Mechanistic studies of ionizing radiation and oxidative mutagenesis: genetic effects of a single 8-hydroxyguanine (7-hydro-8-oxoguanine) residue inserted at a unique site in a viral genome

• Published in: Biochemistry (Easton) Vol. 29; no. 30; pp. 7024 - 7032
• Main Authors: Wood, Michael L, Dizdaroglu, Miral, Gajewski, Ewa, Essigmann, John M
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 31-07-1990
• Subjects: 560120 - Radiation Effects on Biochemicals, Cells, & Tissue Culture; ADDUCTS; AMINES; AROMATICS; AZAARENES; BACTERIA; BACTERIOPHAGES; Base Sequence; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; Biological and medical sciences; BIOLOGICAL EFFECTS; CHROMATOGRAPHY; Coliphages - genetics; DAYS LIVING RADIOISOTOPES; DNA ADDUCTS; ENZYMES; ESCHERICHIA COLI; Fundamental and applied biological sciences. Psychology; GAS CHROMATOGRAPHY; GENE MUTATIONS; Genes, Viral - drug effects; Genes, Viral - radiation effects; GENETIC EFFECTS; Genetic Vectors; GUANINE; Guanine - analogs & derivatives; HETEROCYCLIC COMPOUNDS; HYDROXY COMPOUNDS; ION EXCHANGE CHROMATOGRAPHY; IONIZING RADIATIONS; ISOTOPES; LIGASES; LIGHT NUCLEI; MASS SPECTROSCOPY; MICROORGANISMS; Molecular and cellular biology; Molecular genetics; MUTAGENESIS; Mutagenesis, Insertional; Mutagenesis, Site-Directed; Mutagenesis. Repair; MUTATIONS; NUCLEI; ODD-ODD NUCLEI; Oligodeoxyribonucleotides - chemical synthesis; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; Oxidation-Reduction; PARASITES; PHOSPHORUS 32; PHOSPHORUS ISOTOPES; PHOTOCHEMICAL OXIDANTS; PURINES; RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT; RADIATIONS; RADIOINDUCTION; RADIOISOTOPES; SEPARATION PROCESSES; SPECTRA; SPECTROSCOPY; ULTRAVIOLET SPECTRA; VIRUSES; Purification; Enzyme; Escherichia coli; Site directed mutagenesis; Cytotoxicity; Phage M13; Virus; Transfection; DNA; Nucleotide; Bacteria; Oligonucleotide; Mutation; Polyribonucleotide synthetase (ATP); Enterobacteriaceae; Phage; Phage T4
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00482a011

T4 RNA ligase was used to construct a deoxypentanucleotide containing a single 8-hydroxyguanine (7-hydro-8-oxoguanine; G8-OH) residue, which is one of the putatively mutagenic DNA adducts produced by oxidants and ionizing radiation. The pentamer d(GCTAG8-OH)p was prepared by the ligation of a chemically synthesized acceptor molecule, d(GCTA), to an adducted donor, 8-hydroxy-2'-deoxyguanosine 5',3'-bisphosphate. The acceptor was efficiently converted to the reaction product (greater than 95%), and the final product yield was 50%. Following 3'-dephosphorylation, the pentamer was characterized by UV spectroscopy, by high-pressure liquid chromatography, and by gas chromatography-mass spectrometry of the nucleosides released by enzymatic hydrolysis. Both d(GCTAG8-OH) and an unmodified control were 5'-phosphorylated by using [gamma -32P]ATP and incorporated covalently by DNA ligase into a five-base gap at a unique NheI restriction site in the otherwise duplex genome of an M13mp19 derivative. The ligation product contained G8-OH at the 3' residue of an in-frame amber codon (5'-TAG-3') (genome position 6276) of the phage lacZ alpha gene. The adduct was part of a nonsense codon in a unique restriction site in order to facilitate the identification and selection of mutants generated by the replication of the modified genome in Escherichia coli. Both control and adducted pentamers ligated into the genome at 50% of the maximum theoretical efficiency, and nearly all (approximately 90%) of the site-specifically adducted products possessed pentanucleotides that were covalently linked at both 5' and 3' termini. The G8-OH lesion in the NheI site inhibited the cleavage of the site by a 200-fold excess of NheI.