SINGLE-STRAND BREAKS IN THE DNA OF THE uvrA AND uvrB STRAINS OF ESCHERICHIA COLI K-12 AFTER ULTRAVIOLET IRRADIATION

Abstract— DNA single-strand breaks were produced in uvrA and uvrB strains of E. coli K-12 after UV (254 nm) irradiation. These breaks appear to be produced both directly by photochemical events, and by a temperature-dependent process. Cyclobutane-type pyrimidine dimers are probably not the photoproducts that lead to the temperature-dependent breaks, since photoreactivation had no detectable effect on the final yield of breaks. The DNA strand breaks appear to be repairable by a process that requires DNA polymerase I and polynucleotide ligase, but not the recA, recB, recF, lexA 101 or uvrD gene products. We hypothesize that these temperature-dependent breaks occur either as a result of breakdown of a thermolabile photoproduct, or as the initial endonucleolytic event of a uvrA , uvrB -independent excision repair process that acts on a UV photoproduct other than the cyclobutane-type pyrimidine dimer.     

SYNERGISTIC LETHAL ACTION OF ULTRAVIOLET-VIOLET RADIATIONS AND MILD HEAT IN ESCHERICHIA COLI

Abstract— The lethal interaction of far ultraviolet (254nm), near ultraviolet (334 and 365nm) and violet visible (405nm) radiation treatment with mild heat treatment was studied. Except at 254nm, a strong positive radiation dose-dependent interaction (synergism) was always observed. The efficiency of sensitisation to heat, as a function of dose at each wavelength, was found to be directly correlated with the dose necessary to eliminate the shoulder from the survival curve of a repair proficient strain but was apparently unrelated to the relative near-ultraviolet sensitivities of a repair deficient strain. The interaction was independent of the order of treatments. A radiation dose of 10⁶ Jm^-2 at 365nm slightly sensitised a cell population to 45°C incubation (normally non-lethal) and strongly sensitised the cells to 48°C treatment (normally 80 percent survival after 2 hours). It is proposed that in addition to DNA damage, both heat treatment and near ultraviolet treatment interfere with DNA recovery mechanisms so that the combination of the two agents inevitably leads to a strong positive interaction.     

SENSITIVITY OF DNA REPAIR-DEFICIENT STRAINS OF ESCHERICHIA COLI K-12 TO VARIOUS FUROCOUMARINS AND NEAR-ULTRAVIOLET RADIATION

Abstract— Survival curves were obtained for DNA repair-deficient strains of Escherichia coli K-12 ( polA1, uvrB5 , and recA56 ) exposed to near-ultraviolet radiation [black light (BL)] in the presence of the DNA cross-linking agent 8-methoxypsoralen (8-MOP) or in the presence of photosensitizers forming primarily monoadducts with DNA [angelicin; 3-carbethoxypsoralen (3-CPs); 5,7-dimethoxycoumarin (DMC)], and after exposure to blue light (BluL) in the presence of 8-MOP or 3-CPs. An interpretation of these data suggests that DNA polymerase I is required for the major pathway of monoadduct repair, but appears to play little or no role in the repair of 8-MOP cross-links. The uvrB and recA strains were very sensitive, both to the cross-linking agent and to the monoadduct formers. The markedly different results for BL plus DMC or 3-CPs compared to angelicin suggests that the DMC and 3-CPs monoadducts are repaired by a different mechanism than are the angelicin monoadducts, or else DMC and 3-CPs undergo photochemical side reactions that produce DNA lesions other than the expected monoadducts. From photochemical evidence, we predicted that fewer 8-MOP monoadducts should be converted to cross-links by BluL vs BL; this appears to be the case. 3-CPs showed dramatically different biological results when irradiated with BL vs BluL, suggesting that 3-CPs may form more types of photoproducts than the expected monoadducts; BluL, however, appears to favor monoadduct formation.     

PHOTOREACTIVATION OF ULTRAVIOLET RADIATION DAMAGE IN DARK REPAIR DEFICIENT PHR MUTANTS OF Escherichia coli K-12

There is uncertainty in the literature concerning the genetic control of photoreactivation in E. coli. Two genetic loci, phrA and phrB have been proposed, and two photolyase molecules have been isolated, but in vivo evidence for the activity of the former is controversial. We have studied photoreactivation after 254 nm UV in a dark-repair-deficient phrB mutant and in a strain deleted at the proposed phrA locus. We show apparent photoenzymatic repair in the phrB mutant, which is abolished when the mutation is transduced into the proposed phrA deletion mutant. We conclude that there is a gene in the region of the proposed phrA locus which affects photoenzymatic repair.     

