The alkaline hydrolysis of phosphotriesters in alkylated mammalian DNA.

Isolated DNA was alkylated with N-[14C]methyl-N-nitrosourea or N-[14C]ethyl-N-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 stability of methylated purines and of methylphosphotriesters in the DNA of V79 cells after treatment with N-methyl-N-nitrosourea.

V79-379A cells growing in suspension culture were treated with N-methyl-N-nitrosourea at concentrations of 0.6 and 1.2 mM. After incubation for periods from 1 to 48 h DNA was isolated from the cells and the concentrations of 7-methylguanine, O6-methylguanine, 3-methyladenine and methyl phosphotriesters were determined. After correction for dilution resulting from DNA synthesis during the incubation it was found that no loss of O6-methylguanine or methylphosphotriesters occurred; 7-methylguanine disappeared with a half-life of 22 h and 3-methyladenine was detectable only immediately after the initial treatment. The results show that these cells eliminate 7-methylguanine and 3-methyladenine from DNA by a repair process but are unable to excise or repair O6-methylguanine or methyl phosphotriesters.

Repair processes and the response of dividing and non-dividing cells to methyl methanesulphonate and dimethyl sulphate.

A method for producing a viable non-dividing population of Chinese hamster V79 cells in suspension is described and the characteristics of the population outlined. The stationary population is more sensitive to methylating agents than a similar but exponentially growing population, the increased sensitivity arising from the loss of the shoulder from the survival curve. The extent of reaction of the agent with cellular macromolecules is similar in both cases. The repair capabilities of the two populations was examined. Non-semiconservative DNA repair synthesis occurs whether the cells are in a growth or no-growth condition when insulted. Repair of single-strand breaks, which arise following methylation, also proceeds up to the size of the replicon. The relationship of this stationary population to other no-growth conditions and its utility as a model for carcinogenesis studies is discussed.

Biomethylation of deoxyribonucleic acid in cultured human tumour cells (HeLa). Methylated bases other than 5-methylcytosine not detected.

1. Incorporation of methyl groups from [methyl-(14)C]methionine into DNA of dividing HeLa cells was investigated, essentially by the procedures of Culp, et al. (1970). 2. Contrary to the report of the latter, but in agreement with other work on biomethylation of mammalian DNA, 5-methylcytosine was the sole methylated base detected. 3. Chromatographic separations of 3-methylcytosine from 5-methylcytosine and purines are discussed.