A STUDY OF THE RELATIONSHIP BETWEEN SURVIVAL AND REPAIR SYNTHESIS'' OF DNA AFTER ULTRAVIOLET LIGHT EXPOSURE

Abstract— The influence of amino acid prestarvation on both the resistance to u.v. and the postirradiation repair synthesis of E. coli 15 T^- 555-7 thy meth arg trp and E. coli B/r (HCR⁺) was followed. Prestarvation increased the number of survivors about 30–100 fold in both strains at doses 600-1200ergs/mm². In contrast to survival no increase in repair synthesis was observed. Thus, the increase in survival has to be brought about by a mechanism which seems to be independent of additional repair synthesis.     

PATTERNS OF NUCLEIC ACID AND PROTEIN SYNTHESIS DURING MODIFICATION OF LETHAL ULTRAVIOLET LESION

Abstract— We have shown previously that the viability of E. coli B.U.^- and E. coli B. try^-could be restored after u.v.-irradiation if post-irradiation incubation took place in the presence of chloramphenicol or in the absence of an essential metabolite. By way of contrast, the viability of the u.v.-irradiated E. coli K.₁₂ meth^- decreased rapidly during post-irradiation incubation in the presence of chloramphenicol or in the absence of methionine. During the study of the biosynthesis of nucleic acids and protein in the above strains we made the following observations.
Protein synthesis was permanently inhibited in both amino acid requiring strains during post-irradiation incubation in the absence of the respective essential metabolite. In E. coli B.U.^- a slow synthesis of protein could be observed even if post-irradiation incubation took place in the absence of uracil.
DNA synthesis was completely and permanently inhibited in both E. coli B. try^- and E. coli K.₁₂ meth^- during post-irradiation incubation in the absence of the respective essential amino acid. In E. coli B.U.^-, however, DNA synthesis resumed after a prolonged lag even if the bacteria were incubated after u.v.-irradiation in the absence of uracil.
RNA synthesis was completely and permanently inhibited in both E. coli B. mutants, the uracil requiring and the tryptophan requiring, if they were incubated after irradiation in the absence of uracil and tryptophan respectively. In E. coli K.₁₂ meth^- a small amount of RNA is synthesized during post-irradiation incubation in the absence of the essential amino acid.     

INTERACTION OF RESTORATION PROCESSES IN UV IRRADIATED ESCHERICHIA COLZ CELLS

Abstract— An attempt was performed to estimate survival and course of DNA synthesis in Escherichia coli B/r hcr' and hcr^- cells in relation to the amount of unexcised dimers.
In exponential growing hcr⁺ cells irradiated with 30 Jm^-2, dimers were almost completely excised and survival of cells was equal to about 3%. In the hcr⁺ cells prestarved for amino acid and thymine and irradiated by the same fluence, survival of cells was almost equal while two thirds of dimers remained unexcised and could be detected in the hybrid DNA consisting of parental and daughter chains. In exponentially growing hcr^+- cells irradiated with 20Jm^-, the same amount of dimers was produced which remained unexcised in the prestarved hcr⁺ cells. However, their survival was equal to about 0.02%.
Despite the great differences in dimer contents, about one third of DNA was replicated after UV in both exponentially growing and prestarved hcr+ cells producing well defined HL-hybrid peak, and the newly synthesized DNA was normal-sized. In hcr⁺ cells which contained approximately the same amount of dimers as in hcr⁺ prestarved cells, the amount of replicated DNA was too low to form a detectable density labelled hybrid peak, and the newly synthesized DNA was in short pieces.
Thus, when hcr⁺ and hcr^+- cells contain the same number of residual dimers, they have different levels of tolerance to these dimers.     

THYMINE DIMERIZATION IN L-STRAIN MAMMALIAN CELLS AFTER IRRADIATION WITH ULTRAVIOLET LIGHT AND THE SEARCH FOR REPAIR MECHANISMS

Abstract— The formation of thymine dimers in the DNA of L -strain mammalian cells after irradiation with ultraviolet light has been demonstrated. The amount of dimer formed rises with the dose of u.v. light.
In the course of post-irradiation incubation the thymine dimers remain in the TCA insoluble fraction and diminish as did the other thymidine-H³ derivatives with increasing incubation time. The dimer is not found in the soluble fraction. Thus, dimer excision (i.e. its liberation into the soluble fraction) as an expression of repair of radiation damage analogous to dark repair in E. coli was not found in these experiments.     