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     

THE ACTION OF ULTRAVIOLET RADIATION ON THE CELL PROLIFERATION OF TWO-CELL MOUSE EMBRYOS

Abstract— The two-cell mouse embryo has a unique cell cycle of a short DNA synthesis (S) phase and an extremely long post-DNA synthesis (G₂) phase. An attempt was made to investigate the radiation biology of the long G₂ phase using UV radiation as a probe. Two cell mouse embryos, at various positions in the cell cycle, were UV-irradiated in phosphate-buffered saline. The embryos were cultured for a few hours to 3 days to assay for their cell proliferative characteristics. The embryos were most sensitive to the killing action of UV radiation in the late G₂ phase. The embryos divided more than two times after low UV fluences before dying and experienced G₂ phase delays.
These results can be contrasted to the situation in somatic cells, in which the action of UV radiation is S phase selective. One possibility is that the target for the action of UV radiation is different in two-cell mouse embryos from that in somatic cells and that the target is similar to that for X-ray effects.     

PROTECTION OF ESCHERICHIA COLI CELLS AGAINST THE LETHAL EFFECTS OF ULTRAVIOLET AND X IRRADIATION BY PRIOR X IRRADIATION: A GENETIC AND PHYSIOLOGICAL STUDY

Abstract— When log phase cells of wild-type E. coli K-12 were maintained in growth medium after X irradiation, they became progressively more resistant to a subsequent exposure to UV or X radiation. The time to achieve maximum resistance was about 60 min. The uvrB, uvrD, polA and certain exrA strains (W3110 background) also demonstrated this X ray-induced resistance to subsequent UV or X irradiation but recA, recB, lex (AB1157 or W3110 backgrounds) and other exrA strains (AB1157 background) did not. The resistance induced in wild-type, uvrB and uvrD cells was characterized by the production or enhancement of a shoulder on the survival curves obtained for the second irradiation, while the resistance induced in the W3110 exrA strains was expressed only as a change in slope. The induction of resistance in the W3110 exrA strain was not inhibited by the presence of chloramphenicol, but that in the wild-type cells appeared to be. The production or enhancement of a shoulder on the survival curves of the rec ⁺ lex ⁺ exr ⁺ cells is consistent with the concept of the radiation induction of repair enzymes. Alternative explanations, however, are discussed.     

ULTRAVIOLET RADIATION-INDUCED MUTABILITY OF uvrD3 STRAINS OF ESCHERICHIA COLI B/r AND K-12: A PROBLEM IN ANALYZING MUTAGENESIS DATA

Abstract— The involvement of the uvrD gene product in UV-induced mutagenesis in Escherichia coli was studied by comparing wild-type and uvrA or uvrB strains with their uvrD derivatives in B/r and K-12(W3110) backgrounds. Mutations per survivor (reversions to prototrophy) were compared as a function of surviving fraction and of UV fluence. While recognizing that both methods are not without problems, arguments are presented for favoring the former rather than the latter method of presenting the data when survival is less than 100%. When UV-induced mutation frequencies were plotted as a function of surviving fraction, the uvrD derivatives were less mutable than the corresponding parent strains. The B/r strains exhibited higher mutation frequencies than did the K-12(W3110) strains. A uvrB mutation increased the mutation frequency of its parental K-12 strain, but a uvrA mutation only increased the mutation frequency of its parental B/r strain at UV survivals greater than ˜ 80%. Both the uvrA and uvrB mutations increased the mutation frequencies of the uvrD strains in the B/r and K-12 backgrounds, respectively. Rather different conclusions would be drawn if mutagenesis were considered as a function of UV fluence rather than of survival, a situation that calls for further work and discussion. Ideally mutation efficiencies should be compared as a function of the number of repair events per survivor, a number that is currently unobtainable.     

