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 T4 and T7 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.
This work was supported in part by Grant CA 7175 from the National Cancer Institute, NIH, and Grant BC-2C from the American Cancer Society.
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