The nature of single-strand breaks in DNA following treatment of L-cells with methylating agents

Abstract

DNA from untreated L-cells had a weight average molecular weight (Mw) of 5.7 ± 0.58·10 8 daltons as measured by sedimentation in an alkaline sucrose gradient. This value was reduced by one half after the cells were treated for 1 h with 8 μg/ml of N-methyl- N-nitrosourea (MNUA), 34 μg/ml of methyl methanesulfonate (MMS) or 0.16 μg/ml of N-methyl- N′-nitro- N-nitrosoguanidine (MNNG). That dose of MNUA produced 52 methylations per 5.7·10 8 daltons DNA. 20% of these were not purine derivatives and were assumed to contain some phosphotriesters. That dose of MMS (above) produced 290 methylations per 5.7·10 8 daltons DNA and about 14% of these were not purine derivatives. The rates of loss of methylated purines from DNA were 2.3% per hour for 7-methylguanine (7-MeG), 7.4% per hour for 3-methyladenine (3-MeA) and no detectable loss of O 6-methylguanine (O 6-MeG) over a 12 h period. Since phosphotriesters are alkali-labile the single-strand breaks probably arose from this structure and did not form within the cell. This conclusion is supported by the following considerations. MNUA was more effective than MMS at reducing the molecular weight of DNA, as measured in alkaline medium. The greater SN 1 character of MNUA would cause a greater formation of phosphotriesters than would MMS.