We have examined the time course over a period of days of repair of chromosomal single-strand breaks (SSB) induced by a single dose of JANUS fission-spectrum neutrons in the DNA of human P3 epithelial teratocarcinoma cells. When the cells are allowed a period of repair incubation the breaks are totally sealed by 7 hours. But then following these initial repair the DNA is dismantled as evidenced by the reappearance of SSBs. This secondary breakage is almost as extensive as that caused by the original neutron exposure, with a maximum at 16-18 hours. Finally, the DNA is rejoined, regaining its original size by 40 hours after irradiation. The secondary repair phenomenon may have an editing function, or it many represent the processing of residual damage left unrepaired during the initial rejoining of the backbone breaks.

The ability of scavengers of hydroxyl radical (OH radical) to modulate the photosensitized relaxation (induction of the first single-strand break) of supercoiled plasmid DNA with UVA photoactivated 4'-aminomethyl4,5',8-trimethylpsoralen was examined by comparing the dose reduction factor (DRF: the ratio of fluence required to induce the same degree of relaxation in the absence to the presence of OH radical scavengers). The addition of mannitol, azide, acetate, or formate at concentrations inversely proportional to the value of the rate constants for the scavenging of OH radicals partially attenuated the supercoiled DNA relaxation. The degrees of protection afforded by the four scavengers in the presence of AMT photoactivated by either 334 nm or 365 nm monochromatic photons were similar, giving an average DRF of about 0.25 in all cases. Given the diverse chemical nature of the scavengers and their wide range of concentrations utilized, these findings are evidence for the involvement of a Type I photosensitization in the induction of DNA single-strand breaks by photoactivated AMT.

Action spectra (365–520nm) for the formation of DNA single‐strand breaks (SSB) and slowly developing alkali‐labile sites (SDALS) in human teratocarcinoma P3 cells in culture were determined. Induction of SDALS results from the absorption of blue‐ and green‐light photons. The spectrum has a broad peak that is maximal between 400 nm to 500 nm and declines sharply above and below these wavelength regions. Negligible yields of SDALS were produced by photons at wavelengths of 365 nm or shorter and at 520 nm or longer, whereas for SSB, the action increases with shorter wavelength throughout the whole spectral range studied. The configuration of the SDALS action spectrum suggests that the primary chromophore, and therefore possibly the photosensitizer, is a mixture of porphyrin and flavin residues.

Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at 100 degrees C. It is not conceivable that these organisms could survive with genomic DNA that was subject to thermal destruction, yet the mechanisms protecting the genomes of this and other hyperthermophiles against such destruction are obscure. We have determined the effect of elevated temperatures up to 110 degrees C on the molecular weight of DNA in intact P. furiosus cells, compared with the effect of elevated temperatures on DNA in the mesothermophilic bacterium Escherichia coli. At 100 degrees C, DNA in P. furiosus cells is about 20 times more resistant to thermal breakage than that in E. coli cells, and six times fewer breaks were found in P. furiosus DNA after exposure to 110 degrees C for 30 min than in E. coli DNA at 95 degrees C. Our hypothesis for this remarkable stability of DNA in a hyperthermophile is that this hyperthermophile possesses DNA-binding proteins that protect against hydrolytic damage, as well as other endogenous protective mechanisms and DNA repair enzyme systems.

High enolase activity, as measured by the conversion of 2-phosphoglycerate to phosphoenolpyruvate, was found in the cytoplasm of Pyrococcus furiosus (an anaerobic, hyperthermophilic archaeon that grows optimally at 100 degrees C). In this organism, the enzyme probably functions in a sugar fermentation pathway. The enzyme was purified to homogeneity. It had a temperature optimum of > 90 degrees C and a pH optimum of 8.1. The enzyme was extremely thermostable with a time for 50% inactivation at 100 degrees C of 40 min. In contrast, an enolase from yeast was totally inactivated in 1 min at 88 degrees C. Both the P. furiosus and yeast enzymes required a metal ion for activity, but whereas the yeast enzyme has an absolute requirement for Mg2+, the P. furiosus enolase was equally active in the presence of Mn2+. Both enzymes were competitively inhibited by citrate. P. furiosus enolase, as for mesophilic enolases, probably has a homodimeric structure with subunit M(r) greater than 45,000. A highly conserved sequence of eight amino acids in the N-terminal region was found in enolases from P. furiosus and a wide range of other organisms including bacteria, yeast, birds, and mammals. Substantial differences in the thermal properties of the hyperthermophilic enzyme compared with that from less extreme thermophiles and mesophiles might be due to a substantially enhanced composition of hydrophobic amino acids.

