INDUCED REPAIR IN ESCHERICHIA COLI: INDUCTION BY ULTRAVIOLET LIGHT AT-79°C

Two of the phenomena associated with induced (S.O.S.) repair, namely induced inhibition of post radiation DNA degradation and induced radioresistance have been shown to be elicited by 245 nm radiation applied to E. coli cells in the frozen state at -79°C. The effect of radiation in this condition is considerably less photoreactivable than similar effects produced by exposure in the wet state. Since protein-DNA crosslinks are believed to be formed under these conditions, such consequences of UV radiation appear to be a potent inducer of induced repair.     

INHIBITION OF DNA REPAIR AND PRODUCTION OF SINGLE-STRAND BREAKS IN ESCHERICHIA COLI BY REDUCTONE

Abstract— Reductone (HOCH₂COCHO), a keto-aldehyde produced by thermal degradation of some sugars, at alkaline pHs, blocks the excision repair of DNA lesions in uv-irradiated wild type Escherichia coli. This probably occurs as a result of inhibition of the exonucleolytic activity of DNA polymerase I. In addition, reductone alone induces DNA single-strand breaks. Repair of this damage is mainly dependent on the polA gene products.     

PHOTOREACTIVATION OF ESCHERICHIA COLI Bs-3 AFTER IN ACTIVATION BY 313 nm RADIATION IN THE PRESENCE OF ACETONE

Abstract— The colony-forming ability of E. coli B_s-3 can be inactivated by light of 313 nm wavelength in an acetone-sensitized photochemical reaction. This ability can subsequently be restored quantitatively by illumination with photoreactivating light. A small fraction of the population cannot be inactivated; this is assumed to be due to a complete dark repair of the lesions, whatever the dose of radiation has been. Thus, such triplet energy-transfer experiments can successfully be applied to whole cells. Since thymine dimers are formed almost exclusively, this suggests a new way of studying these lesions in relation to the biologically observable effects.     

REMOVAL OF THYMINE-CONTAINING PYRIMIDINE DIMERS FROM UV LIGHT-IRRADIATED DNA BY S1 ENDONUCLEASE

Abstract— S₁ endonuclease was shown to remove thymine-containing pyrimidine dimers from UV-irradiated human DNA, although efficient removal could be demonstated only by using long digestion times, relatively high enzyme concentrations, and irradiation sufficient to yield dimer substitutions in DNA of 1 per 1W300 (dimers/base pair). Neutral and alkaline sucrose gradient analysis of strand break induction by S, of UV-irradiated DNA suggests that recognition of the dimer by S, is the limiting factor in its removal and dimer removal usually results from attack on the dimer containing DNA strand without the induction of a double-strand break.     

DNA TURNOVER IN BUFFER-HELD ESCHERICHIA COLI AND ITS EFFECT ON REPAIR OF UV DAMAGE

Abstract— Continuous DNA degradation and resynthesis, without a net change in cellular DNA content, were observed in buffer-held, non-irradiated E. coli B/r. This constant DNA turnover probably involves most of the genome and reflects random sites of DNA repair due to the polA-dependent excision-resynthesis repair pathway. Under these non-growth conditions, it appears that at any given time there is a minimum of one repair site per 6.5 × 10⁶ daltons DNA, each of which is at least 160 nucleotides long.
While the amount of DNA degradation is not influenced by prior exposure to UV radiation, the synthetic activity decreases with increasing UV fluence. We suggest that when sites of DNA turnover occur opposite to cyclobutyl dipyrimidines in UV-irradiated cells, repair of the latter damage can be prevented. This implies that both beneficial and deleterious processes take place in irradiated buffer-held cells, and that cell survival depends on the delicate balance between DNA turnover and repair of UV-damage. Based on these findings, we propose a model to explain the limited repair observed during post-irradiation liquid-holding and to account for the large difference in cell survival between irradiation at low fluence rates (fluence-rate dependent recovery) and at high fluence rates followed by liquid-holding (liquid-holding recovery).     

DNA DAMAGE IN RAT 9L CELLS TREATED WITH 8-METHOXYPSORALEN AND NEAR-UV LIGHT ASSAYED BY VISCOELASTOMETRY AND S1 NUCLEASE

–The techniques of viscoelastometry and S1 nuclease digestion were applied to the analysis of DNA damage in rat 9L cells treated with the combination of 8-MOP (8-methoxysporalen) and near-UV light. Treatment of cells with near-UV light alone resulted in a decrease in the viscoelastic retardation time under both denaturing and nondenaturing conditons. Exposure of cells to 8-MOP alone yielded a maximum in the plot of retardation time vs dose under nondenaturing conditions, similar to that found with ionizing radition. This observation suggests that treatment with 8-MOP alone leads to DNA strand breaks. Viscoelastic analysis of cell lysates under denaturing conditions demonstrated that treatment of cells with 8-MOP and UV radiation led to substantial increases in both the viscoelastic retardation time and recoil, consistent with formation of DNA interstrand cross-links. Viscoelastic analysis of cell lysates under nondenaturing conditions showed that exposure to long wavelength UV light in the presence of 8-MOP produced a decrease in retardation time. This decrease reflects the combined effect of strand breaks and interstrand cross-links. Results from the S1 nuclease assay confirmed these observations and permitted quantitation of DNA damage arising from single-strand breaks and DNA interstrand crosslinks. The importance of including the effect of strand breaks in the quantitation of cross-link formation is discussed.     

