The specificity of different classes of ethylating agents toward various sites in RNA.

The alkyl products of neutral in vitro ethylation of TMV-RNA by [14C]diethyl sulfate, [14C]ethyl methanesulfonate, and [14C]ethylnitrosourea have been determined and found to differ significantly depending on the ethylating agent. Diethyl sulfate and ethyl methanesulfonate ethylate the bases of TMV-RNA in the following order: 7-ethylguanine greater than 1-ethyladenine, 3-ethylcytidine greater than 7-ethyladenine, 3-ethyladenine, O6-ethylguanosine, 3-ethylguanine. Ethyl methanesulfonate was more specific for the 7 position of guanine, and other derivatives were found in lesser amounts than with diethyl sulfate. Neither reagent caused the formation of detectable amounts (smaller than 0.26 percent) of 1-ethylguanine, 1,7-diethylguanine, N2-ethylguanine, N6-ethyladenine, N4-ethylcytidine, or 3-ethyluridine. Identified ethyl bases account for over 85% of the total radioactivity of [14C]ethyl methanesulfonate and [14C]diethyl sulfate treated TMV-RNA. Phosphate alkylation accounts for about 13 and 1%, respectively, In contrast, [14C]ethylnitrosourea-treated TMV-RNA, while reacting to a similar extent (15-70 ethyl groups/6400 nucleotides), is found to cause considerably more phosphate alkylation. Upon either U4A RNase or acid hydrolysis up to 60% of the radioactivity is found as volatile ethyl groupw in the form of [14C]ethanol, and a further 15% appears to be primarily ethyl phosphate and nucleosides with ethylated phosphate. Of the remaining radioactivity, half is found as O6-ethylguanosine, the major identified ethyl nucleoside. Other ethyl bases found in ethylnitrosourea-treated TMV-RNA are 7-ethylguanine greater than 1-ethyladenine, 3-ethyladenine, 7-ethyladenine, 3-ethylcytidine, and 3-ethylguanine. It appears that ethylnitrosourea preferentially alkylates oxygens, and that formation of phosphotriesters is by far the predominant chemical event. Since the number of ethyl groups introduced into TMV-RNA by ethylnitrosourea is similar to the number of lethal events, one may conclude that phosphate alkylation leads to loss of infectivity. None of the three ethylating agents studied are strongly mutagenic on TMV-RNA or TMV. The role of phosphate alkylation in regard to in vivo mutagenesis and oncogenesis remains to be established. At present it appears possible that the extent of this reaction may correlate better with the oncogenic effectiveness of different ethylating agents, than the extent of any base reaction. Unfractionated HeLa cell RNA is ethylated primarily in acid labile manner even by diethyl sulfate and ethyl methanesulfonate, a fact that is attributed to its high content of low molecular weight trna rich in terminal phosphates which alkylate readily.     

Function and organization of the G region of bacteriophage Mu; Host specificity determined by gene splicing

Chinese hamster ovary (CHO) cells were exposed to [³H]ethyl nitrosourea (ENU) or [³H]ethyl methanesulfonate (EMS) and the following DNA ethylation products were quantitated: 3- and 7-ethyladenine, O²-ethylcytosine, 3-, 7- and O⁶-ethylguanine, O²- and O⁴-ethyldeoxythymidine and the representative ethylated phosphodiester, deoxythymidylyl (3′–5′)ethyl-deoxythymidine. When mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus induced by these same treaments were compared with the observed ethylation products, mutations were found to correlate best with 3- and O⁶-ethylguanine. EMS induced approximately twice as many sister-chromatid exchanges (SCEs) as ENU at doses yielding equal mutation frequencies. When SCEs were indirectly compared with DNA ethylation products, 3-ethyladenine and ethylated phosphodiesters related best to SCE formation. Because mutation and SCE induction appear, at least in part, to be related to different DNA adducts, SCE induction by simple ethylating agents may not be a quantitative indicator of potentially mutagenic DNA damage.     