INACTIVATION OF THE LACTOSE PERMEASE OF ESCHERICHIA COLI BY MONOCHROMATIC ULTRAVIOLET LIGHT

Abstract— The lactose permease of E. coli is inactivated exponentially by seven wavelengths of monochromatic UV light. An action spectrum reveals that the shorter wavelengths (243, 290 and 313 nm) are much more efficient than longer wavelengths. Inactivation at 290 nm is most efficient and is not due to generalized membrane damage. The rate of counterflux of intracellular β-galactoside in response to externally added β-galactoside was slowed by 290 nm irradiation, indicating destruction of the facilitated diffusion mechanism. The induction of β-galactosidase and β-galactoside permease was co-ordinate both with and without pre-irradiation by 290 nm light. The β-galactosidase is approximately 26-fold more resistant to 290 nm than the permease. These results are discussed in terms of a greater sensitivity of membrane proteins to 290 nm light, which may be due to the role of aromatic amino acids in conferring stability to the permease in the membrane.     

LETHAL EFFECTS OF NATURAL SOLAR ULTRAVIOLET RADIATION IN REPAIR PROFICIENT AND REPAIR DEFICIENT STRAINS OF ESCHERICHIA COLI: ACTIONS AND INTERACTIONS

Abstract— The inactivation of repair proficient ( Escherichia coli K12 AB 1157, E. coli B/r) and repair deficient ( E. coli K12 AB 1886 uvrA , AB 2463 recA and AB 2480 uvrA recA ) strains of bacteria by noon sunlight has been measured. The use of biological dosimetry based on an ultraviolet (UV) sensitive strain of Bacillus subtilis spores has allowed a quantitative comparison of bacterial inactivation by solar, 254 and 302 nm radiations. Our analysis indicates that: (1) uvrA and recA gene products are involved in repair of a substantial portion of the solar DNA damage, (2) 302 nm is a more appropriate wavelength than 254 nm to represent the DNA-damaging action of sunlight and that (3) repair proficient strains are inactivated by sunlight more rapidly than expected from the levels of DNA damage induced. When populations of repair proficient bacteria are exposed to noon sunlight for 20 min, they become sensitive to the lethal action of far-UV (254 nm), MMS (0.1 M ) and to a lesser extent, mild heat (52°C).     

EFFECTS OF ACRIDINE PLUS NEAR ULTRAVIOLET LIGHT ON ESCHERICHIA COLI MEMBRANES AND DNA IN VIVO

Results from a variety of experiments indicate that photodynamic damage to E. coli treated with the hydrophobic photosensitizer acridine plus near-UV light involves both cell membranes and DNA. Split-dose survival experiments with various E. coli mutants reveal that cells defective in rec A, uvr A, or pol A functions are all capable of recovery from photodynamic damage. Alkaline sucrose gradient analysis of DNA from control and treated cells revealed that acridine plus near-UV light treatment converts normal DNA into a more slowly sedimenting form. However, the normal DNA sedimentation properties are not restored under conditions where split-dose recovery is effective. Several lines of evidence suggest that membrane damage may be important in the inactivation of cells by acridine plus near-UV light. These include (a) a strong dependence of sensitivity on the fatty acid composition of the membranes; (b) a strong dependence of sensitivity on the osmolarity of the external medium; and (c) the extreme sensitivity of an E. coli mutant having a defect in its outer membrane barrier properties. Direct evidence that acridine plus near-UV light damages cell membranes was provided by the observations that (a) the plasma membrane becomes permeable to o-nitrophenyl-ß-D-galactopyranoside and (b) the outer membrane becomes permeable to lysozyme after treatment. A notable result was that cells previously sensitized to lysozyme by exposure to acridine plus near-UV light lose that sensitivity upon subsequent incubation. This strongly suggests that E. coli cells are capable of repairing damage localized in the outer membrane.     

PYRIMIDINE DIMERS INDUCED IN ESCHERICHIA COLI DNA BY ULTRAVIOLET RADIATION PRESENT IN SUNLIGHT

Abstract— Escherichia coli DNA was irradiated with various wavelengths of monochromatic UV light from 254 to 320 nm, and the relative yields of the different cyclobutane pyrimidine dimers determined. Cytosine–thymine dimers (C < > T) were more frequent than thymine dimers (T < > T) at low fluences of 300 and 313 nm light, whereas the reverse was true at either longer or shorter wavelengths. Thus, in the solar UV range deemed responsible for skin cancer (i.e. 295–315 nm), C < > T are probably more important than T < > T.     

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.     