High enolase activity, as measured by the conversion of 2-phosphoglycerate to phosphoenolphyruvate, was found in the cytoplasm of Pyrococcus (an anaerobic, hyperthermophilic archaeon that grows optimally at 100{degree}C). In this organism, the enzyme probably functions in a sugar fermentation pathway. The enzyme was purified to homogeneity. It had a temperature optimum of >90 {degree}C, and a pH optimum of 8.1. The enzyme was extremely thermostable with a half time for inactivation at 100{degree}C of 40 min. In contrast, an enolase from yeast was inactivated in 1 min at 88{degree}C. Both the P. furiosus and yeast enzymes required a metal ion for activity, but whereas the yeast enzyme has an absolute requirement for Mg{sup ++} the P. furiosus enolase was equally active in the presence of Mn{sup ++}. Both enzymes were competitively inhibited by citrate. P. furiosus enolase, as for mesophilic enolases, probably has a homodimeric structure with subunit M{sub r} greater than 45,000. A highly conserved sequence of eight amino acids in the N-terminal region was found in enolases from P. furiosus and a wide range of other organisms including bacteria, yeast, birds, and mammals.

The nucleoside 4‐thiouridine, present in some bacterial tRNA species, is known to be a chromophore and a target for near‐UV light‐induced growth delay and also mediates both photoprotection and near‐UV cell killing in various bacterial strains. To investigate the photoreaction of 4‐thiouridine with DNA or its precursors, we irradiated aqueous mixtures of thymine and 4‐thiouridine with 334 nm light and then separated photoproducts using two or more stages of reversed‐phase high performance liquid chromatography. The two equally abundant major photoproducts were analyzed by UV absorbance spectrophotometry, fast‐atom bombardment and electron‐impact mass spectrometry, and 1H‐ and 13C‐NMR spectroscopy, and have been identified as two diastereomers of 6‐hydroxy‐5‐[1‐(β‐D‐erythro‐pentofuranosyl)‐4′‐pyrimidin‐2′‐one]dihydrothymine (o6hThy[5‐4]Pdo), of molecular weight = 370.32. These two diastereomers, although stable at room temperature or below, are interconvertible by heating (90d̀C for 5 min) in aqueous solution. The possible biological significance of this photoproduct is discussed, and an application as a crosslinker for oligonucleotides to selectively block replication is suggested.

An action spectrum for the relaxation of supercoiled plasmid DNA (induction of the first single-strand break) by photoactivated 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) has been determined using monochromatic UV photons from 254 to 405 nm. The spectrum of AMT-induced plasmid DNA relaxation fits closely with the absorbance spectrum of AMT in the spectral region between 313 nm and 405 nm but deviates at wavelengths shorter than 313 nm. This assay also reveals that the psoralen photosensitization reaction with DNA also produces piperidine-labile sites. Addition of mannitol and azide partially quenches the supercoil relaxation reaction, evidence for a role of Type II photosensitization pathway.

The induction by 60Co gamma-rays of DNA breaks, revealed by relaxation (single-strand breaks, SSBs) and linearization (double-strand breaks, DSBs) of supercoiled plasmid DNA, was measured under three irradiation conditions, the DNA being in a dry, humid, or aqueous state in the absence of oxygen, at 25 or -196 degrees C (77 K). Yields of strand breaks (3.0 x 10(-10) SSB/Gy.Da and 2.6 x 10(-11) DSB/Gy.Da) in DNA exposed to a stream of humidified nitrogen were higher than those in the dry condition (5.7 x 10(-11) SSB/Gy.Da and 3.2 x 10(-12) DSB/Gy.Da), but both these yields were markedly lower than those measured for DNA in aqueous solution at a concentration of 73 micrograms/cm3 (1.14 x 10(-7) SSB/Gy.Da and 5.4 x 10(-9) DSB/Gy.Da). Over 100-fold fewer SSBs were observed in the frozen aqueous system compared with the non-frozen liquid state, whereas in the dry and humid states, freezing did not affect the yield as much. The same trend was observed for DSBs. However, the induction of SSBs was more affected than that of DSBs by freezing in the aqueous systems. An interesting reverse relationship was observed in humid systems. The observed linearity of DSB induction with radiation dose supported a single-event mechanism. A comparison of G values for humid systems revealed that the role of bound water in radiation damage becomes significant in the nonfrozen state. Based on these and other measurements of strand breaks under different conditions, the significance of bound and free water on the yields of DNA strand breaks by gamma-rays is discussed, and the relevance of these results to the in vivo situation outlined.

