Quantitative aspects of the repair of alkylated DNA in cultured mammalian cells: II. Non-semiconservative DNA synthesis (‘repair synthesis’) in HeLa and Chinese hamster cells following treatment with alkylating agents

Abstract

Non-semiconservative DNA synthesis (‘repair synthesis’) ∗ was followed in hamster and HeLa cells after treatment with various concentrations of N-methyl- and N-ethyl- N′-nitro- N-nitrosoguanidine, N-methyl- N-nitrosourea, methyl and ethyl methanesulphonate and mustard gas. ‘Repair synthesis’ following treatment with any of the methylating agents was directly proportional to the amount of overall DNA methylation despite considerable differences in the survival relative to a given amount of alkylation registered by the two cell lines. At higher concentrations ‘repair synthesis’ was either inhibited by other cellular reactions or the repair mechanism became overtaxed. The extent of ‘repair synthesis’ after methylation, ethylation and mustard gas alkylation was compared quantitatively, and was again directly proportional to the overall level of DNA alkylation rather than to extents of the reactions which produced similar lethal effects. Repair replication continued at an approximately linear rate for up to 9 h following methylation of hamster cells with MNNG, and could be detected for up to 12 h subsequent to mustard gas alkylation of HeLa cells. Loss of labelled methyl groups from methylated DNA following alkylation of hamster cells with [ 3H]MNNG was determined during the course of 9 h. The similarity in the kinetics of the loss of alkyl groups and of ‘repair synthesis’ was regarded as further evidence that these two processes are directly related. The occurrence of ‘repair synthesis’ in both strands of DNA which had been shown to be equally alkylated was regarded as supporting evidence that ‘repair synthesis’ was related to the initial alkylation of DNA and was not a non-specific response to cellular alkylation. Approx. 100 nucleotides were built into DNA by ‘repair synthesis’ for every methyl group eliminated. Substitution of BUdR for thymidine in DNA did not inhibit repair replication. ‘Repair synthesis’ following methylation of HeLa cells by MMS was not inhibited by hydroxyurea. All mammalian cell types, including primary embryo and tumour lines, so far examined, exhibit ‘repair synthesis’ subsequent upon alkylation. Mixtures of 5-bromo-2′-deoxyuridine and [ 3H]thymidine or [ 3H]deoxycytidine or [ 3H]cytidine but not 5-bromo-2′-deoxyuridine and labelled purines were found to be equivalent to [ 3H]BUdR for the detection of ‘repair synthesis’ in mammalian cells.