The stability of methyl and ethyl phosphotriesters in DNA in vivo

C57BL male mice were injected with N-methyl-N-nitrosourea (MNUA) or N-ethyl-N-nitrosourea (ENUA) and the concentration of alkyl phosphotriesters in the DNA of lung, liver, brain, kidney, spleen and thymus determined from the extent of degradation induced in isolated DNA by alkali. The same total dose of reagent was given either as a single injection (i.p.) or by weekly injections carried out over 5-20 weeks. Methyl phosphotriesters induced in liver, lung and kidney by the single injection were lost with a half-life of about 7 days, in brain the loss was more rapid, t1/2 = 2-3 days. During the multiple injections the observed t1/2 was 16 days. Ethyl phosphotriesters formed in the DNA of lung, liver, kidney and brain were much more stable than the methyl derivatives, t1/2 = 10-15 weeks. Phosphotriesters formed in the DNA of spleen and thymus disappeared very quickly after the single injection presumably as a result of dilution due to DNA replication. No accumulation of phosphotriesters occurred in the DNA of these tissues during the multiple injections. The general pattern of the results suggests that phosphotriesters are not excised by cellular repair systems.     

Removal of minor methylation products 7-methyl adenine and 3-methylguanine from DNA ofescherichia coli treated with dimethyl sulphate

Persistence of methylpurines in DNA methylated in vitro and in vivo inEscherichia coli WP2 cells, by dimethyl sulphate (DMS) was studied, with particular reference to the minor products 7-methyladenine and 3-methyl-guanine, not previously investigated in this respect, but known to be removed from DNA in vitro by spontaneous hydrolysis at neutral pH.The half-life of 7-methyladenine in vivo was relatively short (2.6 ± 0.2 h) but not significantly shorter than in vitro at pH 7.2, 37°C. The half-life of 3-methylguanine was 3.6 ± 0.3 h in vivo, markedly shorter than in vitro, where its stability was somewhat greater than that of 7-methylguanine. Enzymatic excision of 3-methylguanine was therefore indicated to occur inE. coli.Previous findings that 7-methylguanine is probably not enzymatically excised from DNA in vivo, whereas 3-methyladenine is rapidly removed, were confirmed, and additional support for the concept of enzymatic removal of 3-methyladenine was obtained by showing extensive inhibition of its removal from cells treated with iodoacetamide prior to methylation.It is suggested that methylations of adenine or guanine in DNA at N-3 constitute blocks to template activity of DNA and stimulate a “repair” response of enzymatic removal of 3-methylpurines. Possible valence bond structures for 3-methylpurine residues in DNA are discussed, leading to the suggestion that ionized forms with positively charged amino groups may be the most effective blocks to template activity.     

The alkaline hydrolysis of phosphotriesters in alkylated mammalian DNA

Isolated DNA was alkylated with $\text{N-[}{}^{14}\text{C]methyl}\text{-N-}\text{nitrosourea}$ or $\text{N-[}{}^{14}\text{C]ethyl}\text{-N-}\text{nitrosourea}$. Sedimentation analysis of the alkylated DNA before and after alkaline hydrolysis was used to determine the number of single-strand breaks introduced by hydrolysis of the triesters. Vacuum distillation from alkylated DNA solutions before and after alkaline hydrolysis was used to determine the numbers of triesters hydrolysing to the alcohol.     

The effect of ionising radiation and chemical methylation upon the activity and accuracy of E. COLI DNA polymerase I

The activity of $\text{E. coli}$ DNA polymerase I decreases on treatment with γ-rays, methylnitrosourea or dimethyl sulphate. In the case of the first two agents the decrease in activity is accompanied by a decrease in the accuracy of the enzyme in an $\text{in vitro}$ assay. There is no detectable change in the ratio of DNA polymerase activity to 3′→5′ exonuclease activity on treatment.     

Some chemical aspects of dose-response relationships in alkylation mutagenesis

Alkylation of DNA can lead to induction of potentially miscoding groups (promutagenic) or potentially template-inactivating groups (lethal). The proportions of these are found to vary with the chemical nature of the alkylating agent. Agents of low Swain and Scott s factor (or those tending to Ingold's S_Ni type) react relatively more extensively at O-atom sites in DNA, and yield relatively more of the miscoding O₆-alkylguanine residues. Phosphotriester formation is also relatively more extensive with S_Ni agents.Inactivation of DNA can result from depurinations, strand breakage, and cross-linkage.Both promutagenic and lethal lesions are subject to repair; 3 principal enzymatic systems appear to exist; one for excision and repair of cross-links or aralkyl groups resembles the uvr system; others for repair of single-strand breaks parallel repair of X-ray-induced breaks (exr, rec systems); another, less well defined at present, recognizes certain methylated bases, and depurinated sites (probably Goldthwait's endonuclease II).These factors can be shown to influence dose-response in alkylation mutagenesis. This, broadly, can be classified as linear with the promutagenic group-inducing or directly miscoding agents, and is independent of cytotoxicity; whereas with other agents non-linear response parallels the occurrence of “shouldered” survival curves, and reflects mutation induction by “repairs errors”.Additionally, alkylation of cellular constituents other than DNA, e.g. repair enzymes, may influence dose response, and will again depend on chemical reactivity of the agent.     