The inactivation of bacteriophage R17 by ethylating agents: the lethal lesions.

The biological inactivation of bacteriophage R17 by ethyl methanesulphonate (EMS) and N-ethyl-N-nitrosourea (ENUA) has been studied. At the mean lethal dose for the first compound 8 moles ethyl are bound/mole RNA and with the nitroso compound 3.5 moles ethyl are bound. Analysis of the amounts of the different ethylated derivatives formed shows that the toxicity of the sulphonate can be accounted for by the formation of 3-ethylcytosine, O6-ethylguanine, 1-ethyladenine and chain breaks produced on the hydrolysis of ethyl phosphotriesters. With the nitroso derivative on the other hand, the sum of chain breaks and of bases alkylated on a position involved in specific hydrogen bonding between base pairs only accounts for 65% of the observed toxicity. The possibility that 3-ethyladenine may constitute a lethal lesion is discussed.     

Kinetics of formation of O6-ethylguanine in,and its removal from liver DNA of rats receiving diethylnitrosamine

The ethylation of rat liver DNA by a single dose of diethylnitrosamine and the stability of O⁶-ethylguanine in vivo were studied. Whereas the dose response relations for 7-ethylguanine, 3-ethyladenine, the pyrimidine oligonucleotide fraction containing ethylphosphotriesters and an as yet unreported Fraction X corresponded with a first-order process of formation, the results suggested a steeper dose-response relation for O⁶-ethylguanine formation. In the dose range 0.5–10 mg/kg diethylnitrosamine, the O⁶-ethylguanine/7-ethylguanine ratio increased progressively with the dose, under conditions in which the in vivo stability (removal rate) of O⁶-ethylguanine was not affected. This led to the hypothesis that the formation of O⁶-ethylguanine, but not that of the other ethylated products, was facilitated by some dose-dependent process or condition. Support for this view was obtained by the markedly enhanced O⁶-[¹⁴C]ethylguanine content of DNA following pretreatment of the rats with non-radioactive diethylnitrosamine which was allowed to be metabolized completely prior to the administration of a tracer dose of [¹⁴C]diethylnitrosamine. Since neither the amounts of the other ethylation products nor the stability of the labelled O⁶-ethylguanine were affected by the pretreatment, changes in carcinogen metabolism or excision rate could be excluded as causes of the observed increase in O⁶-ethylguanine content. The half-life of the condition that facilitates O⁶-ethylguanine formation following pretreatment, may approximate that of O⁶-ethylguanine itself. The nature of the facilitating process and the possible role of O⁶-alkylguanine in hepatocarcinogenesis are discussed.     

Studies of the ethylation of rat liver transfer ribonucleic acid after administration of l-ethionine

1. The ethylated nucleosides present in tRNA isolated from the livers of rats treated with 0.5g of l-ethionine/kg body wt. were investigated. Evidence that this tRNA contained N(2)-ethylguanine, N(2)N(2)-diethylguanine, N(2)-ethyl-N(2)-methylguanine, 7-ethylguanine, two ethylated pyrimidines and ethylated ribose groups was obtained. 2. Ethylation of bacterial tRNA was catalysed by extracts containing tRNA methylases prepared from rat liver by using S-adenosyl-l-ethionine as an ethyl donor, but the rate of ethylation was 20 times less than the rate of methylation with S-adenosyl-l-methionine as a methyl donor. 3. The principal product of such ethylation in vitro was N(2)-ethylguanine and traces of the other ethylated guanines and pyrimidines found in tRNA isolated from rats treated with ethionine in vivo were also found. 1-Ethyladenine was not formed, although 1-methyl-adenine is a major product of methylation of bacterial tRNA by these extracts, and 1-ethyladenine was not present in the rat liver tRNA isolated from ethionine-treated animals. 4. After injection of actinomycin D (15mg/kg body wt.) or l-methionine (1.0g/kg body wt.) before the ethionine, ethylation of tRNA was diminished by about 80% but not completely abolished. Administration of 1-aminocyclopentanecarboxylic acid (2.5g/kg body wt.) to inhibit the formation of S-adenosyl-l-ethionine inhibited ethylation of tRNA by 44%. 5. These results suggest that not all of the ethylation of tRNA that occurs in the livers of rats treated with ethionine is mediated by the action of tRNA methylases acting with S-adenosyl-l-ethionine as a substrate, but that this pathway does occur and accounts for a major part of the observed ethylation. 6. The results are discussed with reference to ethionine-induced hepatocarcinogenesis.     

