Site-specific mutagenesis by a propanodeoxyguanosine adduct carried on an M13 genome.
The spectrum of mutations induced upon in vivo replication of an M13 genome containing a site-specifically located propanodeoxyguanosine (PdG) adduct was determined. PdG was used as a model for the major deoxyguanosine adduct produced on reaction of DNA with the endogenous genotoxin malondialdehyde. PdG was introduced at position 6256 of M13MB102 by ligating the oligodeoxynucleotide 5'-GGT(PdG)TCCG-3' into an 8-base gap in the (-)-strand of duplex M13MB102. Replication of the adducted strand was maximized by incorporation of uracil into the unadducted (+)-strand. Following replication of dG-containing and PdG-containing M13MB102 genomes in Escherichia coli JM105, frameshift mutations were detected as phenotypic changes in the lacZ alpha marker gene. Base pair substitutions were detected by differential hybridization using 32P-labeled 13-mers bearing different bases opposite position 6256. Neither frameshift nor base pair substitution mutations were detected following replication of PdG-adducted genomes in non-SOS-induced JM105. However, PdG-->T transversions and PdG-->A transitions were detected following transformation of PdG-adducted M13MB102 into SOS-induced JM105. Both types of mutations were detected at comparable frequencies, and the total mutation frequency was approximately 2%. The results indicate that PdG is an efficient premutagenic lesion in E. coli strains in which the SOS response is induced.
Burcham
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PC
A. B. Hancock, Jr. Memorial Laboratory for Cancer Research, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Marnett
L J
LJ
D017104:Bacteriophage M13 / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D018736:DNA Adducts; D004249:DNA Damage; D017931:DNA Primers; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000235:genetics; D016679:Genome, Viral; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009153:Mutagens; D009693:Nucleic Acid Hybridization
D018736:DNA Adducts; D017931:DNA Primers; D009153:Mutagens; C060934:1,N(2)-propanodeoxyguanosine; D003849:Deoxyguanosine