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 lacZ alpha region, by carrying out in vitro limited extension of primed phage DNA. We 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 mutations took place at those positions where N4-aminocytosine residues were originally present.