Analysis of phage M13mp2 mutants produced from transfection of phage DNA having N⁴-aminocytosines at defined sequence positions

N⁴-Aminocytidine is mutagenic in various organisms. In the cell, this cytidine analog is metabolized into N⁴-aminodeoxycytidine 5′-triphosphate, which will then be incorporated into DNA and mutation will result during the replication of the DNA. To prove that the N⁴-aminocytosine residue in DNA is indeed the site of mutagenesis, we prepared a series of phage M13mp2 DNA samples that bear N⁴-aminocytosine residues at a few defined positions in the lacZα region, by carrying out in vitro limited extension of primed phage DNA. Wh then transfected the DNAs to Escherichia coli and examined the progeny phages for the forward mutations. The M13mp2 DNAs bearing N⁴-aminocytosines produced mutant phages at high frequencies. Furthermore, DNA sequencing of the resulting mutants demonstrated that both AT-to-GC and GC-to-AT mutation took place at those position where N⁴-aminocytosine residues were originally present.