SENSITIVITY OF STRAINS OF ESCHERICHIA COLI DIFFERING IN REPAIR CAPABILITY TO FAR UV,NEAR UV AND VISIBLE RADIATIONS*

Abstract— In stationary phase, strains of Escherichia coli deficient in excision (B/r Her) or recombination repair (K.12 AB2463) were more sensitive than a repair proficient strain (B/r) to monochromatic near-ultraviolet (365 nm) and visible (460 nm) radiations. The relative increase in sensitivity of mutants deficient in excision or recombination repair, in comparision to the wildtype, was less at 365 nm than at 254 nm. However, a strain deficient in both excision and recombination repair (K12 AB2480) showed a large, almost equal, increase in sensitivity over mutants deficient in either excision or recombination repair at 365 nm and 254 nm. All strains tested were highly resistant to 650 nm radiation. Action spectra for lethality of strains B/r and B/r Her in stationary phase reveal small peaks or shoulders in the 330–340, 400–410 and 490–510 nm wavelength ranges. The presence of 5μg/ml acriflavine (an inhibitor of repair) in the plating medium greatly increased the sensitivity of strain B/r to radiation at 254, 365 and 460 nm, while strains E. coli B/r Her and K12 AB2463 were sensitized by small amounts. At each of the wavelengths tested, acriflavine in the plating medium had at most a small effect on E. coli K.12 AB2480. Acriflavine failed to sensitize any strain tested at 650 nm. Evidence supports the interpretation that lesions induced in DNA by 365 nm and 460 nm radiations play the major role in the inactivation of E. coli by these wavelengths. Single-strand breaks (or alkali-labile bonds), but not pyrimidine dimers are candidates for the lethal DNA lesions in uvrA and repair proficient strains. At high fluences lethality may be enhanced by damage to the excision and recombination repair systems.     

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).     

DAMAGE TO URACIL- AND ADENINE-CONTAINING BASES, NUCLEOSIDES, NUCLEOTIDES AND POLYNUCLEOTIDES: QUANTUM YIELDS ON IRRADIATION AT 193 AND 254 NM

Abstract Photoreactions, such as base release and decomposition of the base moiety, induced by either 20 ns laser pulses at 193 nm or continuous 254 nm irradiation, were studied for a series of uracil and adenine derivatives in neutral aqueous solution. The quantum yield of chromophore loss (φ) depends significantly on the nature of the nucleic acid constituent and the saturating gas (Ar, N₂O or O₂). In the case of polynucleotides the destruction of nucleotides was measured by high-performance liquid chromatography after hydrolysis; the quantum yields (φ) are comparable to those of chromophore loss or larger. The φ_cl and aφ_dn of 0.04–0.1 for poly(U) and poly(dU), obtained for both wavelengths of irradiation, are due to processes originating from the lowest excited singlet state, i.e. formation of photohydrates and photodimers, and a second part from photoionization using λ_irr= 193 nm. Irradiation at 193 nm effectively splits pyrimidine dimers and thus reverts them into monomers. The quantum yield for release of undamaged bases (φ_br) from nucleosides, nucleotides and polynucleotides upon irradiation at 254 nm is typically φ_br= (0.1–1) × 10^?4 Breakage of the N-glycosidic bond is significantly more efficient for λ_irr=193 nm, e.g. φ_br= 1.1 × 10^?3, 0.8 × 10^?3, 4.3 × 10^?3 and 0.5 × 10^?3 for poly(A), poly(dA), poly(U) and poly(dU) in Ar-saturated solution, respectively. Enhanced φ values for λ_irr= 193 nm, essentially for adenine and its derivatives, are caused by photo-processes that are initiated by photoionization.     

