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The DNA of coliphages T4 and T1 was labelled with 125I-iododeoxyuridine. 125I decay is known to cause severe molecular damage via vacancy cascades (the Auger effect). We have compared the induction of both single- and double-strand breaks (SSBs and DSBs) in 125I-labelled T4 DNA stored at - 196 degrees C during decay, either as intact phage or as free DNA. These comparative experiments indicate that, in addition to one DSB which apparently results directly from the Auger effect, each decay in an intact phage also give rise to an additional 0-05 DSBs, as well as 1-6 SSBs, as a result of ionizing radiation absorbed in the same phage particle where the decay occurs. An examination of T4-killing by 125I decay reveals a two-phase survival curve, whose initial slope corresponds to a lethal efficency per 125I decay of 0-95 +/- 0-05, which is considerably higher than values previously determined. The results for phage T4, and of a more limited comparison of 125I suicide and DNA damage in phage T1, support the hypothesis that the vacancy cascades which accompany each 125I decay in DNA result in a double-strand break at the decay site and that each such break is a lethal event.

The time scale for rejoining of radiation-induced deoxyribonucleic acid (DNA) single-strand breaks was measured in the presence and absence of oxygen. The involvement of DNA polymerase I in this repair process was studied. Formation and rejoining of DNA strand breaks were measured in lambda DNA infecting lysogenic pol(+) and polA1 strains of Escherichia coli irradiated by 4 MeV electrons under identical conditions. Irradiation and transfer to alkaline detergent could be completed in less than 180 ms. The initial yields of DNA strand breaks were identical in pol(+) and polA1 host cells and four- to fivefold higher in the presence of oxygen than in nitrogen anoxia. Evidence for the existence of a very fast repair process, independent of DNA polymerase I, was not found, since no rejoining of radiation-induced DNA strand breaks was observed during incubation from 45 ms to 3 s. In pol(+) host cells most of the strand breaks produced in the presence of oxygen were rejoined within the first 30 to 40 s of incubation, whereas no rejoining could be detected within the same period of time in anoxic cells. Since no rejoining of broken lambda DNA molecules was observed in polA1 host cells, it is concluded that the synthetase activity of DNA polymerase I is involved in the rejoining of DNA breaks induced by radiation in the presence of oxygen.

A method was developed in which E. coli cells were irradiated with four MeV electrons and transferred to alkaline detergent within a fraction of a second. This technique minimizes the amount of repair of radiation damage before analysis without the necessity of using physical or chemical treatments to inhibit repair and alter the physiological condition of the cells. The yield of DNA strans breaks formed in covalent circular superhelical lambda DNA molecules superinfecting E. coli lysogens was about 4-fold greater when the cells were irradiated in oxygen than when they were irradiated under nitrogen anoxia. The same yields were obtained in phosphate buffer at 3 degrees and 22 degrees as well as in growth medium at 37 degrees, and the yields were not altered by the polA1 mutation. When E. coli lysogenic cells superinfected with lambda were irradiated with doses sufficient to introduce at least seven breaks in the phage DNA, the chromosomal DNA and the superinfecting phage DNA sedimented similarly in alkaline sucrose gradients, indicating that both DNAs were broken to a similar extent during irradiation. However, the yield of breaks calculated for chromosomal DNA in similar experiments was greater than the yield calculated from the first break introduced into covalent circular lambda DNA molecules. These apparently contradictory results are explicable either if the initial break in a superhelical molecule occurs with an efficiency different from that for subsequent breaks, or if the pulsed electron radiation produces a high proportion of double-strand breaks.

An endonuclease acting on DNA exposed to ultraviolet light or gamma-rays has been extensively purified from calf thymus. The enzyme has a pH optimum at pH 7.0-7.5, acts with equal efficiency in the presence of EDTA or divalent cations (Mg-2+ or Ca-2+), is inhibited by NaCl and tRNA and is inactivated by incubation at 50 degrees C. Its molecular weight, determined by Sephadex chromatography or sodium dodecylsulfate gel electrophoresis, is approx. 30 000. The enzyme catalyzes the formation of breaks with 5'-phosphate termini in double-stranded DNA irradiated with ultraviolet or gamma-rays. It does not act on unirradiated DNA or denatured DNA. Since in all these properties the enzymatic activity on ultraviolet- and gamma-irradiated DNA behaved similarly and since the two activities cochromatographed in all systems used during purification, we conclude that they are associated with the same protein. The site of action of the enzyme in ultraviolet-irradiated DNA is a photoproduct other than pyrimidine dimers. Such a photoproduct can also be induced by irradiation of the DNA in vivo, i.e. within the cells.

