Spectrum of mutations induced by methyl and ethyl methanesulfonate at the hprt locus of normal and tag expressing Chinese hamster fibroblasts

Abstract

This work describes the isolation and characterization of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) induced 6-thioguanine-resistant mutants in normal and Escherichia coli tag gene expressing Chinese hamster fibroblast, RJKO, cells. It was previously shown that increased removal of 3-alkylated adenine, effected by 3-methyladenine DNA glycosylase I (Tag), reduces the frequencies of hprt mutations induced by alkylating agents which produce mostly N-alkylation (MMS and EMS) to half the normal rate. In order to identify which type of mutation is suppressed by increased 3-alkyladenine repair we have determined the DNA base sequence changes of the hprt cDNA in 61 independent MMS- and EMS-induced mutant clones. For both cell types and irrespective of the agent used, the majority of mutations were GC to AT transitions originating in the non-transcribed strand. Only 6/55 base substitutions occurred at AT base pairs: five AT to GC transitions and one AT to CG transversion. Six mutations were found to be deletions. These results indicate that 3-alkylated adenines in DNA are not directly premutagenic. The fact that the mutation frequency is reduced by increased 3-alkyladenine removal might be explained by postulating the existence in mammalian cells of an SOS-like response turned on by cytotoxic lesions like 3-alkyladenine, or, alternatively, that increased removal of 3-alkyladenine increases the number of single-strand breaks in DNA, which stalls DNA replication and allows a prolonged time for DNA repair by the alkyltransferase.