OYGEN DEPENDENCE AND REPAIR OF LETHAL EFFECTS OF NEAR ULTRAVIOLET AND VISIBLE LIGHT*

Abstract— –Lethality in a repairable strain (WP2) and an excision repair deficient strain (WP2hcr) of Escherichia coli was studied at wavelengths of 254, 313, 365, and 390–750 nm. Survival curves were empirically fitted to the expression S= 1 - (1-e^-kl)“, where S is the fraction surviving, D is the incident dose in ergs mm^-2, k is the inactivation constant in units of (erg mm^-2)^-1 and n is the ‘shoulder constant’. The repairable sector (k(hcr^-)–k(hcr^-)lk(hcr^-), a conservative estimate of the repair capability of E. coli WP2, was 0.91 at 254 nm, 0.92 at 313 nm, 0.60 at 365 nm, and 0.13 at 390–750 nm. Although there was no oxygen enhancement of inactivation at 254 nm and 313 nm, a strong enhancement was identified at 365 nm and 390–750 nm. These results suggest that oxygen-dependent damage induced by near u.v. (365 nm) can be partially repaired by the excision-repair system in E. coli.     

EVIDENCE AGAINST DNA AS THE TARGET FOR 334 NM-INDUCED GROWTH DELAY IN ESCHERICHIA COLI*

Abstract— Growth delay was induced with near-UV (334 nm) radiation in Escherichia coli K12 bacterial strains followed by attempts at photoreactivation (PR) of this effect at 405 nm. In the UV-sensitive strain AB2480, a small PR of the observed population growth delay occurred after 334 nm irradiation at 35°C and a much larger PR after 334 nm irradiation at 5°C. However, much of the population growth delay in this strain can be explained as being due to killing, and all or most of the observed PR pertains only to this killed fraction of the population. The true cell growth delay (i.e. that of surviving cells) thus appears to be only slightly, if at all, photoreactivable. This conclusion is supported by studies with a wild-type strain KW8, which shows growth delay at non-lethal doses; this growth delay shows no PR, regardless of the temperature during 334 nm irradiation. These findings indicate that photoreactivable lesions (cyclo-butyl pyrimidine dimers) are not an important cause of near-UV-induced growth delay. Strain AB2480 lacks known dark-repair systems for DNA damage induced by far-UV (below 300 nm) radiation, yet shows the same efficiency for 334-nm-induced growth delay as the wild type, which possesses these dark repair systems. This indicates that lesions in DNA that are dark-repairable by the systems not tunctional in AB2480are not responsible for 334-nm-induced growth delay. It is possible, however, that fragmentary repair systems in AB2480 can operate on some DNA lesion that might cause growth delay. Spontaneously decaying lesions are unlikely, since growth-delay damage decays at a very low rate in non-nutrient medium. Since most of the known types of DNA damage and repair are thus eliminated, these considerations suggest that DNA damage is not involved in near-UV-induced growth delay.     

DNA polymerase I-mediated repair of 365 nm-induced single-strand breaks in the DNA of Escherichia coli.

Abstract. Irradiation of closed circular phage Λ DNA in vivo at 365 nm results in the induction of single-strand breaks and alkali-labile lesions at rates of 1.1 × 10^-14, and 0.2 × 10^-14/dalton/J/m², respectively. The sum of the induction rates is similar to the rate of induction of single-strand breaks plus alkali-labile lesions (1 × 10^-14/dalton/J/m²) observed in the E. coli genome. Postirradiation incubation of wild-type cells in buffer results in rapid repair of the breaks (up to 80% repaired in 10 min). No repair was observed in a DNA polymerase I-deficient mutant of E. coli.     

BIOLOGICAL DEACTIVATION AND SINGLE-STRAND BREAKAGE OF PLASMID DNA BY PHOTOSENSITIZATION USING TRIS(2,2''- BIPYRIDYL)RUTHENIUM(II) AND PEROXYDISULFATE

