DNA damage and its repair in transformable mouse fibroblasts treated with N-methyl-N'-nitro-N-nitrosoguanidine.

Abstract

Summary The DNA from asynchronous, oncogenically transformable fibroblasts of the C3H/10T1/2 mouse cell line was studied by sedimentation through alkaline sucrose gradients after the cells were lysed on the gradients. A band that contained 70 to 90% of the total cellular DNA was found to be free of lipids containing choline and proteins containing leucine, and did not aggregate with DNA from other sources. Single-stranded DNA from bacteriophages T 4 and T 7 sedimented linearly through the gradients, as did the 10T1/2 cellular DNA. The patterns of degradation in alkali of the DNA from the bacteriophages and from the 10T1/2 cells were very similar, suggesting that the 10T1/2 cellular DNA sediments as single strands. Breakdown of the DNA in cells treated with low doses (from 10 to 80% lethal dose) of N -methyl- N ′-nitro- N -nitrosoguanidine for 2 hr was easily detected and was dose dependent. Repair of this damage was not complete 48 hr after treatment. The molecular-weight distributions of the DNA were analyzed, and the number of single-strand breaks were estimated. From this, the extent of methylation of the DNA was calculated, by assuming chemical depurination to be the rate-limiting step for breakdown of the DNA, and the calculations agreed well with the extent of methylation determined experimentally.