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.     

Inactivation of bacteriophage λ and λ DNA by nitrogen mustard

Bacteriophage λ and λ DNA were treated with alkylating agents. The survival of phage was assayed by infectivity and that of DNA by infectivity of phage particles assembled from the DNA in vitro. Phage λ were more sensitive to nitrogen mustard (Cl(CH₂)₂NMe(CH₂)₂Cl; HN2) than was λ DNA. The inactivation of λ DNA was biphasic; the second component of the inactivation was sensitive to mutations allelic for recA, polA and uvrB. This behaviour was not shown by pBR322 plasmid DNA treated with HN2 nor by λ DNA treated with monofunctional alkylating agents (or HN2 if the second alkylation reaction was stopped by addition of a mercaptan). From Arrhenius plots, the activation energy for the reactions with DNA and intact phage were found to be different. The activation energy for the inactivation of intact phage was the same as that (measured independently) for the predominant reaction (or class of reactions) in which HN2 cross-links DNA to protein in λ particles. From these data we conclude that the inactivation of λ by HN2 is due, primarily, to DNA-protein cross-linking. The implications for the mode of action of DNA-reactive bifunctional anti-viral and cytotoxic compounds are discussed.     

Multiple Unfolding Events during Native Folding of the Tetrahymena Group I Ribozyme

Despite the ubiquitous nature of misfolded intermediates in RNA folding, little is known about their physical properties or the folding transitions that allow them to continue folding productively. Folding of the Tetrahymena group I ribozyme includes sequential accumulation of two intermediates, termed I_trap and misfolded (M). Here, we probe the structure and folding transition of I_trap and compare them to those of M. Hydroxyl radical and dimethyl sulfate footprinting show that both I_trap and M are extensively structured and crudely resemble the native RNA. However, regions of the core P3-P8 domain are more exposed to solvent in I_trap than in M. I_trap rearranges to continue folding nearly 1000-fold faster than M, and urea accelerates folding of I_trap much less than M. Thus, the rate-limiting transition from I_trap requires a smaller increase in exposed surface. Mutations that disrupt peripheral tertiary contacts give large and nearly uniform increases in re-folding of M, whereas the same mutations give at most modest increases in folding from I_trap. Intriguingly, mutations within the peripheral element P5abc give 5- to 10-fold accelerations in escape from I_trap, whereas ablation of P13, which lies on the opposite surface in the native structure, near the P3-P8 domain, has no effect. Thus, the unfolding required from I_trap appears to be local, whereas the unfolding of M appears to be global. Further, the modest effects from several mutations suggest that there are multiple pathways for escape from I_trap and that escape is aided by loosening nearby native structural constraints, presumably to facilitate local movements of nucleotides or segments that have not formed native contacts. Overall, these and prior results suggest a model in which the global architecture and peripheral interactions of the RNA are achieved relatively early in folding. Multiple folding and re-folding events occur on the predominant pathway to the native state, with increasing native core interactions and cooperativity as folding progresses.     

Mutagenicity of methyl-, ethyl-, propyl- and butylnitrosourea towards Escherichia coli WP2 strains with varying DNA repair capabilities.

Methyl- (MNUA), ethyl- (ENUA), propyl- (PNUA) and butylnitrosourea (BNUA) have been tested for toxicity and mutation in a liquid suspension assay towards Escherichia coli WP2 and some of its repair deficient derivatives. A comparison of survival rates after nitrosourea exposure between WP2 and WP2 uvrA showed no difference between the two strains but a consistent difference in potency between the various nitrosoureas studied. Toxicity increased in the order MNUA less than PNUA less than ENUA less than BNUA. ENUA and PNUA induced a greater number of trp+ revertants in both strains than did MNUA and BNUA, particularly at low survival rates. None of these differences in biological potency could be accounted for by differences in rates of hydrolysis. ENUA, PNUA and BNUA were non-mutagenic towards WP2 lexA, WP2 recA and WP2 uvrA lexA, whereas MNUA did induce mutations. Ethyl methanesulphonate (EMS) was able to mutate WP2 lexA. These results are discussed in the light of current theories regarding the mechanism of action of these compounds.     

