Induced mutations in M13mp2 phage DNA exposed to N-nitrosopyrrolidine with UVA irradiation

It is known that N-nitrosopyrrolidine (NPYR), a carcinogen in rodents, is metabolically activated by microsomal cytochrome P450 to form an alpha-hydroxylated derivative, which induces mutations. The mutations have been demonstrated by use of Salmonella typhimurium and Escherichia coli. We discovered directly acting mutagenicity of NPYR plus ultraviolet light-A (UVA) in bacteria and phage. With an O(6)-alkyltransferase-deficient strain of S.typhimurium, the NPYR plus UVA treatment gave greater mutation frequencies compared to those found with the parent strain. We identified the structure of the direct-acting mutagen as N-nitroso-1-phosphonooxypyrrolidine, and analyzed the spectrum of mutations induced in the DNA of M13mp2 phage. The basepair substitutions GC to TA and GC to AT appear to occur predominantly. Several hotspots were observed. In the conditions where SOS response was induced in the host E.coli, greater varieties of mutations were observed in phage DNA compared to those without the SOS response induction. These results suggest that alkylations of DNA occur by the photoactivated NPYR. The roles of the produced damage to the mutations are discussed.