BIOLOGICAL PROPERTIES OF IMIDAZOLE RING-OPENED N7-METHYLGUANINE IN M13MP18-PHAGE DNA

Guanine residues methylated at the N-7 position (7-MeGua) are susceptible to cleavage of the imidazole ring yielding 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7-MeGua). The presence of Fapy-7-MeGua in DNA template causes stops in DNA synthesis in vitro by E.coli DNA polymerase I. The biological consequences of Fapy-7-MeGua lesions for survival and mutagenesis were investigated using single-stranded M13mp18 phage DNA. Fapy-7-MeGua lesions were generated in vitro in phage DNA by dimethylsulfate (DMS) methylation and subsequent ring opening of 7-MeGua by treatment with NaOH (DMS-base). The presence of Fapy-7-MeGua residues in M13 phage DNA correlated with a significant decrease in transfection efficiency and an increase in mutation frequency in the lacZ gene, when transfected into SOS-induced JM105 E.coli cells. Sequencing analysis revealed unexpectedly, that mutation rate at guanine sites was only slightly increased, suggesting that Fapy-7-MeGua was not responsible for the overall increase in the mutagenic frequency of DMS-base treated DNA. In contrast, mutation frequency at adenine sites yielding A --> G transitions was the most frequent event, 60-fold increased over DMS induced mutations. These results show that treatment with alkali of methylated single-stranded DNA generates a mutagenic adenine derivative, which mispairs with cytosine in SOS induced bacteria. The results also imply that the Fapy-7-MeGua in E.coli cells is primarily a lethal lesion.