Properties of 3-methyladenine-DNA glycosylase from Escherichia coli.

An Escherichia coli enzyme that releases 3-methyladenine and 3-ethyladenine in free form from alkylated DNA has been purified 2800-fold in 7% yield. The enzyme does not liberate several other alkylation products from DNA, including 7-methylguanine,O6-methylguanine, 7-methyladenine, N6-methyladenine, 7-ethylguanine, O6-ethylguanine, and the arylalkylated purine derivatives obtained by treatment of DNA with 7-bromomethyl-12-methylbenz[a]anthracene. The reaction of the enzyme with alkylated DNA leads to the introduction of apurinic sites but no chain breaks (less than one incision per ten apurinic sites), and there is no detectable nuclease activity with native DNA, depurinated DNA, ultraviolet-irradiated DNA, or X-irradiated DNA as potential substrates. The enzyme is termed 3-methyladenine-DNA glycosylase. It is a small protein, Mr = 19 000, that does not require divalent metal ions, phosphate, or other cofactors in order to cleave base-sugar bonds in alkylated DNA.     

Alkylation of deoxyribonucleic acid in vivo in various organs of C57BL mice by the carcinogens N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea and ethyl methanesulphonate in relation to induction of thymic lymphoma. Some applications of high-pressure liquid chromatography.

1. Methods were developed for analysis of alkylpurines, O2-alkylcytosines, and representative phosphotriesters [alkyl derivatives of thymidylyl(3'-5')thymidine], in DNA alkylated in vivo, using high-pressure liquid chromatography. 2. The patterns of alkylation products in DNA in vivo at short times were closely similar to those found for reactions in vitro. Alkylation by the nitrosoureas was complete in vivo within 1 h, but with ethyl methanesulphonate was maximal at 2--4h. 3. The time course of persistence of alkylation products in vivo was determined for several tissues. In addition to the rapid loss of 3- and 7-alkyladenines reported previously for all tissues, a relatively rapid loss of O6-alkylguanines from DNA of liver was found which was more rapid at lower doses. In brain, lung and kidney, excision of O6-alkylguanine was much less marked, but was not entirely excluded by the data. In thymus, bone marrow and small bowel, all alkylated bases were lost with half-lives of 12--24h, at non-cytotoxic doses of alkylation. 4. No evidence for any marked excision of other minor products from alkylated DNA in vivo was found; thus 1-methyladenine, O2-ethylcytosine (found in appreciable amount only with N-ethyl-N-nitrosourea), 3-methylguanine, and dTp(Alk)dT persisted in alkylated DNA, including DNA of liver. 5. The induction of thymic lymphoma was determined over the range of single doses by intraperitoneal injection up to about 60% of the LD50 values, and related to the extent of alkylation of target tissues thymus and bone marrow. With N-methyl-N-nitrosourea over 90% tumour yield was attained at 60 mg/kg, and with N-ethyl-N-nitrosourea up to 52% at 240 mg/kg, but with ethyl methanesulphonate at up to 400 mg/kg only a few per cent of tumours were obtained. 6. The carcinogenic effectiveness of the agents was positively correlated with the extents of alkylation of guanine in DNA of target tissues at the O-6 atom. On the basis that at doses giving equal carcinogenic response these extents of alkylation would be equal, the chemical analyses showed that the ratio of equipotent doses to that for N-methyl-N-nitrosourea would be, for N-ethyl-N-nitrosourea, 5.3 for ethyl methanesulphonate about 21, and for methyl methanesulphonate [Frei & Lawley (1976) Chem.-Biol. Interact. 13, 215--222] about 144. These predictions were in reasonably good agreement with the observed dose-response data for these agents.     

