7 and ribose 2'-O methylations by nonstructural protein 5

Many flaviviruses are globally important human pathogens. Their plus-strand RNA genome contains a 5'-cap structure that is methylated at the guanine N-7 and the ribose 2'-OH positions of the first transcribed nucleotide, adenine (m(7)GpppAm). Using West Nile virus (WNV), we demonstrate, for the first time, that the nonstructural protein 5 (NS5) mediates both guanine N-7 and ribose 2'-O methylations and therefore is essential for flavivirus 5'-cap formation. We show that a recombinant full-length and a truncated NS5 protein containing the methyltransferase (MTase) domain methylates GpppA-capped and m(7)GpppA-capped RNAs to m(7)GpppAm-RNA, using S-adenosylmethionine as a methyl donor. Furthermore, methylation of GpppA-capped RNA sequentially yielded m(7)GpppA- and m(7)GpppAm-RNA products, indicating that guanine N-7 precedes ribose 2'-O methylation. Mutagenesis of a K(61)-D(146)-K(182)-E(218) tetrad conserved in other cellular and viral MTases suggests that NS5 requires distinct amino acids for its N-7 and 2'-O MTase activities. The entire K(61)-D(146)-K(182)-E(218) motif is essential for 2'-O MTase activity, whereas N-7 MTase activity requires only D(146). The other three amino acids facilitate, but are not essential for, guanine N-7 methylation. Amino acid substitutions within the K(61)-D(146)-K(182)-E(218) motif in a WNV luciferase-reporting replicon significantly reduced or abolished viral replication in cells. Additionally, the mutant MTase-mediated replication defect could not be trans complemented by a wild-type replicase complex. These findings demonstrate a critical role for the flavivirus MTase in viral reproduction and underscore this domain as a potential target for antiviral therapy.     

Excision repair functions in Saccharomyces cerevisiae recognize and repair methylation of adenine by the Escherichia coli dam gene.

Unlike the DNA of higher eucaryotes, the DNA of Saccharomyces cerevisiae (bakers' yeast) is not methylated. Introduction of the Escherichia coli dam gene into yeast cells results in methylation of the N-6 position of adenine. The UV excision repair system of yeast cells specifically responds to the methylation, suggesting that it is capable of recognizing modifications which do not lead to major helix distortion. The UV repair functions examined in this report are involved in the incision step of pyrimidine dimer repair. These observations may have relevance to the rearrangements and recombination events observed when yeast or higher eucaryotic cells are transformed or transfected with DNA grown in E. coli.     

O6-methylguanine-DNA methyltransferase and alkylation of liver DNA in mice exposed to dimethylnitrosamine during dietary deficiency of essential amino acids

Male NMRI mice were fed a diet containing a complete mixture of amino acids or a mixture deficient in methionine-cysteine or lysine (30% of the control level) for a period of 6 days. During the feeding period all mice received dimethylnitrosamine in the drinking water ad libitum. The exposure averaged 1 mg dimethylnitrosamine/kg body weight and day. The concentration of O6-methylguanine-DNA methyltransferase was measured in liver extracts. It decreased significantly in the methionine-cysteine deficient mice. When DNA from the liver was analyzed for alkylated purine bases the mice received a single dose of 14C-labeled dimethylnitrosamine (0.5 or 1 mg/kg body weight) at 120 min before sacrifice. The concentration of O6-methylguanine increased significantly over the control level upon feeding the deficient diets and was restored to the concentration of the controls by refeeding lysine for 2 days following 6 days of lysine deficiency. The increased ratio of O6-methylguanine to N-7-methylguanine indicated that methylation of guanine in the N-7 position was not subject to variation by the intake of dimethylnitrosamine during the dietary deficiencies. The results demonstrate the requirement for a balanced composition of amino acids in the diet to maintain a sufficient concentration of O6-methylguanine-DNA methyltransferase in the cells and thus to permit efficient removal of the methyl group from the O-6 position of guanine in DNA after exposure to dimethylnitrosamine.     

