Site-specific frameshift mutagenesis by a propanodeoxyguanosine adduct positioned in the (CpG)4 hot-spot of Salmonella typhimurium hisD3052 carried on an M13 vector.
Malondialdehyde induces frameshift mutations in Salmonella typhimurium strain hisD3052. The ability of propanodeoxyguanosine (PdG), a structural analog of the major malondialdehyde-deoxyguanosine adduct, to induce site-specific frameshift mutations was tested in the (CpG)4 hot-spot of hisD3052 carried on an M13 vector (M13MB102). PdG was introduced at position 6248 of duplex M13MB102 by ligation of the oligonucleotide 5'-CGC(PdG)CGGCATG-3' into a heteroduplex containing an 11-nucleotide gap in the (-)-strand between the SphI and BssHII restriction sites and deoxyuridine in place of thymidine in the (+)-strand. Ligation proceeded with 70% efficiency, and closed circular duplex DNA molecules were isolated in 40% yield. The adducted genome was sensitive to cleavage by SphI but resistant to cleavage by BssHII. Transformation of Escherichia coli strain JM105 with adducted M13MB102 led to 25% reduced survival relative to unadducted M13MB102 and produced frameshift mutations in 2.5% of the progeny phage. All of the mutations were deletions, and 70% occurred by deletion of CpG. Unadducted genomes exhibited a 40-fold lower mutation frequency, and all the mutations were single-base deletions at the sites of ligation of the 11-mer. These results illustrate that PdG, a structural analog of the major malondialdehyde-deoxyguanosine adduct, induces frameshift mutations in M13MB102 and that single-stranded nicks are efficient premutagenic lesions in this recombinant bacteriophage.
Benamira
M
M
A. B. Hancock, Jr. Memorial Laboratory for Cancer Research, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Singh
U
U
Marnett
L J
LJ
D017104:Bacteriophage M13; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004249:DNA Damage; D004269:DNA, Bacterial / Q000235:genetics; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry; D016368:Frameshift Mutation; D005822:Genetic Vectors; D006639:Histidine; D009682:Magnetic Resonance Spectroscopy; D008315:Malondialdehyde / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D045706:Proprotein Convertase 2; D012486:Salmonella typhimurium / Q000235:genetics*; D012697:Serine Endopeptidases / Q000378:metabolism*
D004269:DNA, Bacterial; D009838:Oligodeoxyribonucleotides; C060934:1,N(2)-propanodeoxyguanosine; D006639:Histidine; D008315:Malondialdehyde; D012697:Serine Endopeptidases; D045706:Proprotein Convertase 2; D003849:Deoxyguanosine