Abstract— Single-strand break formation and biological deactivation of plasmid pBR322 DNA in the presence of tris(2,2′-bipyridyl)-ruthenium(II), Ru(bpy)2/3;⁺, and K₂S₂O₈, upon irradiation with visible light(400–500 nm), were studied in aqueous solution at room temperature. Conditions of complete binding of Ru(bpy)2/3;⁺ to the strand were employed. The damage is initiated mainly by the SO^2/3;; radical anion. Under anoxic conditions at a ratio of nucleotide to sensitizer concentrations (N/S) of 18 and S₂O2/8^- concentrations of 0.5 mM the quantum yield of single-strand break (ssb) formation is φ_ssb= 8.4 times 10^-3 while that of biological deactivation (bd) is Ø_bd= 7.6 times 10^-3 (φ_ssb= 5.2 times 10^-36.4 times 10^-3, 6.0 times 10^-3 and φ_bd= 4.2 times 10^-3, 5.2 times 10^-3, 4.8 times 10^-3 at N/S=3, 6, and 9, respectively). The quantum yields are approximately 2.5 times smaller in air-saturated solutions. At N/S = 18 about 33 SO₄^-radical anions are required per one lethal event. φ_bd increases linearly with the S₂Oφ^- concentration (up to 0.5 mM). The damage to DNA is drastically reduced on addition of mono- or divalent salts (e.g. NaC10₄, MgCl₂). These additives cause the release of Ru(bpy)2⁺ from the strand. The observed damage to DNA is thus the result of a site specific reaction. When the phenanthroline analogue, Ru(phen)φ⁺, is used as sensitizer, φ_ssb and φ_bd are three times smaller.     

Liquid-holding effects in U.V.-irradiated phage T3

Abstract— Holding complexes of u.v.-irradiated (254 nm) T3 phage in E. coli B/r cells for several hours at 37°C in buffer, or broth with chloramphenicol, affects the phage survival in at least two different ways: (1) by enhancing excision repair, resulting under certain conditions in liquid-holding recovery (LHR), and (2) by destroying the phage (holding inactivation). LHR is most apparent in buffer containing 20 μg ml^-1 chloramphenicol (CAP). It is expressed by as much as a 10–fold increase in the fraction of complexes that display host-cell reactivation (resulting from excision repair), but the percentage of u.v. lesions repaired within repair-proficient complexes is slightly decreased. LHR is not observed if T3 infects the repair-deficient strain B_s-1. Holding inactivation is readily observed with unirradiated phage complexes in broth containing CAP. The response of irradiated-phage complexes to liquid-holding conditions is more complex: holding inactivation is less effective for irradiated than for unirradiated phage DNA (i.e. the irradiated DNA is to some extent ‘protected’), and processes leading to LHR are superimposed. Thus under certain holding conditions one observes the paradoxical phenomenon that the viable titer of irradiated phage is several times higher than that of unirradiated phage. The nature of holding inactivation is not known, nor is the mechanism by which irradiated DNA is partially protected against it. Holding inactivation does not require protein synthesis; it is rather enhanced at high CAP concentration and seems to be favored by otherwise active cell metabolism. At high CAP concentrations (200–400 μg ml^-1, as compared to 20 μg ml^-1) irradiated-phage complexes show neither LHR nor protection against holding inactivation. Likewise they fail to undergo some step by which the phage DNA becomes insensitive to repair inhibition by caffeine.     

The sensitising effect of a sunscreening agent, p-aminobenzoic acid, on near UV induced damage in a repair deficient strain of Escherichia coli.

Abstract. The use of repair deficient strains of bacteria for detecting mutagenic properties of chemical species is now an established technique. In this paper we present inactivation results obtained with Escherichia coli K12 AB2480 (uvr A rec A) which indicate that p-aminobenzoic acid, a common component of sunscreening formulations, may increase the frequency of lethal damage induced in DNA when cell populations are exposed to near ultraviolet radiation. Preliminary experiments, utilising the selective action of photoenzymatic repair, indicated that the sensitisation to near ultraviolet radiation is partially but not wholly due to increased formation of pyrimidine dimers.     

