Modification of DNA bases in mammalian chromatin in aqueous suspension by ionizing radiation generated free radicals was investigated. Argon, air, N2O, and N2O/O2 were used for saturation of the aqueous system in order to provide different radical environments. Radiation doses ranging from 20 to 200 Gy (J.kg-1) were used. Thirteen products resulting from radical interactions with pyrimidines and purines in chromatin were identified and quantitated by using the technique of gas chromatography/mass spectrometry with selected-ion monitoring after acidic hydrolysis and trimethylsilylation of chromatin. The methodology used permitted analysis of the modified bases directly in chromatin without the necessity of isolation of DNA from chromatin first. The results indicate that the radical environment provided by the presence of different gases in the system had a substantial effect on the types of products and their quantities. Some products were produced only in the presence of oxygen, whereas other products were detected only in the absence of oxygen. Products produced under all four gaseous conditions were also observed. Generally, the presence of oxygen in the system increased the yields of the products with the exception of formamidopyrimidines. Superoxide radical formed in the presence of air, and to a lesser extent in the presence of N2O/O2, had no effect on product formation. The presence of oxygen dramatically increased the yields of 8-hydroxypurines, whereas the yields of formamidopyrimidines were not affected by oxygen, although these products result from respective oxidation and reduction of the same hydroxyl-adduct radicals of purines. The yields of the products were much lower than those observed previously with DNA.
Genetic and biochemical studies indicate that the evolutionarily conserved Swi/Snf complex acts at a subset of genes to help transcriptional activators function on chromatin templates. The mechanism by which this complex is targeted to specific chromosomal loci remains unknown. We show that Swi/Snf is required for expression of the yeast histone HTA1-HTB1 locus because of the role of Hir1p and Hir2p corepressors in negatively regulating transcription. Snf5p, Snf2p/Swi2p, and Swi3p, three components of the yeast Swi/Snf complex, coimmunoprecipitate with each Hir protein, and Snf5p is maximally associated with the HTA1-HTB1 promoter when the Hir-based repression system is intact and the Swi/Snf complex is functional. The data support a role for the Hir repressors in the gene-specific targeting of Swi/Snf.
We investigated the capacity of the hyperthermophile Pyrococcus furiosus for DNA repair by measuring survival at high levels of 60 Co ␥-irradiation. The P. furiosus 2-Mb chromosome was fragmented into pieces ranging from 500 kb to shorter than 30 kb at a dose of 2,500 Gy and was fully restored upon incubation at 95°C. We suggest that recombination repair could be an extremely active repair mechanism in P. furiosus and that it might be an important determinant of survival of hyperthermophiles at high temperatures.Pyrococcus furiosus is a hyperthermophile growing optimally at 100°C. It is a member of the Archaea, a group of prokaryotes which have many molecular features in common with modern eukaryotes, and which are thought by many to be ancestral life forms (25). The exceptional degree of tolerance and resistance of hyperthermophiles to high temperatures must include adaptations that affect all levels of the cellular machinery, including the enzymes that are involved in maintaining the integrity and stability of genomic DNA. Kopylov et al. (14) reported that a closely related member of the Archaea, Thermococcus stetteri, is 12 times more resistant to ␥-irradiation than Escherichia coli but 2 times more sensitive than the bacterium Deinococcus radiodurans. In addition, Peak et al. (20) have shown that at 100°C, the DNA of P. furiosus is 20 times more resistant to thermal breakage in vivo than the DNA from the mesophile E. coli. Thermophilic members of the Archaea have histone proteins (24) known to give partial protection of plasmid DNA to fast neutrons and ␥-photons (12) and to prevent thermal denaturation of DNA (5). However, the protective effect of DNA-binding proteins cannot account for the extreme resistance of these microorganisms to heat and ␥-irradiation (12,14,20), suggesting the presence of very active mechanisms for DNA repair.Among the lesions induced by ionizing radiation in cellular DNA, double-strand breaks (DSBs) are the least efficiently repaired, and their frequency is correlated with cell death (9). Indeed, E. coli and most other organisms cannot survive if more than two or three DSBs are introduced per chromosome, independently of their physiological state (15,21). DSBs are noninformative lesions that affect the DNA double helix at the same site, eliminating intact template for repair and precluding any excision repair processes. However, D. radiodurans has been found to be extremely resistant to ionizing radiation (19). This organism can repair more than 100 DSBs per chromosome, induced by ionizing radiation, without loss of viability (19). Its extreme resistance to ␥-irradiation is attributed to possible adaptation to desiccation (16). RecA-dependent recombinational repair and single-strand annealing are the two mechanisms proposed to account for repair of ionizing-radiation damage in D. radiodurans (16,18).In contrast, there is currently very little information about DNA repair systems in hyperthermophiles, and what information there is consists mainly of the characterization of DNA rep...
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