Biological properties of imidazole ring-opened N7-methylguanine in M13mp18 phage DNA.

Guanine residues methylated at the N-7 position (7-MeGua) are susceptible to cleavage of the imidazole ring yielding 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7-MeGua). The presence of Fapy-7-MeGua in DNA template causes stops in DNA synthesis in vitro by E. coli DNA polymerase I. The biological consequences of Fapy-7-MeGua lesions for survival and mutagenesis were investigated using single-stranded M13mp18 phage DNA. Fapy-7-MeGua lesions were generated in vitro in phage DNA by dimethylsulfate (DMS) methylation and subsequent ring opening of 7-MeGua by treatment with NaOH (DMS-base). The presence of Fapy-7-MeGua residues in M13 phage DNA correlated with a significant decrease in transfection efficiency and an increase in mutation frequency in the lacZ gene, when transfected into SOS-induced JM105 E.coli cells. Sequencing analysis revealed unexpectedly, that mutation rate at guanine sites was only slightly increased, suggesting that Fapy-7-MeGua was not responsible for the overall increase in the mutagenic frequency of DMS-base treated DNA. In contrast, mutation frequency at adenine sites yielding A----G transitions was the most frequent event, 60-fold increased over DMS induced mutations. These results show that treatment with alkali of methylated single-stranded DNA generates a mutagenic adenine derivative, which mispairs with cytosine in SOS induced bacteria. The results also imply that the Fapy-7-MeGua in E. coli cells is primarily a lethal lesion.     

The specificity of different classes of ethylating agents toward various sites of HeLa cell DNA in vitro and in vivo.

The sites and extent of ethyl products of neutral ethylation of HeLa cell DNA by [14-C]diethyl sulfate, [14-C]ethyl methanesulfonate, and [14-C]ethylnitrosourea have been determined in vitro and in vivo, and found to differ significantly depending on the ethylating agents. Diethyl sulfate and ethyl methanesulfonate ethylate the bases of HeLa cell DNA in the following order: 7-ethylguanine greater than 3-ethyladenine greater than 1-ethyladenine, 7-ethyladenine greater than 3-ethylguanine, 3-ethylcytosine, O-6-ethylguanine. Ethyl bases accounted for 84-87% of the total ethyl groups associated with HeLa cell DNA. Ethylnitrosourea, in contrast, has particular affinity for the O-6 position of guanine. It ethylates the bases of HeLa cell DNA in the following order: O-6-ethylguanine, 7-ethylguanine greater than 3-ethyladenine greater than 3-ethylguanine, 3-ethylthymine greater than 1-ethyladenine, 7-ethyladenine, 3-ethylcytosine. Ethylation of the bases only accounts for 30% of the total ethylation in the case of ethylnitrosourea. The remaining 70% of the [14-C]ethyl groups, introduced in vivo and in vitro, are in the form of phosphotriesters which after perchloric acid hydrolysis are found as [14-CA1ethanol and [14-C]ethyl phosphate. In contrast, phosphotriesters amounted to only 8-20% of total ethylation in in vivo or in vitro diethyl sulfate and ethyl methanesulfonate treated HeLa cell DNA, and 25% of the total methylation in in vitro methylnitrosourea treated HeLa cell DNA. Alkylation at the N-7 and N-3 positions of purines in DNA destabilizes the glycosidic linkages. Part of 7-ethylguanine and 3-ethyladenine are found to be spontaneously released during the ethylation reaction. Incorporation of the 14-C of the alkylating agents into normal DNA bases of HeLa cells can be eliminated by performing the alkylations, in the presence of cytosine arabinoside, for 1 hr.     

