Comparison of mutagenesis induced in single- and double-stranded M13 viral DNA by treatment with N-hydroxy-2-aminofluorene

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Abstract
The specificity of mutagenesis in single-stranded and its complementary double-standard DNA of the bacteriophage M13mp8 induced by N-hydroxy-2-aminofluorene (N-OH-AF) was analyzed after transfection into its bacterial host Escherichia coli, strain JM103. In this foward mutation assay, randomly modified DNA with increasing levels of aminofluorene (AF) guanine adducts was transfected into component host JM103 cells with or without prior induction of SOS functions in the host cells. These cells were then screened for mutants of the marker enzyme, .beta.-galactosidease, on a selective medium. In this assay, the mutation frequency was increased up to 10-fold in host cells with induced SOS functions as compared to the control host cells. Transfection of AF-substituted single-stranded DNA gave a 2.5-fold higher mutation frequency as compared to the double-stranded form at similar levels of AF modification and plaque-forming efficiency. DNA sequence analysis of the mutants showed that AF-modified single- and double-stranded DNA induced base substitutions (52-55%), large deletions (i.e. > 300 bp, 25-30%) and frameshifts (16-18%). The mutation sites for 73 base substitutions and frameshifts examined within a limited DNA sequenceof 120 bases (6280-6400) were different in single- and double-stranded DNAs. A possible ''hotspot'' for base substitutions within one of the two GGCG sequences has also been identified in single-stranded but not double-stranded DNA.

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