Structural alterations in deoxyribonucleic acid on chemical ethylation.

Ethylation of DNA by diethyl sulfate gave 7-ethylguanine as the major product. Dimethyl sulfate was much more reactive than diethyl sulfate in forming 7-alkylguanine. The hydrodynamic properties of DNA did not change as a direct consequence of ethylation. On incubation at 37 °C, the viscosity of ethylated DNA decreased at a rate similar to that of methylated DNA. The rate of depurination of 7-ethylguanine from ethylated DNA was the same as that of 7-methylguanine from methylated DNA. These results demonstrate that ethyl groups have identical effects as methyl groups on the secondary structure and stability of DNA.     

Chemical dosimetry of ethyl nitrosourea in the mouse testis

[3H-Et]Nitrosourea was administered to male (101 X C3H) mice by i.p. injection at exposure levels of 10 mg/kg or 100 mg/kg. At intervals from 1 h to 6 days following treatment, the ratio of O6-ethylguanine to N7-ethylguanine in testis DNA averaged 1.13 following the 100 mg/kg exposure and 0.72 following the 10 mg/kg exposure. The amount of O6-ethylguanine recovered after the 100 mg/kg exposure was 40% greater than predicted from a linear extrapolation of the amount of O6-ethylguanine recovered after the 10 mg/kg exposure. We suggest that the high (100 mg/kg) exposure to ethyl nitrosourea results in depletion of the O6-alkylguanine acceptor protein within the testis and permits O6-ethylguanine to persist at higher levels than would be predicted from lower exposure data. W.L. Russell et al. (1982), W.L. Russell (1984) have found that specific-locus mutation frequencies induced in mouse spermatogonial stem cells are 5.8-fold greater after a single 100 mg/kg exposure to ethyl nitrosourea than after 10 weekly exposures to 10 mg/kg. The finding that the corresponding ratio for O6-ethylguanine formed in the testis is only 1.4 may be interpreted in a number of possible ways. If O6-ethylguanine is an important lesion for producing specific-locus mutations, then its formation in the stem cells must be at least 4-fold greater than that for the whole testis as the ENU exposure goes from 10 to 100 mg/kg: alternatively, the rate of repair of this lesion by the stem cells must decrease at least 4-fold relative to the average testicular cell. Other explanations for the difference in mutation response of the stem cells to acute vs. chronic ethyl nitrosourea-exposures include the possibility that other DNA lesions may be responsible for many of the mutations or that two hits on the DNA may be required to produce an effect.     

DNA adduct formation in mouse testis by ethylating agents: a comparison with germ-cell mutagenesis

DNA adduct formation in various organs of mice was determined after i.p. injection with the ethylating agents N-ethyl-N-nitrosourea (ENU), ethyl methanesulfonate (EMS), and diethyl sulfate (DES). The potency of the 3 chemicals to react either at the O6 position of guanine or at the N-7 position of guanine was related to their potency to induce mutations in the specific-locus assay of the mouse. ENU, which produces relatively high levels of O-alkylations (O6-ethylguanine), is primarily mutagenic in spermatogonia of the mouse, whereas EMS and DES, which produce relatively high levels of N-alkylations (7-ethylguanine) in DNA, are much more mutagenic in post-meiotic stages of male germ cells. The relationship between exposure to ENU and the dose, determined as O6-ethylguanine per nucleotide in testicular DNA, is non-linear. However, the relationship between dose and mutation induction in spermatogonia by ENU appears to be linear, which is expected if O6-ethylguanine is the major mutagenic lesion. The relatively high mutagenic potency of EMS and DES in the late stages of spermatogenesis is probably due to the accumulation of apurinic sites which generate mutations after fertilization. A comparison of mutation induction by ENU in spermatogonia and mutation induction in cultured mammalian cells indicates that about 10 O6-ethylguanine residues were necessary in the coding region of a gene to generate a mutation.     

