title: Relative contribution of cytosine deamination and error-prone replication to the induction of propanodeoxyguanosine-->deoxyadenosine mutations in Escherichia coli.
abstract: The role of cytosine deamination as a possible mechanism for PdG-->A transitions induced by propanodeoxyguanosine (PdG) was investigated by site-specific mutagenesis techniques. PdG was placed at position 6256 in the (-)-strand of M13MB102 by ligating the oligodeoxynucleotide 5'-GGT(PdG)TCCG-3' into a gapped-duplex derivative of the vector. Unmodified and PdG-modified M13MB102 genomes containing either uracil or thymine in the (+)-strand were transformed into Escherichia coli strains differing in their ability to excise uracil bases from DNA. After replication of the site specifically modified M13MB102, base-pair substitutions were detected by in situ hybridization using [32P]-labeled probes containing each of the possible mismatched bases opposite position 6256 in the (+)-strand. The ratio of PdG-->A and PdG-->T was unchanged in strains defective in the repair of uracil residues, which suggests that uracil is not an intermediate in the generation of PdG-->A mutations. Similar results were obtained when PdG-M13MB102 was incubated for 14 days prior to transformation in an attempt to increase the extent of deamination. As a control experiment to test the sensitivity of the assay to detect deaminations opposite PdG, uracil-containing M13MB102 with a PdG.T mismatch at position 6256 was transformed into E. coli JM105. Hybridization analysis indicated that approximately 80% of the phage plaques generated after genome replication contained T in the (+)-strand at position 6256. Thus, any deamination of cytosine to uracil would have been easily detected. Adducted and unadducted genomes were also transformed into E. coli LM114 or LM113, which carry a mutant umuD or umuC gene, respectively. Significant and comparable reductions in PdG-->A and PdG-->T were observed, suggesting that both mutations require the active participation of the UmuD and the UmuC proteins in the replication complex. The results of our experiments suggest that the PdG-->A mutations induced by PdG are not caused by cytosine deamination, but arise coincident with PdG-->T mutations during replication of the PdG-containing genomes. Also, the uracil-containing (+)-strand does not appear to be degraded, as is commonly assumed in site-specific mutagenesis experiments, and serves as a template for DNA synthesis when replication of the (-)-strand is blocked by an adduct such as PdG.
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
authors.forename: S P
authors.initials: SP
authors.lastname: Fink
authors.forename: G R
authors.initials: GR
authors.lastname: Reddy
authors.forename: L J
authors.initials: LJ
authors.lastname: Marnett
mesh_terms: D017104:Bacteriophage M13 / Q000235:genetics; D001483:Base Sequence; D003596:Cytosine / Q000378:metabolism* / Q000633:toxicity*; D004260:DNA Repair; D004261:DNA Replication; D003641:Deamination; D003839:Deoxyadenosines / Q000235:genetics*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011336:Probability; D014170:Transformation, Genetic
chemical_list: D003839:Deoxyadenosines; C060934:1,N(2)-propanodeoxyguanosine; D003596:Cytosine; D003849:Deoxyguanosine