EXCISION OF PYRIMIDINE DIMERS FROM THE NUCLEAR DNA OF A HAPLOID RESPIRATION-DEFICIENT (ρ) STRAIN OF SACCHAROMYCES CEREVISIAE

Abstract— A haploid respiration-deficient (ρ^-) mutant of Saccharomyces cerevisiae exhibits a lower ability to excise ultraviolet-induced pyrimidine dimers in various post-irradiation treatments as compared to the original respiration-competent wild-type strain. This decrease in excision may account for the inability of ρ^- strains to show liquid-holding recovery.     

EFFECTS OF DOSE FRACTIONATION ON ULTRAVIOLET SURVIVAL OF ESCHERICHIA COLI

Abstract— Exposure of E. coli B/r and B at low average dose rates of u.v. radiation (2537 Å), produced either by fractionated doses or by continuous irradiation at a very low dose rate (80 ergs/mm²/hr), results in much increased survival compared to single exposure at high dose rate. This increase is attributed to repair taking place during the irradiation period. The effect is small in the repair-deficient strains E. coli B_8-1_, and C syn- , and is absent in phage T1 and T4, which cannot undergo repair in the extracellular state. However, the prolonged time available for repair in these experiments accounts for only a very minor part of the increase in survival. The principal factor apparently is that the number of lesions present at any time remains relatively low. Presumably complete repair, not only the excision step, can occur in buffer during the irradiation period. This interpretation is supported by experiments in which cells were exposed to combinations of highly fractionated irradiation and single-dose irradiation. We therefore propose that mutual interference in repair, possibly by overlapping of repair regions in complementary DNA strands, reduces considerably the repair efficiency if many lesions are present. This hypothesis explains the 'shouldered' survival curves of B/r and possibly other E. coli strains as due to decreasing repair efficiency with increasing u.v. dose     

PHOTOREVERSION VON VIER GRUPPEN UV-INDUZIERTER MUTATIONEN ZUR GIFTRESISTENZ IM NICHTPHOTOREAKTIVIERBAREN E. COLI

Abstract— In the non-photoreaclivable bacterial strain E. coli B/phr^-/MC2 the photoreversion of four groups of u.v.-induced mutations were investigated. They lead to resistance to Chloramphenicol (2 mg/l; "C"), Penicillin (13 or 16 mg/l; "P13" and "P16") or Streptomycin (3 mg/l; "S"). The u.v.-dose curve is concave for the C-mutations (two to three hits), about linear for P13 and S, and they reach peaks and decrease at high u.v.-doses. Though no photoreactivation of killing (PR) is present there is photoreversion of all four types of mutations (PRM). At u.v.-doses below the peaks in average about 43 per cent mutations are photoreversible. At high u.v.-doses the curves with light-post treatment (L) cross the darkcurves (D). In the photoreactivable strain B/r (by the spontaneous mutation MC2 to Mitomycin-resistance strain B/phr^- was made about as u.v.-resistant as B/r is) the photoreversion of the mutation groups C, P13 and P16 (S was not investigated here) was much higher, in average about 77 per cent at low doses. It is assumed that the difference in PRM of about 34 per cent between both strains is due to a PRM-mechanism present in B/r but not in B/phr^-/MC2; this mechanism may be the photoreactivating enzyme that opens thymine-dimers. The PRM in B/phr-/MC2 must then be due to a second mechanism which is probably not the dimer opening enzyme. It may be the same mechanism as in the case of mutations of phage kappa which are induced by u.v. and reversed partially by light, both extra cellularly. The premutations giving this second type of PRM may perhaps be cytosine-hydrate in the DNA. Tn average about 23 per cent mutations of B/r are photostable. Since this ratio decreases with low u.v.-doses in the C-mutations and increases in P13 and in P16 probably two types of photostable premutations seem to exist.     

THE ROLE OF HOST-CELL REPAIR IN LIQUID-HOLDING RECOVERY OF U.V.-IRRADIATED ESCHERICHIA COLI

Abstract— The experiments reported give evidence that liquid-holding recovery (LHR) of u.v. irradiated E. coli cells involves basically the same type of dark repair which causes reactivation of phage and which results in much increased survival of the cells themselves [host-cell reactivation (HCR)]. LHR is very small in the two HCR(-) strains B syn^- and B_s-1, but occurs to larger but different extents in the three HCR(+) strains B, B/r, and B/r (Λ). LHR is inhibited if the liquid contains caffeine or acriflavine, both of which are known to inhibit HCR. The results indicate that most of the LHR effect, if not all, occurs during the liquid holding, rather than under growth conditions after liquid holding. It is assumed that the holding itself allows a prolonged time for, and therefore an enhancement of, HCR. It is thus implicit that LHR can be observed only where otherwise HCR of repairable u.v. damage would be incomplete, and that different extents of LHR, as observed in the three HCR(+) strains, reflect different extents of incompleteness of HCR. It is concluded that the repairable u.v. hits which are not fully repaired by HCR are predominantly those concerned with the extra u.v. sensitivity of the strains B and B/r (Λ), relative to B/r.     