Formation and removal of DNA adducts after treatment of Chinese hamster ovary cells with N-methyl- and N-ethyl-N-nitrosourea

We have studied formation and stability of alkylguanines following treatment of Chinese hamster ovary cells with either N-[3H]methyl-N-nitrosourea (MeNOUr) (applied at 50 microM and 40 microM concentrations) or N-[3H]ethyl-N-nitrosourea (EtNOUr) (applied at 43.1 microM). Analyses of acid hydrolysates of the methylated DNA revealed that 9.3% and 57.0% of the total DNA were O6-methylguanine (m6Gua) and 7-methylguanine (m7Gua), respectively. Analysis of enzymic hydrolysate resulted in 8.2% m6Gua and 50.3% m7Gua. For ethylation, the % of ethylated purines identified as O6-ethylguanine (e6Gua) and 7-ethylguanine (e7Gua) were 20.4% and 31.3%, respectively. Half-lives of the main alkylated purines were determined by analysing DNA of dividing cultures over a time interval of 48 h after treatment with carcinogens. Half-lives measured for methylated DNA bases were: m1Ade, 20.6 h; m3Ade, 25.5 h; m7Ade, 0.9 h; m3Gua, 1.1 h; m6Gua, infinity; m7Gua, 39.1 h. Determinations at the level of deoxyribonucleosides resulted in similar half-lives: m3dA, 15.2 h; m7dA, 2.7 h; m3dG, 2.3 h; m6dG, 224 h; m7dG, 25.6 h. The corresponding values for ethylated purines were: e3Ade, 2.9 h; e7Ade, 7.1 h; e3Gua, 1.4 h; e6Gua, infinity; e7Gua, 42.6 h. The relatively high yields of the premutagenic m6Gua and e6Gua, and their long half-lives (greater than or equal to 224 h) are consistent with the suggestion that these adducts play a dominant role in mutation induction at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus in CHO cells.     

Phosphotriesters in rat liver deoxyribonucleic acid after the administration of the carcinogen NN-dimethylnitrosamine in vivo.

After treatment with NN-di[14C]methylnitrosamine, samples of DNA were isolated from rat livers by a conventional phenol procedure and examined for the presence of phosphotriesters. A method of capable of detecting relatively small amounts of 14C-labelled phosphotriesters was developed and used to establish that these products account for 10-12% of the total methylation pattern found after treatment with this agent in vitro. The significance of the presence of phosphotriesters in DNA is discussed.     

Methylation and ethylation of uridylic acid and thymidylic acid. Reactivity of the ring and phosphate as a function of pH and alkyl group.

At pH 6.8 in aqueous solution (4 hr, 22 degrees), all methylating agents tested, i.e., dimethyl sulfate, methyl methanesulfonate, and methylnitrosourea, react with both the N-3 of the ring and the phosphate of UMP and dTMP. Although the extent of reaction varies from 17 to 76%, the ratio of phosphate/ring methylation is approximately 4. Both the 3-methyl nucleotides and methyl ester of 3-methyl nucleotides are identified, as well as the methyl esters of unmodified UMP and dTMP. At pH 8.2 the extent of total methylation is similar but reactivity of the N-3 is increased and that of the phosphate decreased so that the phosphate/ring ratio is approximately 1. At pH 6 almost all reaction is with the phosphate group. Uridine, under the same conditions, is methylated at pH 6.8 to form 15% 3-methyluridine and, at pH 8.2, the N-3 of uridine and thymidine is methylated to about 50%. Neither uridine nor UMP forms detectable ribose methyl products at any of these pH's. The comparable ethylating agents (diethyl sulfate, ethyl methanesulfonate, and ethylnitrosourea) are less reactive and the total ethylation of UMP or dTMP is about 1/5 that of methylation. There is little ethylation of the N-3 but the phosphate is alkylated to a relatively high extent so that the phosphate/base ratio at pH 6.8 is 10-23, and at pH 8.2 the ratio is 5-8. The fact that ethylating agents have a greater affinity than methylating agents for alkylating phosphates is proposed as the basis for the previously reported analytical data in which ethylating agents, acting on DNA or RNA at neutrality, form more phosphotriesters than the analogous methylating agents.     