EXTERNAL HEAVY ATOM EFFECTS ON THE S1 AND T1 ELECTRONIC STATES OF INDOLE

Abstract— The absorption, fluorescence and phosphorescence spectra along with the phosphorescence decay function of indole perturbed by various amounts of halocarbons have been studied in a 1:1 ethanol-ether glass at 77 K. For carbon tetrachloride. chloroform, halothane and methylene chloride used as quenchers. the biexponential nature of the phosphorescence decay prompted us to assume the existence of a triplet state complex (exciplex) between indole and the halocarbons. On the other hand. propyl bromide gives rise to a non-exponential decay and to an increase in the phosphorescence intensity suggesting the occurrence of a normal external spin-orbital coupling interaction between indole and the propyl bromide molecule.     

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.     

PHOTOREACTIVATION OF THYMINE-STARVED ESCHERICHIA COLIBs-1

Abstract —Thymine starvation prior to 254 nm ultraviolet light (UV) exposures has been found to decrease the level of maximum photoreactivation in Escherichia coli B _s-1. The dark equilibrium level of photoreactivating enzyme-substrate complexes was determined from the levels of photoreactivation obtained with exposures to single flashes of high-intensity light. The kinetics indicate that photoreactivating enzyme concentration does not decrease as a result of thymine starvation. The UV sensitivities of normal and thymine-starved cells are found to be the same. Photoreactivation by sequential flashes shows a lesser number of total photorepairable lesions in starved cells. It is concluded that thymine starvation renders a portion of the dimers inaccessible to the photoreactivating enzyme, thus lowering the level of maximum photoreactivation.     

DNA DAMAGE AND REPAIR: INDUCTION AND REMOVAL OF THYMINE DIMERS IN ULTRAVIOLET LIGHT IRRADIATED INTACT WATER PLANTS

Abstract— The production of UV-induced thymine dimers and their fate upon post-irradiation incubation in the dark was studied in DNA of the intact water plants Wolffia microscopica and Spirodela polyrhiza. The results demonstrate production of thymine dimers, and the ability of the plant cells to remove the dimers from their DNA. The rate of removal is rapid during the first few h of post-irradiation incubation in the dark. It continues at a slower rate for the next 24–48 h, at which time it is essentially complete. The disappearance of thymine dimers in light or in the dark is analogous to the well-known processes in other biological systems, namely, photoreactivation and dark excision.     

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).     

MATRIX EFFECTS ON THE RADIATIONLESS TRANSITION' S1→T1 AND T1→S0 FOR BENZENE AND N, N, N', N'-TETRAMETHYLPARAPHENYLENE-DIAMINE MOLECULES

Abstract— The present study attempts to correlate the phosphorescence life time τ_p at 77°K of a definite solute: tetramethylparaphenylenediamine (TMPD) with various solvents viscosity and polarity. A few experiments with benzene in the same solvents are also reported. The following results have been obtained:

• 1 The measured τ_p vary regularly with the sample immersion time in liquid N₂, reaching a constant value after a few hours. This effect is related to the glass matrix relaxation. The rate constant K_isc (S, ₁→T₁) is also found to vary during relaxation of the solvent.
• 2 In the expression giving the nonradiative rate constant K_nr (T₁→S₀), the bimolecular quenching term appears negligible for high viscosity matrices i.e. for η= 10⁹ poises for benzene and for TMPD. K_nr is found to vary linearly with log η, as well as the intersystem crossing S₁→T₁ rate constant K^isc.
• 3 Both K_nr (T₁→S₀) and K^isc (S₁→T₁), increase with decreasing polarity of the solvent.
• 4 From our own observations and literature data[6] for C₆H₆ it appears that solvent viscosity does not contribute appreciably to the observed temperature effect on the solute τ_p when only a monomolecular triplet deactivation is operative.