Methylation of ribonucleic acid by the carcinogens dimethyl sulphate, N-methyl-N-nitrosourea and N-methyl-N′-nitro-N-nitrosoguanidine. Comparisons of chemical analyses at the nucleoside and base levels

1. The following methods for hydrolysis of methyl-(14)C-labelled RNA, and for chromatographic isolation and determination of the products, were investigated: enzymic digestion to nucleosides at pH6 or 8; alkaline hydrolysis and conversion into nucleosides; hydrolysis by acid to pyrimidine nucleotides and purine bases, or completely to bases; chromatography on Dowex 50 (NH(4) (+) form) at pH6 or 8.9, or on Dowex 50 (H(+) form), or on Sephadex G-10. 2. The suitability of the various methods for determination of methylation products was assessed. The principal product, 7-methylguanosine, was unstable under the conditions used for determinations of nucleosides. 3- and 7-Methyladenine and 3- and 7-methylguanine are best determined as bases; 1-methyladenine and 3-methylcytosine can be isolated as either nucleosides or bases; O(6)-methylguanine is unstable under the acid hydrolysis conditions used and can be determined as the nucleoside; 3-methyluracil was detected, but may be derived from methylation of the ionized form of uracil. 3. Differences between the patterns of methylation of RNA and homopolyribonucleotides by the N-methyl-N-nitroso compounds and dimethyl sulphate were found: the nitroso compounds were able to methylate O-6 of guanine, were relatively more reactive at N-7 of adenine and probably at N-3 of guanine, but less reactive at N-1 of adenine, N-3 of cytosine and probably at N-3 of uridine. They probably reacted more with the ribose-phosphate chain, but no products from this were identified. 4. The possible influences of these differences on biological action of the methylating agents is discussed. Nitroso compounds may differ principally in their ability to induce miscoding in the Watson-Crick sense by reaction at O-6 of guanine. Both types of agent may induce miscoding to a lesser extent through methylation at N-3 of guanine; both can methylate N atoms, presumably preventing Watson-Crick hydrogen-bonding. N-Methyl-N-nitrosourea can degrade RNA, possibly through phosphotriester formation, but this mechanism is not proven.     

Methylation of DNA in various organs of c57b1 mice by a carcinogenic dose of N-methyl-N-nitrosourea and stability of some methylation products up to 18 hours

Female 6–8-week-old C57B1 mice were injected i.p. with N-methyl-N-nitrosourea (MNUA) (¹⁴C or ³H-methyl-labelled) in saline (80 mg/kg) and DNA was isolated from bone marrow, small bowel, kidneys, liver, lungs, spleen and thymus at various times thereafter up to 18 h. Methylation of DNA was found in all organs examined, and by analyses using column or paper chromatography of DNA hydrolysates, the extent of methylation of DNA purines was determined.Methylated guanine residues (at N-3, N-7 and O-6 positions) were stable in DNA up to 18 h, but methylated adenines (at N-3 or N-7) were removed from DNA of all organs examined; the overall half-life of methyladenines was about 3 h, but removal appeared to occur in a biphasic manner, with a proportion of methyladenine remaining relatively stable. This relative stability was somewhat more marked in bone marrow than in other organs.     

Mutagenic activity of ethylene oxide and propylene oxide under XPG proficient and deficient conditions in relation to N-7-(2-hydroxyalkyl)guanine levels in Drosophila