Synthesis and properties of oligodeoxyribonucleotides containing an ethylated internucleotide phosphate

Internucleotide phosphotriesters comprise an important class of DNA lesions produced by carcinogenic alkylating agents. To avoid confusion resulting from the presence of other DNA lesions, synthetically prepared oligonucleotides containing ethylated internucleotide phosphates as the sole form of damage were employed to investigate several chemical and biochemical properties of DNA alkyl phosphotriesters. A total of four oligonucleotides were synthesised for this study, the dimers Tp(Et)T and pTp(Et)T and the decamer d-TpTpTp(Et)TpCpTpApTpTpT together with its unmodified analogue. The dimers were characterized by UV and phosphorus NMR spectroscopy and the decamers by two-dimensional homochromatography, alkali hydrolysis, and variable-temperature circular dichroism (CD). Alkali hydrolysis of the ethylated decamer produced strand breaks in approximately 75% of the molecules. This is in close agreement with data previously obtained for dinucleoside ethyl phosphotriesters and triesters in alkylated cellular DNA. Results from the CD study suggest that the ethyl substituent does not disrupt base stacking within the oligomer. The interactions of two enzymes with the alkylated oligonucleotides were examined. First, it was found that ethylation of the internucleotide phosphate renders TpT inactive as a substrate for T4 polynucleotide kinase, implying that a negative charge is required on the 3'-phosphate group of the nucleotide to be phosphorylated. Hence, postlabeling assays of DNA damage that depend upon enzymatic phosphorylation of modified 3'-nucleotides cannot be applied to dinucleoside alkyl phosphotriesters. Second, both decamers, when annealed to a single-stranded plasmid template, were able to prime DNA synthesis, catalyzed by Escherichia coli DNA polymerase I, with equal effectiveness. The use of this reaction as a means of site-specifically incorporating phosphotriesters into viral vectors is recognized.     

Repair of O6-ethylguanine DNA lesions in isolated cell nuclei. Presence of the activity in the chromatin proteins

Cell nuclei prepared from rat liver were alkylated in vitro with ethylnitrosourea; the nuclear DNA was found to lose O6-ethylguanine and 7-ethylguanine during a subsequent incubation at 37 degrees C. The rate of O6-ethylguanine loss is comparable to that observed in vivo, indicating that no cytoplasmic component is needed for the repair; no free O6-ethylguanine was found in the incubation medium of the ethylated nuclei. The rate of 7-ethylguanine loss is higher than the spontaneous depurination in vitro and an amount of free 7-ethylguanine equivalent to that lost by the nuclear DNA was found in the incubation medium; these results suggest that this DNA lesion is excised by a DNA glycosylase. The proteins of the chromatin prepared from the isolated nuclei induced the disappearance of O6-ethylguanine from an added ethylated DNA. No free O6-ethylguanine was released indicating that the repair is not catalyzed by a DNA glycosylase; no oligonucleotides enriched in O6-ethylguanine were released either, indicating that the disappearance of O6-ethylguanine from DNA is not the result of the cooperative action of a specific endonuclease and an exonuclease. Activities capable of removing O6-ethylguanine from DNA were found in other cell compartments; most of it, however, is in the nucleus where the main location is chromatin. A pretreatment of the rats with daily low doses of diethylnitrosamine during 3 or 4 weeks increased 2-3-times the repair activity of the chromatin proteins.     