The protective effect of proflavine on gamma-irradiated bacteriophage lambda and its isolated DNA was investigated under conditions of predominantly indirect or direct effects. In both conditions addition of small amounts of the dye during irradiation of phage or DNA was shown to enhance their biological activity. Protection against indirect effects results probably from extensive scavenging of radioinduced water radicals within the medium. On the other hand the results obtained at minus 196 degrees C, with irradiated DNA-proflavine complexes, imply the existence of a long-range transfer of the primary radiation damage of DNA towards the intercalated molecules of proflavine. A mechanism for the protective effect of proflavine against the direct effect of ionizing radiation on biologically active DNA is suggested.

The radiation chemical reactivity of thymine residues has been compared for single-stranded phi chi 174 DNA free in solution and in the intact bacteriophage, as well as for native E. coli DNA in solution and inside the cell. The gamma-ray-induced release of [3H]H2O from thymine-methyl[3H] was measured to assess the reactivity of the thymine methyl group. Formation of ring-saturated products of the 5,6-dihydroxy-dihydrothymine type (see article) was determined by an alkali-acid degradation assay. It was observed that the formation of that the formation of [3H]H2O was suppressed by a factor of 9 in intact phi chi 174 bacteriophage compared to phi chi 174 DNA, whereas the formation of (see article) was only slightly suppressed in the phage. For E. coli cells the formation of [3H]H29 was reduced 790-fold compared with free E. coli DNA, and (see article) formation was reduced 140-fold. The extents of survival after gamma-irradiation of phi chi 174 phage plaque-forming ability and E. coli colony-forming ability were also determined. Under the conditions used in these experiments, a linear relationship was observed between the loss of biological activity and the formation of thymine damage for both phi chi 174 phage and E. coli cells.

Bacteriophage T7 was irradiated in a protecting medium under nitrogen and oxygen with 60Co gamma-rays. Double-strand breaks were measured by sucrose-gradient sedimentation and by boundary-sedimentation analysis. Both methods showed that the presence of oxygen during irradiation enhanced the production of double-strand breaks. This is at variance with a recent report which suggests that boundary-sedimentation analysis does not show an effect of oxygen. The discrepancy must be ascribed to differences in interpretation of the sedimentation data.

Since superoxide radicals are involved in many metabolically important as well as in some other, detrimental cellular processes, the reactivity of gamma-ray-induced superoxide radicals and its dismutation products singlet molecular oxygen and hydrogen peroxide with DNA have been studied. Superoxide dismutase which removes superoxide radicals and inhibits the formation of singlet oxygen in the solution protects the biologically active replicative form of DNA (from bacteriophage theta X174) against inactivation by ionizing radiation. Catalase which removes hydrogen peroxide also protects the DNA. Attempts with various chemical sources of singlet oxygen to determine whether this species inactivates DNA did not give an unequivocal answer. It is concluded from the presented experiments that a combination of the protonated form of the superoxide radical (HO-2) and H2O2 do inactivate DNA.

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.

An enconuclease activity that reacts with x-irradiated DNA is present in extracts of E. coli. By using centrifugal methods to monitor the conversion of the supercoiled, circular double-stranded DNA for phage phi-x-174 (replicative form) or PM2 to the relaxed circular form it was possible to quantitate the rate of radiation induced endonuclease-sensitive sites in the DNA. For every single-strand break induced by x-rays under aerobic irradiation conditions, there is approximately one induced site sensitive to this endonuclease activity. Under irradiation conditions (addition OF Potassium iodide) that dramatically reduce rates of single-strand breaks and "alkalilabile" lesions, the number of endonuclease-sensitive sites relative to single-strand breaks increase approximatley 4-fold. This nuclease is present in several strains of E. coli B and K12, including mutants deficient in DNA polymerase I, recombination gene products (rec mutants), ultraviolet light incision enzyme (uvr A mutant), and endonuclease II. It is suggested that this endonuclease may be involved in an excision repair process for damages incurred in DNA by ionizing radiation.

Alkali-labile sites produced by gamma-irradiation in dry RF-DNA of bacteriophage phi chi 174 were found to be lethal lesions with respect to the plaque-forming ability.