THE ROLE OF PYRIMIDINE DIMERS AND NON-DIMER DAMAGE IN THE INACTIVATION OF ESCHERICHIA COLI BY UV RADIATION

Abstract— We have quantitated the role of pyrimidine dimers and non-dimer damage in the inactivation of Escherichia coli by far-UV radiation, near-UV radiation, and triplet state sensitized near-UV radiation. The extent of photoreactivation in vivo of an excision and postreplication repair-deficient strain of E. coli after the different radiation treatments has been correlated with the relative proportion of pyrimidine dimers and non-dimer lesions produced. Using an excision deficient strain of E. coli, the susceptibility to recA ⁺ -dependent repair of the damage produced by the different radiation treatments has also been quantified.     

CHANGE IN THE BASE COMPOSITION OF MESSENGER RNA OF ESCHERICHIA COLI BY ULTRAVIOLET IRRADIATION AND ITS RECOVERY

Abstract— The base composition of messenger RNA in Escherichia coli B/r and B _8–1 irradiated with ultraviolet (u.v.) light has been examined. The experimental results are as follows: (1) the synthesis of rapidly labeled RNA does not stop in ultraviolet irradiated bacteria. (2) The rapidly labeled RNA in irradiated cells shows a change in base composition corresponding to the formation of pyrimidine dimers in DNA molecules. The mole per cent of adenine component is increased with ultraviolet dose. The ratio of purine/pyrimidine becomes larger and the GC content smaller. (3) The base composition of the rapidly labeled RNA in irradiated bacteria reversed to that in unirradiated cells, when the irradiated cells were reactivated by experimental procedures for photoreactivation or dark reactivation. The reversion in the base composition corresponds well to the decrease in the amount of thymine dimers in DNA molecules. (4) The mechanism of the change in the base composition of rapidly labeled RNA caused by ultraviolet irradiation is discussed.     

DIFFERENTIAL, LETHAL AND MUTAGENIC ACTION OF 254 nm AND 320–400 nm RADIATION ON SEMI-DRIED BACTERIA

Abstract— The effect of culture conditions on the lethal and mutagenic action of 254 nm (u.v.) and 320–400 nm (b.l.) light has been examined. Ten strains of Escherichin coli were used in these investigations. It was found that semi-dehydration in aerosols greatly enhanced the lethal and mutagenic actions of both U.V. and b.l., Mutations induced by U.V. were found to be of a random kind, while those produced by b.l. were specific and of a particular biochemical type depending on the strain of cell and its stage of development. The presence of oxygen during irradiation enhanced b.l. effects but had no effect on U.V. damage while anaerobic growth endowed the cells with added resistance to b.l. and u.v., Stationary phase cells of E. coli B/r were found to be mutated by b.l. specifically at a thymine locus and to be more sensitive than E. coli B to the inhibition by b.l. of respiration. Some mutations induced by b.l. in E. coli B/r were found to hinder the cells ability to carry out the photoreversal of U.V. damage. It is suggested that b.1. affects a specific piece of DNA which is in contact with the cytochrome chain of the cytoplasmic membrane and that this contact point between the cytochrome chain and DNA alters sequentially as the cell proceeds through its life cycle.     

Action of photoreactivating light on pyrimidine heteroadduct in bacteria

Abstract— In stationary-phase Escherichia coli B/r, photoreactivation (PR) at 313 nm of ultraviolet (u.v.) killing is inefficient compared with PR at 405 nm, and can be explained solely by photoenzymatic reversal of pyrimidine dimers. In Staphylococcus epidermidis, PR shows a maximum at 313 nm, suggesting that this organism shows the Type III PR proposed by Jagger et al.[5] for Streptomyces strains. Reversal of pyrimidine dimers is not sufficient to explain this PR. The mechanism of Type III PR remains unknown. With both S. epidermidis and E. coli B/r, the amount of uracil–thymine heteroadduct in DNA hydrolysates decreases if the cells are given a post-u.v. treatment at 313 nm, but no decrease is observed if the post-u.v. treatment is at 405 nm. The biological significance of this adduct and of its removal is not clear. It may play a role in Type III PR.     