An assay for phosphotriester formation in the reaction of alkylating agents with deoxyribosenucleic acid in vitro and in vivo.

Preliminary studies in vitro using bacteriophage T7-DNA have shown that breaks formed in the DNA on the alkaline hydrolysis of apurinic sites and phosphotriesters can be distinguished from each other by measuring the extent of degradation of the DNA immediately after adding NaOH to 0.1 M and after incubating for 1 h in 0.5 M NaOH. This method has then been applied to the study of the formation and stability of phosphotriesters invivo. Methyl phosphotriesters formed in liver DNA following injection of mice with N-methyl-N-nitrosourea (MNUA) disappear with time (50% in 4-5 days). The concentration of ethyl phosphotriesters in liver DNA formed by injecting mice with N-ethyl-N-nitrosourea (ENUA) does not appear to decrease with time. Results of experiments on injecting methyl methane-sulphonate (MMS), ethyl methanesulphonate (EMS) and dimethyl sulphate (DMS) are also reported. The method described does not require the use of radioactively labelled reagents.     

Ethylation of nucleophilic sites in DNA by N-ethyl-N-nitrosourea depends on chromatin structure and ionic strength

Depending on ionic strength, chromatin can assume either a condensed, supranucleosomal conformation or the form of an extended nucleosomal fiber. Using sedimentation velocity analysis, both types of structures could be identified in chromatin prepared from cell nuclei of fetal rat brain. When the ionic strength was reduced from 60 to 10 mM NaCl, the average S-value of a defined chromatin fiber fraction (12–15 nucleosomes in size) decreased dramatically from 72 S to 55 S, reflecting the unfolding of condensed chromatin to an extended conformation. Correspondingly, the average S-value of histone H1-depleted chromatin (Ch⁻) was 54 S at 60 mM NaCl and did not change significantly at lower NaCl concentrations. Ch⁻ contains only the core histones and is, therefore, relaxed into an extended form.

Using a monoclonal antibody (ER-6) specific for O⁶-ethyldeoxyguanosine, we studied the influence of chromatin conformation on the formation of O⁶-ethylguanine (O⁶-EtGua) in the DNA of chromatin exposed to the carcinogen N-ethyl-N-nitrosourea (EtNU; 1 mg/ml, 37°C, 20 min) in vitro. When the NaCl concentration during incubations with EtNU was varied between 0 and 100 mM, the amount of O⁶-EtGua formed in the DNA of complete chromatin (Ch⁺) was highest at 0 mM NaCl, then decreased exponentially with increasing ionic strength, and remained approximately constant at values 50 mM NaCl. A similar dependence on ionic strength was found for the formation of O⁶-EtGua in the DNA of Ch⁻ and in native DNA. The frequency of O⁶-EtGua was highest in native DNA, followed by the DNA of Ch⁻, and lowest in the DNA of Ch⁺. At each salt concentration, the O⁶-EtGua content of Ch⁺ DNA relative to the corresponding values for Ch⁻ DNA and native DNA, remained unchanged (0.70±0.03 S.D. and 0.42±0.03 S.D., respectively). In addition to O⁶-EtGua, the formation of 7-ethylguanine (7-EtGua; major groove of the DNA double helix) and 3-ethyladenine (3-EtAde; minor groove) was analysed after exposure to [1-¹⁴C]EtNU. 7-EtGua was the most frequently formed ethylation product, followed by O⁶-EtGua and 3-EtAde. As in the case of O⁶-EtGua, the frequencies of 7-EtGua and 3-EtAde were dependent on ionic strength, and decreased in the order: native DNA, Ch⁻ DNA, and Ch⁺ DNA. Compared with native DNA (relative value, 100), the frequencies of O⁶-EtGua and 7-EtGua in DNA were reduced to a similar extent in Ch⁻ (rel. values 62.1 and 61.2, respectively) and in Ch⁺ (rel. values for both products, 43.9). The corresponding values for 3-EtAde were slightly lower in both types of chromatin fibers (rel. values 56.7 and 39.5, respectively). Thus, the core histones generally protect DNA from ethylation by EtNU. While nucleophilic sites in the major groove and in the base-pairing region of the DNA double helix are protected to about the same degree, the N-3 position of adenine in the minor groove is slightly less accessible to the ethyldiazonium ion generated from EtNU. In all cases the highest degree of protection is obtained when histone H1 is present in chromatin.     