Ethylene oxide (EO) and propylene oxide (PO) are direct acting mutagens with high Swain-Scott s-values, which indicate that they react preferentially with ring nitrogens in the DNA. We have previously described that in the X-linked recessive lethal (RL) assay in Drosophila postmeiotic male germ cells EO is, per unit exposure dose, 5-10 times more mutagenic than PO. Furthermore, at the higher dose range of EO tested, 62.5-1000 ppm, up to 20-fold enhanced mutation rates were measured in the absence of maternal nucleotide excision repair (NER) compared to repair proficient conditions. The lower dose range of EO tested, 2-7.8 ppm, still produced a small increased mutation rate but without a significant elevated effect when the NER system is being suppressed. The lowest dose of PO tested, 15.6 ppm, produced only in NER- condition an increased mutation rate. The aim of the present study was to compare the mutagenic effect of EO and PO in the RL assay under XPG proficient and deficient conditions with the formation of N-7-(2-hydroxyethyl)guanine (7-HEG) and N-7-(2-hydroxypropyl)guanine (7-HPG), respectively, the major DNA adducts formed. The formation of 7-HEG and 7-HPG was investigated in Drosophila males exposed to EO and PO as a measure of internal dose for exposures ranging from 2 to 1000 or 2000 ppm, respectively, for 24h. Analysis of 7-HEG and 7-HPG, using a highly sensitive 32P-postlabelling assay, showed a linear increase of adduct levels over the entire dose range. The non-linear dose-response relationship for mutations could therefore not be explained by a reduced inhalation or increased detoxification at higher exposure levels. In analogy with the four times higher reactivity of EO the level of N-7-guanine alkylation per ppm was for EO 3.5-fold higher than that for PO. Per unit N-7-guanine alkylation EO was found to be slightly more mutagenic than PO, whereas PO was the more potent clastogenic agent. While this research has not identified the DNA lesions that cause the increase in repair deficient flies, it supports the hypothesis that efficient error-free repair of some N-alkylation products can explain why these agents tend to be weakly genotoxic or even inactive in repair-competent (premeiotic) germ cells of the mouse and the Drosophila fly.     

Tet repressor-tet operator contacts probed by operator DNA-modification interference studies

Contacts between tet operator DNA and Tet repressor protein are characterized by modification interference studies. The modified DNA fragments are separated into fractions with high, intermediate and low affinities for Tet repressor by polyacrylamide gel electrophoresis. Ethylation of the phosphates with N-ethylnitrosourea reveals 12 contacts of a repressor dimer to tet operator. Eight of these contacts appear to be important for Tet repressor binding, as judged by the strong interference at these positions, while four contacts are probably less important. All of the phosphate contacts are located on the same side of the B-DNA structure. The sequences of tet operators proposed to interact with the recognition alpha-helix of Tet repressor are TCTATC in three cases and CCTATC in one case. After methylation of N-7 with dimethylsulfate, strong interference is observed at the guanine residues at positions +/- 2. None of the N-7 functions of other guanine residues seems to be involved in tight contacts to Tet repressor. Tet repressor subunits form identical phosphate and guanine N-7 contacts with each half side of the two tet operators indicating twofold dyad symmetry of the complexes. Attempts to analyze the methylation interference at the adenine N-3 sites reveal different results for the operators. Modification of DNA fragments with diethylpyrocarbonate yields hypersensitive sites in the tet operators, indicating different local DNA structures. Carbethoxylation interference studies confirm the contacts at the purines found by methylation interference. All of the sequence-specific protein-DNA contacts detected in this study are centered at the inside four base-pairs in each tet operator half side. The contacts are discussed with respect to the structure of the repressor-operator complex.     

Effects of alkylation by dimethyl sulfate, nitrogen mustard, and mitomycin C on DNA structure as studied by the ethidium binding assay.

The extent of alkylation of DNA by dimethyl sulfate, nitrogen mustard, and the antibiotic mitomycin C is related to the resulting decrease in the fluorescence of intercalated ethidium. The fluorescence losses due to the first two types of reagents show a marked pH dependence, with greater losses of fluorescence being observed at alkaline pH values. At pH 11.6 the fluorescence shows a slow recovery, so that with low levels of methylation (4% deoxyguanosine residues modified) one observes complete return of fluorescence. We postulate that these phenomena are due to conversion of 7-methyldeoxyguanosine to the zwitterionic form, and partial denaturation of the DNA duplex with loss of ethidium binding sites. Hydroxide-ion-catalyzed imidazole ring opening, and the removal of the positive charge permits reannealing with concomitant return of the ethidium intercalation sites. This conclusion is substantiated by enzymatic hydrolysis of 14C-labelled methylated DNA and identifiions of the ethidium assay. The distinctly different behavior of mitomycin C confirms previous conclusions that its alkylation, preferentially on guanine, does not take part at the N-7 position.     