     

EFFECT OF PHOTOREACTIVATING LIGHT ON LETHAL AND PRE-MUTATIONAL UV LESIONS IN ESCHERICHIA COLI WP2s CARRYING THE R46 MUTATOR PLASMID

Abstract—An excision-deficient E. coli strain carrying the R46 mutator plasmid showed a different response towards photo-reactivation after UV irradiation than the same strain without plasmid. While the photoreactivation of lethal lesions was comparable in both strains, the number of UV-induced mutants per 10⁶ survivors was slightly reduced for the plasmid bearing strain by photoreactivating light at UV fluences below 60 mJ/m² but increased at higher fluences. To explain this it is proposed that some UV photoproduct(s) of DNA other than cyclobutane dipyrimidine dimers are pre-mutational lesions for error-prone DNA repair by the plasmid, P-repair, but not for SOS-repair.     

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.     

THERMOLUMINESCENCE EVIDENCE FOR CHLORIDE REQUIREMENT IN THE S2→S3 TRANSITION OF THE WATER-SPLITTING SYSTEM

Abstract— The role of chloride in photosynthetic oxygen evolution was investigated by means of thermoluminescence measurements. It was found that chloride depletion in isolated chloroplasts almost completely abolished the B₁ thermoluminescence band (S₃Q_B⁻ recombination) but diminished only slightly the amplitude of the B₂ band (S₂Q_B⁻ recombination). The B₂ band could be excited to full intensity by the first flash of a flash series and subsequent flashes caused no further change in the amplitude of the band. These observations suggest a block in the S₂→S₃ transition of the water-splitting system in chloride-depleted chloroplasts. Readdition of chloride provided evidence that the inhibitory effect of chloride removal is reversible.     

LETHAL INTERACTION BETWEEN ULTRAVIOLET-VIOLET RADIATIONS AND METHYL METHANE SULPHONATE IN REPAIR PROFICIENT AND REPAIR DEFICIENT STRAINS OF ESCHERICHIA COLI

Abstract— The lethal interaction between monochromatic radiation at various wavelengths and methyl methane sulphonate was tested in strains of Escherichia coli proficient and deficient in DNA repair. In the repair proficient wild-type strain K12 AB1157, the efficiency of sensitization to MMS as a function of dose (at 334 nm, 365 nm and 405 nm) was found to be directly correlated with the dose necessary to remove the shoulder from the survival curve at the wavelength employed. The 365 nm: MMS interaction was also observed in other repair proficient E. coli strains (W3110 and B/r) but was absent in a recA and a polA strain. Pre-treatment of AB1157 with MMS leads to a much larger interaction than pre-irradiation with 365 nm. It is concluded that dose-dependent damage to DNA repair by the near-UV radiation is involved in the interaction and possibly that MMS causes irreversible damage 10 repair enzymes.     

DIFFERENCE SPECTRA OF THE OXIDIZED INTERMEDIATES IN THE PHOTOSYNTHETIC OXYGEN-EVOLVING SYSTEM: EVIDENCE FOR A SMALL S0→S1 ABSORPTION CHANGE

A consensus has emerged in the recent literature on the fact that the UV difference spectrum of the first oxidation step (S₀→ S₁) of the photosynthetic oxygen-evolving complex is significantly different and generally smaller than the spectra of the higher oxidation steps (S₀→ S₁and S₂→ S₃). Discrepancies still persist, however, notably in the 300 nm region where the S₀→ S₁ change was either reported to be markedly smaller than the other changes, or, at variance, to have a similar amplitude. A novel approach is proposed here for estimating the ratio of these changes, requiring no estimate of the Kok model parameters, such as the initial S₀/S₁ratio, or damping coefficients. This was achieved by comparing the absorption difference between two fully deactivated states, differing only in their S₀/S₁, distribution, with the flash-induced changes measured from these states. The results show that, at two wavelengths around 300 nm, the S₀→ S₁ change is at least 4 times, and probably 5–6 times smaller than the S₀→ S₁change.     

INDUCTION OF FREE RADICALS IN DNA BY PROFLAVINE AND VISIBLE LIGHT: INFLUENCE OF OXYGEN AND IONIC STRENGTH

Abstract —The photosensitization of native DNA is observed as an induction of free radicals in the DNA moiety of proflavine-DNA complexes. The intensity of the electron paramagnetic resonance spectra (at 77 K) is a measure of the number of free radicals present in frozen solutions of DNA-proflavine complexes after irradiation with visible light (Λ > 320 nm). In the absence of O₂, the photosensitization is significant but very low; it increases slightly with increasing NaCl ionic strength; it appears to be due to intercalated dye molecules and the qualitative analysis of the spectra obtained shows that mainly thymidine is involved. The reaction measured after saturation with O₂ is the same as the reaction in air but is quantitatively higher; the free radicals observed are peroxides. This induction of free radicals appears to be due to the intercalated dye molecules, each molecule acting independently. The important observation is a very sharp and large (around a hundred-fold) increase in the photosensitizing efficiency of the bound dye molecules occurring in NaCl between μ, # 0–25 and μ= 0–5 and in MgCl₂ between μ# 0–01 and μ=0–1.