Characterization of clones displaying enhanced resistance to methylating agents in O6-methyl-guanine-DNA methyltransferase-proficient CHO cells

Abstract

DNA from untreated L-cells had a weight average molecular weight (Mw) of 5.7 ± 0.58·108 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 5.7·108 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 5.7·108 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 O6-methylguanine (O6-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 SN1 character of MNUA would cause a greater formation of phosphotriesters than would MMS.