Eighty percent of the single-strand DNA breaks induced by y-irradiation were prevented by the hydroxyl radical (-OH) scavenger dimethyl sulfoxide (Me2SO); CH4 was generated in the process as a product of the interaction of -OH and Me2SO. In contrast, Me SO completely blocked DNA nicking by an iron/ H202 system which produces -OH but smaller amounts of CH4 from Me2SO. Because Me2SO prevented DNA breaks from the more efficient iron/H202 system but only blocked 80% of irradiation-mediated nicking, the results sugest that -OH is responsible for 80% ofthe DNA single-strand breaks and the remaining 20% is due to interactions not involving OH.vents the formation of 80% of the single-strand breaks in DNA introduced by ionizing radiation in the presence of oxygen. During both of these processes, CH4, a product of the interaction of OH and Me2SO (8,10), is generated. The results indicate that at least 80% ofsingle-strand breaks introduced in DNA by ionizing radiation are due to an indirect effect and that 80% ofbreaks are probably generated by -OH; 20% ofbreaks are due to some process other than -OH, but our results do not indicate whether this process is direct or indirect.Lethal damage to cells exposed to ionizing radiation has been attributed mostly to effects on the structure of cellular DNA. For this reason the radiochemistry of DNA and its component parts has been studied extensively in the past 3 decades (for reviews, see refs. 1 and 2). Damage to DNA from ionizing radiation might occur directly ifenergy were transferred, without intermediates, to ionize or to excite components of the DNA. Damage might also develop indirectly if irradiation of water generated toxic products which then reacted with the DNA. One recent analysis ofthese processes based on theoretical considerations concluded that both direct and indirect damage to DNA occurs and that about 45% ofthe damaged nucleotides are derived from the direct interaction (3). This is important because radioprotective agents, which are believed to act by scavenging the toxic products such as -OH and other free radicals, are likely to interfere only with the indirect processes. Experiments with many such agents have found that about 70% of the single-strand breaks introduced into DNA by ionizing radiation can be eliminated, suggesting that only 30% of the breaks result from direct action of radiation of the DNA (4, 5). In these studies the frequency ofradiation-induced strand breaks was estimated from the sedimentation rate of damaged DNA on alkaline sucrose gradients. It is known that alkali-labile lesions in irradiated DNA can be converted into interruptions in the DNA phosphodiester backbone (6, 7). Measurements in alkali therefore overestimate the number ofbreaks. We have reinvestigated this question using procedures that assess the direct formation ofsingle-strand breaks more accurately. We also have studied in detail the radioprotective effect ofthe -OH scavenger dimethyl sulfoxide (Me2SO) (8-11). Animals (12) or cells (13) irradiated in the presence ...

Cells of Escherichia coli containing the plasmid F were 7-irradiated with various doses to introduce determined numbers of single-strand breaks in the F DNA. The cells were then incubated to permit repair of the breaks while DNA gyrase was inhibited with coumermycin to limit restoration of any relaxed supercoils. Repaired, covalently continuous F DNA was isolated and its superhelical density was measured by two different methods. Both indicated that a major part (50-60%) of the negative superhelical turn's were maintained in the repaired molecules, suggesting that the supercoils are restrained in vivo.In eukaryotic chromatin and chromosomes, the first order of DNA packaging seems to be attained via the nucleosome structure (for reviews, see refs. 1 and 2). The double-helical DNA in this particle is coiled through its interaction with the core histones into about 13/4 toroidal superhelical turns per core nucleosome (3, 4). Prokaryotic DNA has superhelical densities similar to those found in eukaryotes (5, 6), but thereis no clear evidence for restraint of the supercoils in structures analogous to the nucleosome. Histone-like proteins are found in prokaryotes (7,8), and their mode of interaction with DNA in prokaryotic chromosomes may have some similarity with nucleosomic histones (8, 9). Also, beaded structures having the appearance of nucleosomes have been seen in an electron microscope study of DNA from partially lysed bacteria (10).However, pure DNA can spontaneously form beaded structures in certain solvents (11), and electron microscopic evidence alone cannot be considered sufficient to establish the existence of the prokaryotic equivalent of a nucleosome.DNA gyrase seems to play a vital role in forming DNA supercoils in prokaryotic chromosomes. This enzyme can catalyze an ATP-driven reaction that has the topological effect of decreasing the linking number between the two continuous strands of a DNA double helix, thereby introducing negative superhelical turns into the DNA (12). When the DNA gyrase is inhibited by coumermycin (or oxolinic acid) in intact Escherichia coli cells, X DNA molecules, which are converted in vivo to the covalently circular form, attain only a small fraction of their normal superhelical density (13,14). This result indicates that the DNA gyrase activity is necessary for the formation (or the maintenance or both) of at least part of the DNA supercoils in prokaryotic cells. Thus, it seems possible that, in viwo, supercoils in prokaryotic DNA may not be stabilized by a nucleosome-like structure and that the Major reason for both the formation and maintenance (15) of DNA supercoils is the activity of DNA gyrase. Other alternatives include the possibility that DNA supercoils catalyzed by the DNA gyrase are subsequently restrained in a nucleosome-like structure by interactions involving molecules other than DNA gyrase (9). These two alternatives differ in that in the former case the double helix (17). After irradiation, the 32p-labeled cells kept at 0C were divided into several p...

Sturgeon muscle glyceraldehyde-3-phosphate de-hydrogenase has been isolated and purified to maximal activity. The purified enzyme contains four unusually reactive cysteine sulfhydryls per 145,000 daltons. This highly selective

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