This document presents information on the possible mechanisms of carcinogenesis caused by UVA (ultraviolet radiation in the 320--400 nm region). Most studies showing the carcinogenic effects of ultraviolet light have concentrated on UVB (280--320 nm). UVA had been considered harmless even though it penetrates biological tissues better than UVB. Recently, it has become apparent that UVA is also capable of causing damage to cellular DNA. This was unexpected because the DNA UV absorption spectrum indicates a negligible probability that photons of wavelengths longer than 320 nm will be directly absorbed. The most common defects induced in DNA by UVB are pyrimidine photoproducts, such as thymidine dimers. UVA photons produce defects resembling those caused by ionizing radiations: single- and double-strand breaks, and DNA-protein crosslinks. This paper also discusses the role of DNA repair mechanisms in UVA-induced defects and the molecular mechanisms of UVA damage induction. 38 refs. (MHB)

Natural solar radiation (5 min of midday exposure in mid July, latitude 42-degrees-N) induces protein kinase C mRNA almost two-fold in human epithelioid P3 cells in culture. This response is the same as that following tumor promotion by chemicals. The result indicates a possible role of promotion by solar UV radiation.

The dose-response for the induction of initial double-strand breaks (dsb) in DNA of human epithelioid cells by JANUS 0.85 MeV fission-spectrum neutrons was parabolic as assayed by a calibrated neutral filter elution technique. The relative biological effectiveness (RBE) of these neutrons relative to 60Co gamma-rays was unity. The kinetics of repair after a 60 Gy gamma-ray exposure were biphasic. About 65% of these dsb were rapidly repaired (T 1/2 of approximately 2 min), and the remainder were almost completely removed after 150 min at a slower rate (T 1/2 = 30 min). After the same dose of JANUS neutrons, the rapid repair component was markedly reduced (possibly not a significant repair component), and the bulk of the dsb were sealed more slowly (T 1/2 = 90 min). After 150 min, 25% remained unsealed. Even after a lower neutron dose (20 Gy), a proportion of the dsb were refractory to repair. Thus, unrepaired (or irreparable) dsb induced by high energy neutrons might explain the high RBE of neutrons for cell killing.

Cell survival parameters and the induction and repair of DNA single-strand breaks were measured in two Chinese hamster ovary cell lines after irradiation with monochromatic UVA radiation of wavelength 365 nm. The radiosensitive mutant cell line EM9 is known to repair ionizing-radiation-induced single-strand breaks (SSB) more slowly than the parent line AA8. EM9 was determined to be 1.7-fold more sensitive to killing by 365-nm radiation than AA8 at the 10% survival level, and EM9 had a smaller shoulder region on the survival curve (alpha = 1.76) than AA8 (alpha = 0.62). No significant differences were found between the cell lines in the initial yields of SSB induced either by gamma-radiation (as determined by alkaline sucrose gradient sedimentation) or by 365-nm UVA (as determined by alkaline elution). For measurement of initial SSB, cells were irradiated at 0.5 degrees C to minimize DNA repair processes. Rejoining of 365-nm induced SSB was measured by irradiating cells at 0.5 degrees C, allowing them to repair at 37 degrees C in full culture medium, and then quantitating the remaining SSB by alkaline elution. The repair of these breaks followed biphasic kinetics in both cell lines. EM9 repaired the breaks more slowly (t1/2 values of 1.3 and 61.3 min) than did AA8 (t1/2 values of 0.9 and 53.3 min), and EM9 also left more breaks unrepaired 90 min after irradiation (24% vs 8% for AA8). Thus, the sensitivity of EM9 to 365-nm radiation correlated with its deficiency in repairing DNA lesions revealed as SSB in alkaline elution.(ABSTRACT TRUNCATED AT 250 WORDS)

