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

Abstract

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.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulated human leukocytes cause activating mutations in the K-ras proto-oncogene

“…OH-modi®ed DNA bases, including 8 hydroxydeoxyguanosine (Kuchino et al, 1987;Shibutani et al, 1991;Wood et al, 1990;Moriya et al, 1991;Cheng et al, 1992;Moriya, 1993), cytosine glycol (Hayes et al, 1988), thymine glycol (Basu et al, 1989), and 8 hydroxydeoxyadenosine (Guschlbauer et al, 1991), to mispair with each of the four DNA bases. The spectrum of mutations observed in our .…”

Effects of base excision repair proteins on mutagenesis by 8-oxo-7,8-dihydroguanine (8-hydroxyguanine) paired with cytosine and adenine

“…In addition to dCTP, dATP is incorporated opposite 8-oxo-Gua in template DNA by the action of DNA polymerases, resulting in an 8-oxo-Gua:A mispair [6]. Thus, the formation of 8-oxo-Gua:C in DNA leads to G:C → T:A transversions in cells [7][8][9][10][11][12][13][14][15]. The incorporation of 8-oxo-dGTP opposite A is catalyzed by a variety of DNA polymerases, and the 8-oxo-Gua:A pairs thus formed induce A:T → C:G transversions [8,[16][17][18].…”

Translocation of human ribosomal protein S3 to sites of DNA damage is dependant on ERK-mediated phosphorylation following genotoxic stress

“…Among the numerous DNA lesions formed under conditions of oxidative stress, 7, 8-dihydro-8-oxoguanine (8-oxoG) is notable as it is mutagenic if not repaired [1][2][3][4] where its persistence in the genome can lead to a number of pathological states. The primary means to remove 8-oxoG from DNA is via the action of N-glycosylases that participate in the base excision repair (BER) pathway.…”