Lesion selectivity in blockage of lambda exonuclease by DNA damage.

Various kinds of DNA damage block the 3' to 5' exonuclease action of both E. coli exonuclease III and T4 DNA polymerase. This study shows that a variety of DNA damage likewise inhibits DNA digestion by lambda exonuclease, a 5' to 3' exonuclease. The processive degradation of DNA by the enzyme is blocked if the substrate DNA is treated with ultraviolet irradiation, anthramycin, distamycin, or benzo[a]-pyrene diol epoxide. Furthermore, as with the 3' to 5' exonucleases, the enzyme stops at discrete sites which are different for different DNA damaging agents. On the other hand, digestion of treated DNA by lambda exonuclease is only transiently inhibited at guanine residues alkylated with the acridine mustard ICR-170. The enzyme does not bypass benzo[a]-pyrene diol epoxide or anthramycin lesions even after extensive incubation. While both benzo[a]-pyrene diol epoxide and ICR-170 alkylate the guanine N-7 position, only benzo[a]-pyrene diol epoxide also reacts with the guanine N-2 position in the minor groove of DNA. Anthramycin and distamycin bind exclusively to sites in the minor groove of DNA. Thus lambda exonuclease may be particularly sensitive to obstructions in the minor groove of DNA; alternatively, the enzyme may be blocked by some local helix distortion caused by these adducts, but not by alkylation at guanine N-7 sites.     

Nitrosamine-induced carcinogenesis. The alkylation of nucleic acids of the rat by N-methyl-N-nitrosourea, dimethylnitrosamine, dimethyl sulphate and methyl methanesulphonate

1. N[(14)C]-Methyl-N-nitrosourea, [(14)C]dimethylnitrosamine, [(14)C]dimethyl sulphate and [(14)C]methyl methanesulphonate were injected into rats, and nucleic acids were isolated from several organs after various time-intervals. Radioactivity was detected in DNA and RNA, partly in major base components and partly as the methylated base, 7-methylguanine. 2. No 7-methylguanine was detected in liver DNA from normal untreated rats. 3. The specific radioactivity of 7-methylguanine isolated from DNA prepared from rats treated with [(14)C]dimethylnitrosamine was virtually the same as that of the dimethylnitrosamine injected. 4. The degree of methylation of RNA and DNA produced in various organs by each compound was determined, and expressed as a percentage of guanine residues converted into 7-methylguanine. With dimethylnitrosamine both nucleic acids were considerably more highly methylated in the liver (RNA, about 1% of guanine residues methylated; DNA, about 0.6% of guanine residues methylated) than in the other organs. Kidney nucleic acids were methylated to about one-tenth of the extent of those in the liver, lung showed slightly lower values and the other organs only very low values. N-Methyl-N-nitrosourea methylated nucleic acids to about the same extent in all the organs studied, the amount being about the same as that in the kidney after treatment with dimethylnitrosamine. In each case the RNA was more highly methylated than the DNA. Methyl methanesulphonate methylated the nucleic acids in several organs to about the same extent as N-methyl-N-nitrosourea, but the DNA was more highly methylated than the RNA. Dimethyl sulphate, even in toxic doses, gave considerably less methylation than N-methyl-N-nitrosourea in all the organs studied, the greatest methylation being in the brain. 5. The rate of removal of 7-methylguanine from DNA of kidneys from rats treated with dimethylnitrosamine was compared with the rate after treatment of rats with methyl methanesulphonate. No striking difference was found. 6. The results are discussed in connexion with the organ distribution of tumours induced by the compounds under study and in relation to the possible importance of alkylation of cellular components for the induction of cancer.     

