Single-strand Breaks in Gamma-irradiated ØX174 DNA Induced by Exposure to Alkali

Achey, Phillip M.; Billen, Daniel; Beltranena, H.P.

doi:10.1080/09553007114551411

Cited by 21 publications

(5 citation statements)

References 11 publications

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“…19,20 Some indirect damage, including "alkali-labile damage," leads to breaks in the DNA at elevated temperatures or alkaline conditions. [21][22][23][24] Alkali-labile damage, which can be distinguished by comparing the mobility of fragments in alkali and neutral agarose gels, 25 could have some nonrandom selectivity. 26 As the detailed mechanism of damage to DNA differs for these different damaging agents, it is not clear whether dosage responses would scale with length.…”

Section: Introductionmentioning

confidence: 99%

Tests of the Single-hit DNA Damage Model

Spangler

Goddard

Spangler

et al. 2009

Journal of Molecular Biology

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Section: Introductionmentioning

confidence: 99%

Tests of the Single-hit DNA Damage Model

Spangler

Goddard

Spangler

et al. 2009

Journal of Molecular Biology

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“…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.…”

mentioning

confidence: 99%

Dimethyl sulfoxide prevents DNA nicking mediated by ionizing radiation or iron/hydrogen peroxide-generated hydroxyl radical.

Repine¹,

Pfenninger²,

Talmage³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

168

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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 ...

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“…This evidence is indirect and is based on the fact that NaBFU or NH2OH added at the end of the preincubation period prevented the appearance of three-fourths of the alkali-catalyzed spontaneous breaks and that the number of single strand breaks observed in alkali is greater than those observed in formamide. The production of alkali-labile bonds after irradiation has been observed by a number of groups Kessler et al, 1971;Achey et al, 1971;Ward and Kuo, 1973). The increase in bond breakage due to alkali varied from 1.4to 3-fold in these reports.…”

Section: Discussionmentioning

confidence: 76%

Endonuclease II of Escherichia coli. Degradation of γ-irradiated DNA

Kirtikar¹,

Slaughter²,

Goldthwait³

1975

Biochemistry

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Irradiation of DNA in a nitrogen atmosphere with 60Co gamma-radiation produces at least two types of damage. The first type leads to single strand breaks in the DNA observed after exposure to alkali. This type of alkali-labile bond will be designated a spontaneous break. The second type of damage to DNA is an alteration which makes the DNA susceptible to phosphodiester bond hydrolysis by a 1600-fold purified preparation of endonuclease II of Escherichia coli and is designated an enzyme-sensitive site. This site is not alkali-labile. After irradiation, preincubation of the DNA either for days at 0 degrees or for 4 hr at 37 degrees increases both the spontaneous breaks and the enzyme sensitive sites. There is a greater increase of spontaneous breaks when the preincubation is in O2 compared to N2. The increase of enzyme sensitive sites due to the preincubation is not altered significantly by O2. The increase of spontaneous breaks during the preincubation is almost completely prevented by addition of either NaBH4 or NH2OH after the irradiation. The treatment can be before or after the preincubation. This effect indicates that these breaks are due to alkali-labile bonds possibly produced by depurination or depyrimidination reactions. That the spontaneous breaks are due primarily to alkali-labile bonds is supported by an experiment in which formamide gradients were used. Neither NaBH4 nor NH2OH has any effect on the enzyme sensitive sites. Addition of beta-mercaptoethanol (0.5 M) at the start of the preincubation prevents in part the appearance of both spontaneous breaks and enzyme-sensitive sites. It has no effect when added at the end of the preincubation. Catalase added before the preincubation has no effect on either type of damage. It is postulated that the spontaneous breaks occur because purine or pyrimidine radicals are formed (possibly hydroxyl radicals) which can then interact with oxygen to produce unstable intermediates. The intermediates then undergo either depurination or depyrimidination. The subsequent alkali catalyzed beta-elimination reaction of depurinated or depyriminidinated DNA is prevented by NaBH4 or NH2OH. An alternative hypothesis would involve damage to the sugar rather than to bases. The enzyme-sensitive sites represent another form of base damage which is not oxygen dependent. The chemical nature of either form of primary damage is not known.

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Single-strand Breaks in Gamma-irradiated ØX174 DNA Induced by Exposure to Alkali

Cited by 21 publications

References 11 publications

Tests of the Single-hit DNA Damage Model

Tests of the Single-hit DNA Damage Model

Dimethyl sulfoxide prevents DNA nicking mediated by ionizing radiation or iron/hydrogen peroxide-generated hydroxyl radical.

Endonuclease II of Escherichia coli. Degradation of γ-irradiated DNA

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