ENHANCED SENSITIVITY TO THE LETHAL AND MUTAGENIC EFFECTS OF PHOTOSENSITIZING ACTION OF CHLORPROMAZINE IN ETHYLENEDIAMINETETRAACETATE-TREATED ESCHERICHIA COLI

Abstract— Ethylenediaminetetraacetate (EDTA) treatment of Escherichia coli H/r30 (Arg_-) enhanced cell sensitivity to the lethal and mutagenic effects of the photosensitizing action of chlorpromazine (CPZ). The most obvious effect of EDTA on the fluence-survival curve was an elimination of the shoulder. In the absence of EDTA, CPZ plus near-UV radiation did not induce the reversion from arginine-auxo-troph to autotroph of E. coli H/r30. However, when EDTA (5 mM)-treated cells were subjected to CPZ plus near-UV radiation, the induced reversion frequency increased with time of irradiation. It is concluded that the enhanced penetration of CPZ into E. coli cells by EDTA facilitates the drug binding to DNA within the cells upon near-UV irradiation and that this is the cause for the enhanced photosensitized lethal and mutagenic effects of CPZ.     

POSTREPLICATION REPAIR IN uvrA AND uvrB STRAINS OF ESCHERICHIA COLI K-12 IS INHIBITED BY RICH GROWTH MEDIUM

Abstract Escherichia coli K-12 uvrA or uvrB strains grown to logarithmic phase in minimal medium showed higher survival after ultraviolet (UV) irradiation (254 nm) if plated on minimal medium (MM) instead of rich medium. This'minimal medium recovery'(MMR) was largely blocked by additional recA56 (92% inhibition) or lexA101 (77%) mutations, was partially blocked by additional recB21 (54%), uvrD3 (31%) or recF143 (22%) mutations, but additional polA1 or polA5 mutations had no effect on MMR. When incubated in MM after UV irradiation, the uvrB5 and uvrB5 uvrD3 strains showed essentially complete repair of DNA daughter-strand gaps (DSG) produced after UV radiation fluences up to ∼ 6 J/m² and ∼1 J/m², respectively, and then they accumulated unrepaired DSG as a linear function of UV radiation fluence. However, when they were incubated in rich growth medium after UV irradiation, they did not show the complete repair of DSG and unrepaired DSG accumulated as a linear function of UV radiation fluence. The fluence-dependent correlation observed for the uvrB and uvrB uvrD cells between UV radiation-induced killing and the accumulation of unrepaired DSG, indicates that the molecular basis of MMR is the partial inhibition of postreplication repair by rich growth medium. Rich growth medium can be just MM plus Casamino Acids or the 13 pure amino acids therein in order to have an adverse effect on survival, regardless of whether the cells were grown in rich medium or not before UV irradiation.     

THE EFFECTS OF ULTRAVIOLET AND VISIBLE RADIATION ON MOUSE ASCITES TUMOUR CELLS

Abstract— Effects of ultraviolet and visible radiation on the viability of Landschutz ascites tumour cells have been tested by growing control and treated tumour samples in adult mice. The tumour cells were irradiated as a dilute suspension in isotonic buffered salt solution, and were equilibrated at 0°C with oxygen or with nitrogen before irradiation.
Tumour cell proliferation was measured by a variety of techniques. The preferred assay-method was the growth of solid tumours in the axillae and groins of mice after sub-cutaneous inoculation of varying dilutions of treated or control ascites tumour cells. The immune response of the mice to the injected cells was reduced by whole body irradiation with a 300r dose of x-rays two days before inoculation. Results were calculated from parallel line assays using the reciprocal of the delay in appearance of the solid tumours up to 30 days post-innoculation. This reciprocal (1/T) was linearly related to the logarithm of the number of cells inoculated.
Photoreactivation has been demonstrated for this system, in which both U.V. and visible radiations were absorbed by the same cells. Light delivered alone in oxygen or in nitrogen was without effect on cell-viability, but it increased cell-survival after u.v.-irradiation in nitrogen and decreased survival after u.v.-irradiation in oxygen. Ultraviolet radiation alone was not significantly more lethal in oxygen than in nitrogen. A further observation in this work was an interaction between irradiated and control tumour cells injected into the same animal.
It is suggested that the radiation used may affect the antigenic character of the tumour cells as well as their reproductive capadity.