The yield of single-strand breaks in lambda DNA within lysogenic host bacteria was measured after exposure to 4-MeV electrons (50 msec) and rapid transfer (45 msec) to alkaline detergent. In nitrogen anoxia the yield was 1.2 X 10(-12) DNA single-strand breaks per rad per dalton, and under full oxygenation the yield increased to 5 X 10(-12) breaks per rad per dalton. A search for the presence of fast repair of strand breaks operating within a fraction of a second. Strand breaks produced in the persence of oxygen were repaired in 30-40 sec, while breaks produced under anoxia were rejoined even slower. A functional product from the po[A] gene was needed for the rejoining of the broken molecules. Intermediate levels of DNA strand breakage seen at low concentrations of oxygen are dependent on the concentration of cellular sulfhydryl compounds, suggesting that in strand breakage oxygen donors compete for reactions with radiation-induced transients in the DNA. Intercomparisons of data on radiation-induced lethality of cells and single-strand breaks in episomal DNA allow the distinction between two classes of radiation-induced radicals, R-1 and R-2, with different chemical properties; R-1 reacts readily with oxygen and N-oxyls under formation of potentially lethal products. The reactivity of oxygen in this reaction is 30-40 times higher than that of TMPN. R-2 reacts 16 times more readily that R-1 with oxygen under formation of single-strand breaks in the DNA. R-2 does not react with N-oxyls.

14C-2-thymidine was incorporated into the DNA of E. coli B/r and of coliphage T4. The labelled organisms were stored for several years at -196 degrees C. Both were periodically assayed for loss of viability, and the coliphage also for the appearance of double-strand breaks (DSBs) in DNA. E. coli B/r exhibited a survival curve with a substantial initial shoulder, extrapolation number 5-2 +/- 2-3, and a final exponential portion corresponding to a lethal efficiency per 14C decay per 2-5 X 10(9) daltons of DNA, of 0-009 +/- 0-002. For coliphage T4, our best estimate for the lethal efficiency per 14C decay is 0-03 +/- 0-04, and that for the DNA breakage efficiency is -0-002 +/- 0-004. The large standard errors result from the very small number of 14C decays occurring in each phage. These results suggest that 14C decay in the DNA of micro-organisms does not cause DSBs but does cause potentially lethal damage to the thymine bases in which decay occurs, and that wild-type E. coli can repair a large number of such DNA lesions.

The cell-free extract from blue-green alga Anacystis nidulans contains enzymatic activities which repair in vitro transforming DNA of bacteriophage T4 damaged by UV light or X-rays. The repair effect of the extract was observed with double-stranded irradiated DNA but not with denatured irradiated DNA. The level of restoration of the transforming activity depends on the protein concentration in the reaction mixture and on the dose of irradiation. A fraction of DNA lesions induced by X-rays is repaired by a NAD-dependent polynucleotide ligase present in the extract. The repair of UV-induced lesions is the most efficient in the presence of magnesium ions, NAD, ATP and the four deoxynucleoside triphosphates. The results indicate that the repair of UV-irradiated DNA is performed with the participation of DNA polymerase and polynucleotide ligase which function in the cell-free extract of the algae on the background of a low deoxyribonuclease activity.

The supertwisted, double-stranded, replicative intermediate of øX174 DNA (RFI) has been used to determine whether one of the two strands of the double helix is uniquely sensitive to induction of single-strand breaks by ionizing radiation. This could result from a particularly sensitive base sequence or a transfer of energy to a specific location of the DNA molecule. The results indicate that both strands of the double helix are equally broken, even though their base compositions are significantly different. If there are "hot spots" in the strands, then they are present in equal amounts in each strand.

The behaviour of the modifications due to ultraviolet (UV) radiation on denatured phagic DNA has been investigated by looking at the changes of density in CsCl gradient, after irradiation with increasing doses of monochromatic light. The spectral range studied is 2300-2900 A, with a wavelength resolution Deltalambda = +/-20 A. The observed effect is a gradual shift toward higher densities as the UV absorbed dose increases. The experimental results show an exponential law and the analysis of the action spectra indicates that thymine is the main factor responsible for the observed effect. An evaluation of the quantum yield for the thymine-dimers formation gives a constant value of (18 +/- 3) x 10(-3) quanta(-1).