Disinfection and Mechanistic Insights of Escherichia coli in Water by Bismuth Oxyhalide Photocatalysis

This study demonstrates the potential of a new BiOCl_0.875Br_0.125 photocatalyst to disinfect Escherichia coli in water under simulated solar irradiation. Photocatalytic efficiency was examined for different photocatalyst loadings, solar wavelengths, exposure times, photocatalyst concentration × contact time (Ct) concept and with the use of scavengers. To elucidate the inactivation mechanism, we examined DNA damage, membrane damage, lipid peroxidation and protein release. Both photolysis and photocatalysis were negligible under visible irradiation, but enhanced photocatalytic activity was observed under solar UVA (λ > 320 nm) and UVB (λ > 280 nm), with 1.5 and 3.6 log inactivation, respectively, after 40 min of irradiation. The log inactivation vs Ct curve for E. coli by UVA/BiOCl_0.875Br_0.125 was fairly linear, with Ct = 10 g L^?1 × min, resulting in 2 log inactivation. Photocatalytic treatment led to membrane damage, but without lipid peroxidation. Accordingly, protein was released from the cells after UVA or UVA/BiOCl_0.875Br_0.125 treatment. Photocatalysis also increased endonuclease‐sensitive sites vs photolysis alone, by an unknown mechanism. Finally, E. coli inactivation was not influenced by the addition of tert‐butanol or l ‐histidine, implying that neither hydroxyl radicals nor singlet oxygen reactive species are involved in the inactivation process.     

DIFFERENTIAL SENSITIVITY TO INACTIVATION OF NUR AND NUR+ STRAINS OF ESCHERICHIA COLI AT SIX SELECTED WAVELENGTHS IN THE UVA,UVB AND UVC RANGES*

The radiation response of stationary-phase cells of Escherichia coli strains RT4 (nur⁺) and RT2 (nur) was measured at 6 selected wavelengths between 254 and 405 ran. The relative response of the nur⁺. and nur strains was almost the same at 254 and 290 nm. However, the differential sensitivity of the RT4 and RT2 strains (ratio of the initial F₃₇ values of the nur⁺ to the nur strains) was 2.7 at 313 nm, 3.2 at 334 nm, 3.1 at 365 nm, and 2.3 at 405 nm. Thus, the fluence enhancing effect of the nur genotype extends over the wavelength range of approximately 300 to 420 nm. The substantial effect of nur at 313 nm strongly suggests that the increased sensitivity of the nur strain is the consequence of a repair deficiency that reduces the efficiency of mending DNA lesions produced by UVA (320–400 nm) and UVB (290–320 nm), but not UVC (200–290 nm) radiation.     

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.     

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.     

PRIMARY INTERMEDIATES IN THE PULSED IRRADIATION OF RETINOIDS

Abstract Laser flash photolysis and pulse radiolysis have led to the characterisation of several shortlived intermediates formed after irradiation of retinoic acid and retinyl acetate in hexane or methanol. For retinoic acid, the triplet state, wavelength maximum 440 nm, extinction coefficient 7.3 × 10⁴ dm³ mol^?1 cm^?1, decay constant 6.2 × 10⁵ s^?1, is formed with a quantum yield of 0.012 for 347 nm excitation. The radical cation, absorption maximum 590 nm, extinction coefficient ～7 × 10⁴ dm³mol^?1 cm^?1, is formed in a biphotonic process. The radical anion, absorption maximum 510nm in hexane, 480 nm in methanol where its extinction coefficient is 1.2 × 10⁵ dm³mol^?1 cm^?1, appears to decay partially in methanol into another longer-lived neutral radical, wavelength maximum 420 nm, by loss of OH^?. For retinyl acetate, the triplet state, absorption maximum 395 nm, extinction coefficient 7.9 × 10⁴dm³mol^?1 cm^?1, decay constant 1.2 × 10⁶s^?1 is formed with a quantum yield of 0.025 for 347 nm excitation. Monophotonic photoelimination of OCOCH₃? in methanol produces the retinylic carbenium ion, wavelength maximum 590 nm, whose decay is enhanced by ammonia, k ～ 2 × 10⁶ dm³ mol^?1 s^?1 and retarded by water. The radical cation also has a wavelength maximum at 590 nm, its extinction coefficient being ～ 1.0 × 10⁵ dm³mol¹ cm^?1. The long-lived transient absorption with maximum at 385 nm, extinction coefficient 1.0 × 10⁵ dm³mol^?1 cm^?1, obtained from the reaction of the solvated electron with retinyl acetate in methanol may be due to either the radical anion itself or more likely the radical resulting from elimination of OCOCH₃? from this anion. These results suggest that skin photosensitivity caused by retinyl acetate might be greater than that due to retinoic acid.     