Neutral elution is a sensitive and convenient method for measuring double-strand breaks in cellular DNA, but results obtained with this method are controversial, particularly as regards form of dose-response relationship. We pointed out in a recent publication (1) that explanation for different results obtained by neutral elution compared with neutral sedimentation remains obscure. The possibility exists that types of radiation damage other than double-strand breaks might affect neutral elution behavior. In a recent Letter to Editor, Hutchinson (2) stated that single-strand breaks might alter neutral elution because of configurational changes in DNA and/or increased susceptibility to shear but the necessary experiments to determine effect of single-strand breaks on neutral elution have not been performed. These necessary experiments must test whether single-strand breaks can modulate elution behavior of DNA containing a significant number of double-strand breaks, and we have performed such experiments. We induced double-strand breaks in DNA of human P3 epithelioid cells using 125I decay exactly as described previously (3), then, after allowing for accumulations of specific numbers of decays, we induced various numbers of single-strand breaks in cells by using treatments known to produce many single-strand breaks and few or no double-strand breaks: H202 (4) and bromodeoxyuridine photolysis (5), as well as y radiation. In no case (three measurements for each single-strand breakinducing treatment at different levels of induced singleand double-strand breaks) did extra single-strand breaks have any measurable effect on elution profiles (manuscript in preparation). Thus we have experimental evidence that neutral elution is not affected by concomitant singlestrand breaks, and this explanation for discrepancy between neutral sedimentation and neutral elution appears to be invalid. With respect to effect of protein crosslinked to DNA, more rigorous DNA lysis and cleaning procedures were described by Okayasu and Iliakis (6), who obtained double-strand break dose responses without a shoulder and, in some cases, straight elution profiles following these treatments. We have repeated those experiments as closely as possible using P3 cells, but saw neither exponential profiles nor dose responses without shoulders that Okasayu and Iliakis obtained. Therefore, DNA-to-protein crosslinking does not appear to be a determinant of neutral elution behavior in all types of cells. Thus question of different form of dose response measured by elution and sedimentation is still not resolved. Hutchinson's critique of neutral elution did not mention several positive aspects of assay or generally negative aspects of neutral sedimentation covered very thoroughly by van der Schans (7). The validity of elution assay is supported by fact that singleto doublestrand break ratios caused by different agents (which may differ by more than two orders of magnitude) detected by neutral elution correspond well with results obtained by other methods. Neutral sedimentation is beset with problems of irreproducibility caused in part by susceptibility of DNA to shear (7), as well as insensitivity. (Equivalent sensitivity to that of neutral elution has been achieved (8) but only by using extraordinarily laborious sample-handling procedures and 4-day sedimentation times.) Although neutral sedimentation, unlike neutral elution, is based upon mathematical theory, extent to which sedimentation theory is both untested and problematic in range of molecular weights relevant to low-dose studies in mammalian cells (9) should not be ignored. Despite unresolved probms, neutral elution remains only practical means of determining double-strand breaks at relatively low radiation doses.

Abstract— A covalently closed, supercoiled plasmid was irradiated with 334‐nm ultraviolet radiation in the presence of the naturally occurring photosensitizer 2‐thiouracil (s2Ura). After irradiation, some DNA samples were treated to reveal labile sites. Agarose gel electrophoresis was then used to resolve the unrelaxed supercoils from the relaxed forms, and the DNA bands were quantitated by fluorescence scanning. Irradiation of the plasmid in the absence of s2Ura induced small numbers of frank DNA strand breaks (FSB), alkali‐labile sites (ALS), and piperidine‐labile sites (PLS). The induction of each of these lesions was enhanced 30 times when s2Ura was present during aerobic irradiation. Anoxia, as well as the hydroxyl radical scavengers acetate and formate, inhibited the formation of all three lesion types. The relative proportions of the three lesion types produced by several DNA damaging treatments were measured. Hydrogen peroxide, γ‐irradiation, and s2Ura photosensitization produced nearly identical damage proportions, with PLS: FSB ratios of 1.25:1, 0.78:1, and 0.84:1, respectively. Treatment with singlet oxygen [data from Blazek et al. (1989) Photochem. Photobiol. 48,607–613] produced much different proportions, with a PLS:FSB ratio of 4.1:1. These results may indicate a role for hydroxyl radical in s2Ura‐photosensitized DNA damage.

— Neutral filter elution at pH 7.2 and 9.6 was used to measure the induction of DNA lesions in human P3 teratocarcinoma cells by monochromatic 254‐, 270‐, 313‐, 334‐, 365‐, and 405‐nm radiation and by 60 gamma rays. In this assay DNA double‐strand breaks (dsb) increase the rate of elution of DNA from cell lysates on a filter. Yields of dsb as measured by this procedure were determined by using a calibration of the assay that correlates elution parameters with number of dsb caused by disintegration of 125 I incorporated into the DNA. Analysis of fluence responses obtained by using the calibrated assay indicated that the number of dsb induced per dalton of DNA as measured by this assay is proportional to the square of the fluence at all the energies of radiation studied, implying that the induction of these lesions may be a two‐hit event. Analysis of the relative efficiencies for the induction of dsb by ultraviolet radiation, corrected for quantum efficiency, revealed a spectrum that coincided closely with that for the induction of single‐strand breaks (ssb) in the same cells, having a close fit with the spectrum of nucleic acid in the UVC and UVB region below 313 nm, and a shoulder in the UVA region. It was calculated, however, that there may be too few ssb for dsb to result from randomly distributed closely opposed ssb.