The nature of single-strand breaks in DNA following treatment of L-cells with methylating agents

DNA from untreated L-cells had a weight average molecular weight (Mw) of 5.7 ± 0.58·10⁸ daltons as measured by sedimentation in an alkaline sucrose gradient. This value was reduced by one half after the cells were treated for 1 h with 8 μg/ml of N-methyl-N-nitrosourea (MNUA), 34 μg/ml of methyl methanesulfonate (MMS) or 0.16 μg/ml of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). That dose of MNUA produced 52 methylations per 5.7·10⁸ daltons DNA. 20% of these were not purine derivatives and were assumed to contain some phosphotriesters. That dose of MMS (above) produced 290 methylations per 5.7·10⁸ daltons DNA and about 14% of these were not purine derivatives. The rates of loss of methylated purines from DNA were 2.3% per hour for 7-methylguanine (7-MeG), 7.4% per hour for 3-methyladenine (3-MeA) and no detectable loss of O⁶-methylguanine (O⁶-MeG) over a 12 h period. Since phosphotriesters are alkali-labile the single-strand breaks probably arose from this structure and did not form within the cell. This conclusion is supported by the following considerations. MNUA was more effective than MMS at reducing the molecular weight of DNA, as measured in alkaline medium. The greater SN₁ character of MNUA would cause a greater formation of phosphotriesters than would MMS.     

Evidence for removal at different rates of O-ethyl pyrimidines and ethylphosphotriesters in two human fibroblast cell lines.

The potent carcinogen, ethylnitrosourea, has been shown to ethylate oxygens, in preference to nitrogens, in the DNA of cultured cells. We have now studied the removal of seven ethyl derivatives in replicating cells. The following findings are reported. 1) The absolute amounts of 02-EtT, 04-EtT and 02-EtC are decreased in cellular DNA after correction for cell growth. However the rate of decrease diminishes after approximately 20 hr and after more than two cell doublings 20--40% of each derivative persists. This decrease is presumed to be due to enzymes since these derivatives are stable in isolated DNA. 2) The amount of ethyl phosphotriesters remains almost unchanged during 72 hr of cell culture. 3) The unstable purine derivatives, 7-EtG and 3-EtA, are both removed from cellular DNA with a rate faster than can be accounted for by the lability of the glycosyl bond. 4) Both GM 637 fibroblasts and Xeroderma pigmentosum fibroblasts (12-RO) (XP-12) have similar ability to remove ethyl products, except for O6-ethyl G which persists to a greater extent in XP12 cells. 5) The implications of the in vivo persistence of ethylated bases is discussed in regard to recent demonstrations that O2-EtT, O4-ET, O2-EtC and O6-EtG are all mutagenic.     

Difference in the action of ethyl and methyl methane sulfonates on DNA template activity for RNA synthesis in vitro.

RNA produced in vitro from alkylated T7 DNA has been characterized by polyacrylamide gel electrophoresis. Methylation of T7 DNA by methyl methane sulfonate reduces RNA chain length. In contrast, ethylation of T7 DNA by ethyl methane sulfonate, while reducing RNA synthesis to the same extent, does not alter chain length.