Site-directed mutagenesis in single cells: transitions produced by DNA carrying a single O6-alkylguanine residue

Using a single burst assay based on a Poisson Distribution, I have determined the mutant virus frequency in single spheroplasts transfected with phi X174 form I' DNA carrying an O6-methyl-, ethyl-, n-propyl- or n-butylguanine residue at position 2401 of the minus strand. One set of experiments was performed with spheroplasts derived from Escherichia coli AB1157, which has normal DNA-repair systems. Of the cells examined after transfection with DNA carrying a methylguanine moiety, 30% produced mutant virus and 12% contained only mutants; with ethylguanine, 55% of the cells had mutants and 41% produced only mutants; with butylguanine, 6% of the cells had mutants and 3% contained only mutants; with propylguanine no mutants were detected in the 33 cells examined. In similar experiments carried out with spheroplasts defective in excision repair (E. coli AB1157 uvrA6) the percentage of cells producing mutant phage after transfection with DNA carrying an O6-butylguanine residue increased from 6 to 21%, and the percentage of cells producing only mutants increased from 3 to 8%; with DNA carrying an O6-methylguanine moiety, the percentage of cells producing mutants decreased from 30 to 6% and the percentage of cells producing only mutants fell from 12% to 0. In order for an individual uvrA cell to produce exclusively mutant phage from a single O6-alkylguanine residue some form of selection must occur during replication because one strand of the transfecting DNA is wild-type, and excision repair, which could lead to a homoduplex with the transition in both strands, is defective in these cells. This selection must also occur in cells with normal DNA repair. The first event in the selection process is critical; if replication of the alkyl-DNA occurs first and if a mutation is produced, then there is a significant probability that the cell will produce only mutant virus regardless of whether or not repair occurs in subsequent events, but the frequency one observes is influenced strongly by the status of the repair systems in the cell.