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

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 (>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 [γ-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 lacZα 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 (~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. Transformation of E. coli strain DL7 with the uniquely modified single-stranded genome resulted in ~0.5-1.0% of the progeny phage showing the G → T transversion mutation at the original position of G8−OH; no such mutations were observed from control genomes containing guanine in place of G8−OH. The vector containing G8−OH also transformed 50-90% as efficiently as the unmodified control, indicating that the adduct can be both weakly cytotoxic and mutagenic to the phage genome. © 1990, American Chemical Society. All rights reserved.