CARBOXYLATION OF FORMIC ACID IN AQUEOUS SOLUTIONS UNDER THE INFLUENCE OF U.V.-LIGHT

Abstract— Carboxylation of various organic substances by dissolved carbon dioxide or bicarbonate is achieved by the action of U.V. -Light of 184.9 and 253.1 mμ on water. The transformation of formic acid to oxalic acid was investigated in more detail as a function of u.v.-dose, pH-values and the formate concentration. For an irradiation period of 12 hr of 0.1 M formate, a yield of about 2 m mol.1^--¹ oxalic acid was obtained. A reaction mechanism for the photochemical carboxylation process is presented. The results are of some interest in respect of the synthesis of organic compounds under the primitive earth conditions.     

PHOTOLESIONS IN DNA UPON 193 nm EXCITATION

Aqueous solutions of plasmid (pBR322 and pTZ18R) and calf thymus DNA were excited by 20 ns laser pulses at 193 nm. The quantum yields of single- and double-strand break formation, interstrand cross-links, locally denatured sites, (6–4)photoproducts and biological inactivation (Φ_ssb, Φ_dsb, Φ_icl, Φ_ids, Φ_6–4 and Φ_ina, respectively) were measured. The quantum yields are virtually independent of intensity, demonstrating a one-quantum process. The obtained values in aerated neutral solution in the absence of additives are Φ_ssb= 1.5 × 10^--³, Φ_dsb, = 0.06 × 10^--³ (dose: 10–200 J m^-2), Φ_icl=Φ_Ids= 0.1 × 10³ and Φ_6–4= 0.5 × 10^--³ Both Φ_ssb and Φ_dsb decrease strongly with increasing concentrations of TE buffer (0.01–10 m M ). Biological inactivation of the pTZ18R plasmid was determined from the transformation efficiency of Escherichia coli bacteria strains AB1157, AB1886 uvr and A82480 uvr rec; the Φ_ina values are 1.4 × 10³, 2.1 × 10³ and 3 × 10^--³, respectively. The monoexponential survival curves in all cases show that a single damage site leads to inactivation (one single hit). The biological consequences of different photoproducts are discussed.     

THE RATE OF REMOVAL OF SUNLIGHT-INDUCED PYRIMIDINE DIMERS FROM THE DNA OF CULTURED HUMAN DIPLOID FIBROBLASTS

Abstract The rate of excision of sunlight-induced pyrimidine dimers in DNA of exposed human cells was determined. Two normal excision repair-proficient human diploid fibroblast strains (WS-1 and KD) and a repair-deficient strain (XP12BE, group A) maintained in a nondividing state were exposed to summer noon-time sunlight for times (5 and 20 min) that induced numbers of dimers equivalent to far UV (254 nm) exposures of 1 and 4 J/m². Pyrimidine dimers were quantified in extracted DNA using a U V-endonuclease-alkaline sedimentation assay. The excision rates of these dimers were similar to those observed for the excision of UV-induced pyrimidine dimers. No sunlight-induced inhibition or stimulation of DNA repair was observed in either strain at these low exposures.     

SENSITIZED PHOTOREDUCTION OF CO(EDTA)- IN AERATED AQUEOUS SOLUTION. ITS PHOTO-ELECTROCHEMICAL AND PHOTOBIOLOGICAL APPLICATIONS

Abstract— The reduction of Co(edta)^- in aerated aqueous solution containing 1-hydroxy-5-oxo-5H-pyrido[3,2a]phenoxazine-3-calboxylic acid (HPPC) as sensitizer and ethylenediaminetetraacetate ion(EDTA) as electron donor was studied. Semireduced HPPC, formed in a reaction between triplet HPPC and EDTA, was shown to reduce Co(edta)^- to Co(edta)^2-. The ratio of the rate constant for oxidation of semireduced HPPC by oxygen to that by Co(edta)^- was determined to be 0.45± 0.01 from competition reaction kinetics. A (concentration)half-cell (oxidation-reduction electrode) based on the photoreduction of Co(edta)^- was found to give an electrode potential decrease of 100 mV. Co(edta)^- was also found to be reduced during illumination in aerated suspension of chloroplasts. The rate of oxygen evolution for the Co(edta)^- -chloroplast system was nearly the same as that for the Fe(CN)₆^3- -chloroplast system both in the absence and presence of ammonium ions.     