Studies on the stability of trialkyl phosphates and di-(2'deoxythymidine) phosphotriesters in alkaline and neutral solution. A model study for hydrolysis of phosphotriesters in DNA and on the influence of a beta hydroxyethyl ester group

Various trialkyl phosphates were investigated as model compounds for DNA-phosphotriesters for their stability in neutral or alkaline conditions. The results show that phosphotriesters were highly stable even at strongly alkaline pH, with the exception of diethyl 2-hydroxyethyl phosphate (DHP). The extreme instability of the latter was found to be due to the 2-hydroxy function. In accordance with earlier interpretations the 2-hydroxyethyl group is proposed to participate in the formation of a highly reactive dioxaphospholane ring intermediate which decays rapidly by hydrolysis. Alkylation of 3'- and 5'-deoxythymidine monophosphates with methyl- or hydroxyethylnitrosourea (MNU, HENU) results in practically exclusive phosphate alkylation. In analogy with the model phosphotriesters, di(2'-deoxythymidine) phosphotriesters generated after reaction with MNU or HENU showed extreme dependence of their stabilities on the nature of the alkyl group transferred to phosphate. Whereas the methyl phosphotriester was highly stable, the corresponding hydroxyethyl analogue showed half lives of decay of less than 1 min (pH 12.5), 27 min (pH 9.1) and 60 min (pH 7). Thus the introduction of a 2-hydroxyethyl function into phosphate strongly decreases the stability of the phosphate link of DNA, resulting in DNA single strand breaks, in analogy to RNA phosphotriesters which have been found earlier to be highly unstable because of the presence of the ribose 2'-OH-group.     

Methylation of DNA by 3H-14C-methyl-labelled N-methyl-N-nitrosourea--evidence for transfer of the intact methyl group

The following products have been isolated from methyl-labelled N-methyl-N-nitrosourea (MNUA) and DNA after reaction at pH 8: 1-, 3-, 7-methyladenines, 3-methyldeoxycytidine, 3-, 7- and O⁶-methylguanines, 3- and O⁴-methylthymidines. Comparison of C³H₃ and ¹⁴CH₃ labelling showed that the ratio ${}^3\text{H}{}^{14}\text{C}$ in the products was the same and equal to that in the original reagents, in accord with the concept that the methyl group is transferred intact, and not via diazomethane. In some cases, most notably with O⁶-methyldeoxyguanosine, the ³H-labelled products were found to elute from Dowex 50 (NH₄⁺ form) slightly ahead of ¹⁴C-labelled or unlabelled products.     

DNA ligase activity in crude extracts of fibroblasts and lymphocytes

DNA ligase activity was determined in crude cell extracts using a new assay which measures the retention of double stranded circular phage λ DNA on nitrocellulose filters, and allows accurate determinations of the enzyme activity with cell concentration corresponding to 0.1 μg of proteins. Using this assay, we show that the DNA ligase activity varies greatly among mammalian cell lines. The higher activity is found in actively growing fibroblasts where it is stimulated by dimethyl sulfate pretreatment of the cells, whereas the low activity measured in resting lymphocytes is not modified by dimethyl sulfate. The DNA ligase activity correlates with the cells sensitivity towards ionizing radiations.     

Cytotoxicity of monofunctional alkylating agents methyl methanesulfonate and methyl-N′-nitro-N-nitrosoguanidine have different mechanisms of toxicity for 10T1/2 cells

N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MMS) are directly active alkylating agents that methylate cellular macromolecules by SN₁ and SN₂ mechanisms, respectively. These two chemicals produce similar types of alkylation products in DNA and a similar level of total alkylations on a molar basis, but strikingly different proportions of alkylations of ring oxygen atoms of purines and pyrimidines. Because of this attribute, they have been used in combination to attempt to determine which types of alkylation products are responsible for mutation, transformation, and toxicity. Studies have suggested that the mutation rates produced by these and similar chemicals in cells surviving toxicity correlate well with the number of methyl adducts at the O⁶ position of guanine, but that cytotoxicity (reduced colony-forming efficiency) does not correlate with any single adduct or with the total level of alkylation of DNA. In this study we have investigated the cytotoxic mechanisms of MNNG and MMS in synchronized 10T1/2 cells, using colony-forming ability as a measure of toxicity. Both MNNG and MMS cause dose-dependent reduction in the ability of 10T1/2 cells to produce colonies of more than 50 cells after 2 weeks in culture. MNNG is about 100-fold more toxic than MMS on a molar basis. As indicated by the inability of cells to exclude trypan blue, MMS kills a fraction of the population of treated 10T1/2 cells after a 30-min exposure; the fraction of cells that excludes trypan blue is correlated with dose of MMS and with colony-forming efficiency. Neither the fraction of cells that is permeable to trypan blue nor the relative colony-forming efficiency is affected by the phase of the cycle when 10T1/2 cells are treated with MMS. Furthermore, MMS toxicity for 10T1/2 cells is not potentiated by caffeine, MMS treatment does not delay progress of S phase, and cells that survive acute membrane toxicity complete the cell cycle without significant delay. In contrast, MNNG treatment produces toxicity that is maximal when 10T1/2 cells are exposed during the S phase and the effect of potentiated by caffeine. MNNG treatment delays DNA replication and this delay is reversed by caffeine. In sharp contrast to 10T1/2 cells treated with MMS. MNNG-treated cells are not made permeable to trypan blue, but are blocked in their ability to proliferate. These observations indicate that MNNG and MMS kill 10T1/2 cells by drastically different mechanisms, MNNG producing toxicity mainly by preventing chromosome replication and MMS producing toxicity mainly by damaging cell membranes.