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)
A single 7,8-dihydro-8-oxoguanine (G8-OXO; 8-hydroxyguanine) adduct in the lacZ alpha gene of bacteriophage M13 DNA induces a targeted G-->T transversion after replication in Escherichia coli (Biochemistry, 29, 7024-7031 (1990)). This mutation is thought to be due to the facile formation during DNA synthesis of a G8-OXO.base pair, where G8-OXO is in the syn conformation about the deoxyglycosyl bond. A related modified purine, 7,8-dihydro-8-oxoadenine (A8-OXO; 8-hydroxyadenine), is an abundant product found in irradiated and oxidized DNAs. Similar to G8-OXO, as a mononucleoside A8-OXO assumes the syn conformation. This work has assessed the relative mutagenicities of A8-OXO and G8-OXO in the same experimental system. A deoxypentanucleotide containing A8-OXO [d(GCT-A8-OXOG)] was synthesized. After 5'-phosphorylation with [gamma-32P] ATP, the oligomer was ligated into a duplex M13mp19-derived genome at a unique NheI restriction site. Genomes containing either A8-OXO (at position 6275, [+] strand) or G8-OXO (position 6276) were denatured with heat and introduced into E.coli DL7 cells. Analysis of phage DNA from mutant plaques obtained by plating immediately after transformation (infective centers assay) revealed that G8-OXO induced G-->T transversions at an apparent frequency of approximately 0.3%. The frequency and spectrum of mutations observed in DNA sequences derived from 172 mutant plaques arising from the A8-OXO-modified DNA were almost indistiguishable from those generated from transfection of an adenine-containing control genome. We conclude that A8-OXO is at least an order of magnitude less mutagenic than G8-OXO in E.coli cells with normal DNA repair capabilities.
The mutagenic and genotoxic properties of 1,N6-ethenoadenine (epsilon Ade), 3,N4-ethenocytosine (epsilon Cyt), and 4-amino-5-(imidazol-2-yl)imidazole (beta) were investigated in vivo. The former two modified bases are known DNA adducts formed by the human carcinogen vinyl chloride; beta is formed by pyrimidine ring-opening of epsilon Ade. Chemically synthesized deoxyhexanucleotides containing epsilon Ade and beta, d[GCT-(epsilon A)GC], and d[GCT(beta)GC], respectively, were described previously [Biochemistry (1987) 26, 5626-5635]. epsilon Cyt was inserted into an oligonucleotide, d[GCTAG(epsilon C)], by a mild enzymatic synthetic procedure, which avoided exposure of the base to alkaline conditions. 3,N4-Etheno-2'-deoxycytidine 3',5'-bisphosphate coupled with reasonable efficiency (30-40%) to the 3'-nucleoside of an acceptor pentamer, d(GCTAG), in a reaction catalyzed by T4 RNA ligase in the presence of ATP. Each of the three modified hexanucleotides and an unmodified control were inserted into a six-base gap positioned at a known site in the genome of bacteriophage M13-NheI. A nick was placed in the DNA strand opposite that containing the single DNA lesions, enabling the formation of singly adducted single-stranded genomes by denaturation. After transfection of the adducted phage DNAs into Escherichia coli, each of the adducts was found to be genotoxic. The most toxic lesion was beta, which reduced survival of the genome by 97%. epsilon Cyt and epsilon Ade reduced survival by 90% and 65%, respectively. An examination of the surviving phage populations revealed that each of the three adducts was mutagenic. The least mutagenic lesion was epsilon Ade (0.1% of the survivors were mutant), which showed primarily A-->G transitions.(ABSTRACT TRUNCATED AT 250 WORDS)
Herein, we report a novel intramolecular
ring-closing reaction
of biaryl thioethers that give access to highly functionalized dibenzothiophene
sulfonium salts under mild conditions. The resulting precursors react
regioselectively with [18F]fluoride to give [18F]fluoroarenes in predictable radiochemical yields.
The strategy expands the available radiochemical space and provides
superior labeling efficiency for clinically relevant PET tracers.
Human herpesvirus 6A and 6B, alongside some other herpesviruses, have the striking capacity to integrate into telomeres, the terminal repeated regions of chromosomes. The chromosomally integrated forms, ciHHV-6A and ciHHV-6B, are proposed to be a state of latency and it has been shown that they can both be inherited if integration occurs in the germ line. The first step in full viral reactivation must be the release of the integrated viral genome from the telomere and here we propose various models of this release involving transcription of the viral genome, replication fork collapse, and t-circle mediated release. In this review, we also discuss the relationship between ciHHV-6 and the telomere carrying the insertion, particularly how the presence and subsequent partial or complete release of the ciHHV-6 genome may affect telomere dynamics and the risk of disease.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.