Reduced glutathione inhibits the alkylation by N-nitrosodimethylamine of liver DNA in vivo and microsomal fraction in vitro

The transfer of radioactivity from N-nitroso-[14C]dimethylamine to trichloroacetic acid precipitable macromolecules in the microsomal fraction of rat liver was investigated. This transfer was found to depend on N-nitrosodimethylamine being metabolized. Cytosolic fraction and cytosol enriched with reduced glutathione inhibited the binding of radioactivity to acid insoluble proteins. Depletion of glutathione in rat liver with diethylmaleate prior to i.v. administration of 10 mg N-nitroso-[14C]dimethylamine/kg led to an increase in O6-methylguanine and N-7-methylguanine in DNA. If rats were fed disulfiram for 6 days (2 g/kg feed), glutathione and glutathione S-transferase were enhanced, and the degree of methylation of guanine by N-nitrosodimethylamine was greatly reduced, as was the metabolism of N-nitrosodimethylamine in the intact animal. Fasting rats for 24 h did not change the N-nitrosodimethylamine-demethylase activity in vitro but greatly enhanced the methylation of guanine in vivo, while the glutathione content and glutathione S-transferase activity were not changed compared to fed animals.     

Deoxyribonucleic acid-cytosine methylation by host- and plasmid-controlled enzymes.

Deoxyribonucleic acid (DNA)-cytosine methylation specified by the wild-type Escherichia coli K 12 mec+ gene and by the N-3 drug resistance (R) factor was studied in vivo and in vitro. Phage lambda and fd were propagated in the presence of L-[methyl-3H]methionine in various host bacteria. The in vivo labeled DNA was isolated from purified phage and depurinated by formic acid-diphenylamine treatment. The resulting pyrimidine oligonucleotide tracts were separated according to size and base composition by chromatography on diethylaminoethyl-cellulose in 7 M urea at pH 5.5 and 3.5, respectively. The distribution of labeled 5-methylcytosine in DNA pyrimidine tracts was identical for phage grown in mec+ and mec minus (N-3) cells. For phage lambda the major 5-methylcytosine containing tract was the tripyrimidine, C2T; for both fd-mec minus (N-3) DNA and fd-mec+DNA, C2T was the sole 5-methylcytosine-containing tract. When various lambda DNAs were methylated to saturation in vitro by crude extracts from mec+ and mec minus (N-3) cells, the extent of cytosine methylation was the same. This is in contrast to in vivo methylation where lambda-mec minus (N-3) DNA contains twice as many 5-methylcytosines per genome as lambda-mec+ DNA. Therefore, we suggest that the K12 met+ cytosine methylase and the N-3 plasmid modification methylase are capable of recognizing the same nucleotide sequences, but that the in vivo methylation rate is lower in mec+ cells.     

Adenine N3 is a main alkylation site of styrene oxide in double-stranded DNA

Styrene 7,8-oxide (SO), a major metabolite of styrene, is classified as a probable human carcinogen. In the present work, salmon testis DNA was reacted with SO and the alkylation products were analysed after sequential depurination in neutral or acidic conditions followed by HPLC separation and UV-detection. A novel finding was that the N-3 position of adenine was the next most reactive alkylation site in double-stranded DNA, comprising 4% of the total alkylation, as compared to alkylation at the N-7 position of guanine, 93% of the total alkylation. Both alpha- and beta-products of SO were formed at these two sites. Other modified sites were N2-guanine (1.5%, alpha-isomer), 1-adenine (0.4%, both isomers) and N6-adenine (0.7%, both isomers) as well as 1-hypoxanthine (0.1%, alpha-isomer), formed by deamination of the corresponding 1-adenine adduct. The results indicated that in double-stranded DNA N-7 of guanine and N-3 of adenine account for 97% of alkylation by SO. However, these abundant adducts are not stable, the half-life of depurination in DNA for 3-substituted adenines being approximately 10 and approximately 20 h, for alpha- and beta-isomers, respectively, and 51 h for both isomers of 7-substituted guanines.