Thymine-dimer and action-spectrum evidence for indirect photoreactivation in Escherichia coli

Abstract— Cells of Escherichia coli B and B phr- were labeled with tritiated thymidine, exposed to inactivating ultraviolet radiation at 254 nm, given photoreactivation (PR) treatment immediately thereafter, and then immediately hydrolyzed and assayed for thymine-containing dimers. It was found that (1) PR treatment of strain B phr- does not split thymine dimers and (2) the amount of splitting of thymine dimers in strain B at 334 nm is only 45 per cent of the amount of splitting observed at 405 nm for the same amount of biological PR. These findings show that all of the PR in E. coli B phr-, and part of the PR at 334 nm in E. coli B, is indirect (does not use PR enzyme) and is not due to thymine-dimer splitting. Action spectra for PR in Escherichia' coli strains B_s-1 and B/r were obtained. At wavelengths below 366 nm (the indirect PR region), PR is relatively much more efficient in strain B/r in logarithmic phase than in strain B/r in stationary phase or in strain B_s-l. This is consistent with the expectation that indirect PR would not be exhibited by strains, such as B_s-1 that lack dark-repair ability, or by certain strains in the stationary phase, such as B/r, which, upon plating, have a ‘built-in’ growth delay that can permit optimal dark repair, but indirect PR would be exhibited by intermediate cases, such as in strain B (which is less resistant to u.v. than strain B/r) or in strain B/r in logarithmic phase. These findings support the hypothesis that indirect PR results from enhancement of dark repair.     

QUANTITATIVE EVIDENCE FOR ENZYMATICALLY-INDUCED DNA DOUBLE-STRAND BREAKS AS LETHAL LESIONS IN UV IRRADIATED pol+ AND polAl STRAINS OF E. COLI K-12

Abstract— We have recently reported that DNA double-strand breaks arise enzymatically during the course of excision repair in uvr ⁺ strains of Escherichia coli K-12. Survival curves for ultraviolet (UV) irradiated E. coli K-12 pol⁺ (JG139) and polA1 (JG138) strains have a pronounced shoulder region. The regions of the survival curves at which killing approaches exponential correspond to the fiuences at which DNA double-strand breaks (assumed to be lethal events) accumulate linearly. Reducing the number of UV photoproducts either by photoreactivation or fluence fractionation results in an increase in survival and a decrease in the yield of DNA double-strand breaks in both strains. These data support the hypothesis that enzymatically-induced DNA double-strand breaks may be the lesion ultimately responsible for UV-induced cell killing in the pol⁺ strain of E. coli K-12. and perhaps also in the polA1 strain.     

REPAIR OF UV DAMAGE IN ESCHERICHIA COLI UNDER NON-GROWTH CONDITIONS

Abstract. –A large difference in survival occurs between buffered suspensions of E. coli irradiated with UV radiation at a low fluence rate and those irradiated at a high fluence rate. For sufficiently large fluences, the extent of this fluence rate dependent recovery (FRR) is about two orders of magnitude greater than that which can be brought about by liquid holding recovery (LHR) following high fluence rate irradiation in most of the E. coli strains studied. LHR and FRR occur in excision resynthesis repair proficient (ERR⁺) but not ERR^- strains of E. coli , although its observation can be masked in strains with complete repair potential upon subsequent growth on nutrient plates. Accumulation of DNA strand interruptions and excision of cyclobutyl dipyrimidine occur during LHR and FRR but are more extensive for the latter. Our data suggest mat events beyond incision and excision occur during LHR and FRR, but differences in the extent of ERR during LHR and FRR cannot account for the difference in cell survival between these two phenomena.     

THE INFLUENCE OF NUTRITION ON THE DNA CONTENT OF ESCHERICHZA COLI AND ITS RESPONSE TO ULTRAVIOLET LIGHT

Abstract— The influence of nutrition on the sensitivity of Escherichia coli 15 T- to ultraviolet light (u.v.) and the synthesis of DNA has been studied. Growth in media containing glucose or NH,⁺ has been found to endow cells with a greater resistance to lethal u.v. damage than those grown in media containing succinate or amino acids, respectively. In addition, the sensitivity of the lactose ( lac ) locus of the DNA to mutagenic damage has been found to be altered by changes in the carbon supply but not by changes in the nitrogen source, while the sensitivity of loci controlling amino acid synthesis was altered by changes in the nitrogen source but not in the carbon source. Cells fed with glucose or NH₄⁺ have been found to possess more DNA than cells fed with succinate or amino acids, respectively. The data indicate that the type of carbon and nitrogen supplied to the cells will determine whether or not set regions of the DNA will undergo more than one round of replication. The presence in the cell of identical genetic loci either in duplicate or in multiples, directed by the particular types of carbon and nitrogen supplied, is suggested to be, in part, the reason why an alteration in nutrition is able to influence the sensitivity of bacterial cells to radiation.