Alkylation of Phosphates and Stability of Phosphate Triesters in DNA

Abstract

A method is presented to measure the alkylation of phosphates in DNA after a treatment with an alkylating agent. Using this method, we have shown that phosphate alkylation represents 15O/, of total alkylation when DNA is alkylated with ethyl methanesulfonate and only l o / , of total alkylation when DNA is alkylated with methyl methanesulfonate. Experiments are also presented which show that phosphate triesters resulting from the alkylation of DNA by ethyl methanesulfonate are very stable, most of them remaining intact after heating a t 100 "C for 90 min at pH 7.0.Lett et al. [I] have stated that alkylating agents react primarily with phosphates in DNA and have presented spectrophotometrical evidence for a transalkylation process a t room temperature from triester phosphates to bases in the macromolecule. Epoxides were an exception because they reacted directly with the bases, while methyl methanesulfonate had an intermediate behaviour, alkylating initially both phosphates and bases. On the other hand, the transalkylation process did not occur a t room temperature with ethyl methanesulfonate which, according to these authors, gave onlyethylated DNAphosphates. Lawley and Brookes [Z], using labelled alkylating agents, found that nearly all alkyl groups bound to DNA were liberated by formic acid hydrolysis a t 175 "C as alkylated bases, especially alkylated purines ; they concluded that the alkylation of the phosphates was negligible. As ethyl methanesulfonate was not an exception in their work, it was suggested by Alexader [3] that the formation of ethylated bases was an artifact occurring during acid hydrolysis a t high temperature.Some work has been done using oligonucleotides. Holy and Scheit [4,5], in contrast to Brimacombe et al. [6], have observed phosphate alkylation with diazomethane. Also Rhaese and Freese [7] observed phosphate alkylation with ethyl and methyl methanesulfonates.Because of these contradictions concerning the fundamental question of DNA phosphate alkylation, it is not surprising that little work has been done on the assay of phosphate alkylation in DNA. To achieve this goal, Strauss and Hill [S] used labelled alkylating Abbreviations. PPO, 2,5-diphenyloxazole; dimethyl-POPOP, 1,4-bis-2(4-methyl-5-oxazolyl)-benzene.Enzymes. Alkaline phosphatase (EC 3.1.3.1); phosphodiesterase (EC 3.1.4.1). agents and measured the radioactivity bound to DNA which resisted heating the aqueous soIution to 100°C a t neutral pH. One might except such a treatment to lead to a complete loss of the alkylated purines and, as the alkylation of pyrimidines is very low, it seems that most of the residual radioactivity ought to be due to alkyl groups bound to phosphates. The aim of the first part of our work was to check this assumption using ethyl and methyl methanesulfonates and to develop a reliable method for the estimation of DNA phosphate alkylation.The second part of our work deals with the stability of the phosphate triesters formed by alkylation of DNA. If it seems well established that phosphate alkylation i...

The Interaction of Chromatin with Alkylating Agents

“…Alkylated guanines are known to undergo both cleavage [33] and depurination, even at neutral pH ; this rate increases at lower pH [34,35]. Phosphotriester formation has also been suggested to account for up to 20 % of DNA methylation but is considered to be relatively stable to hydrolysis [36,39]. To test for loss of label, chromatin labeled with the reagent was incubated for several hours under our standard conditions of reaction (Table 2).…”

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

“…Esterification of the phosphate residues in DNA by these alkylating agents produces triesters which are stable under conditions ofmild hydrolysis (Bannon & Verly, 1972;Lawley et al, 1973), but in RNA these products are unstable (Brown & Todd, 1955). Their presence in poly(A) after incubation with ethyl methanesulphonate was indicated by chain breakage, but this was not observed in similar experiments with methyl methanesulphonate (Ludlum, 1969).…”

“…Although it is over 20 years since it was first proposed that phosphotriesters might be present in DNA after reaction with alkylating agents (Elmore et al, 1948), uncertainty surrounded this issue until direct evidence was provided for the existence of alkyl phosphates in DNA, by Bannon &Verly (1972). They found that after treatment with methyl methanesulphonate or ethyl methanesulphonate in vitro alkylation of the phosphate residues accounted for 1 and 15% of the total reaction respectively.…”

“…the N-methyl-or N-ethyl-nitrosoureas and N-methyl-N'-nitro-N-nitrosoguanidine, which give much higher yields of these triesters and 06_ alkylguanine. Ethyl methanesulphonate also falls into this latter category on the basis of these reactions (Bannon & Verly, 1972;Orr, 1973).…”

Characterization of the catalytic activity of U2 and U6 snRNAs

“…Phosphotriesters have long been known to occur in both DNA and RNA as a result of exposure to certain toxins and chemicals (e.g., see Bannon and Verly 1972;Singer et al 1975). Formation of a phosphotriester similar to the type proposed to exist in RNA X was observed to occur as a side reaction during chemical synthesis of DNA (Weimann and Khorana 1962).…”