OXYGEN EFFECT IN THE PROTECTION OF E. COLI AGAINST U.V. INACTIVATION AND MUTAGENESIS BY ACRIDINE ORANGE*

Abstract— Protection by acridine orange against ultraviolet light effects in resting cells of E. coli B/r/1, try^- was studied with special reference to a possible oxygen effect. Dose-response relationships were described by the function S= 1–(1 - e-^kD)ⁿ where S is the surviving fraction and D is the u.v. dose in ergs/mm². For cells suspended in 5 × 10^--⁶M acridine orange (AO) in air, the radiation sensitivity k was reduced from 0.010 (ergs/mm²)^)-1 in the absence of the dye to 0.0053 (ergs/mm²)^-1 in the presence of the dye. Under anoxia at this AO concentration, k was further reduced to 0.0015 (ergs/mm²)^-1. The oxygen effect ratio, kO₂/k_N2, was 3.5 at this concentration of AO. Greater protection was observed in cells suspended in 2 × 10^--⁵M AO, the oxygen effect ratio was unchanged. No oxygen effect was detected in the u.v. response in the absence of the dye. The value of n was reduced from about 12 with no dye to about 5 at dye concentrations of 5 × 10^--⁶M AO or more when oxygen was present. Under anoxia, in the presence of AO, n was further reduced to about 1.3. Atebrin, an efficient u.v. protective agent but an inefficient photodynamic agent, had no oxygen effect for protection against u.v. inactivation. Acridine orange protected against u.v.-induced reversion to tryptophan indepence in E. coli WP2 to about the same extent as it did for inactivation. A similar oxygen effect was observed for both inactivation and mutagenesis.     

Kinetics and mechanism of protection of thymine from phosphate radical anion under anoxic conditions

The rates of photooxidation of thymine in the presence of peroxydiphosphate (PDP) have been determined by measuring the absorbance of thymine at 264 nm spectrophotometrically. The rates and the quantum yields (φ) of oxidation of thymine by phosphate radical anion have been determined in the presence of different concentrations of dithiothreitol (DTT). An increase in DTT is found to decrease the rate of oxidation of thymine, suggesting that DTT acts as an efficient scavenger of PO₄^·2? and protects thymine from it. Phosphate radical anion competes for thymine as well as DTT; the rate constant for the phosphate radical anion with DTT has been calculated to be 2.21 × 10⁹ dm³ mol^?1 s^?1, assuming the rate constant of phosphate radical anion reaction with thymine as 9.6 × 10⁷ dm³ mol^?1 s^?1. The quantum yields of photooxidation of thymine have been calculated from the rates of oxidation of thymine and the light intensity absorbed by PDP at 254 nm, the wavelength at which PDP is activated to phosphate radical anion. From the results of experimentally determined quantum yields (φ_exptl) and the quantum yields calculated (φ_cl), assuming DTT acts only as a scavenger of PO₄^·2? radicals, show that φ_exptl values are lower than φ_cl values. The φ′ values, which are experimentally found quantum yield values at each DTT concentration and corrected for PO₄^·2? scavenging by DTT, are also found to be greater than φ_exptl values. These observations suggest that the thymine radicals are repaired by DTT in addition to scavenging of phosphate radical anions. © 2001 John Wiley & Sons, Inc. Int J Chem Kinet 33: 271–275, 2001     

PHOTOPROTECTION OF E. COLI B/r: RESPIRATION,GROWTH, MACROMOLECULAR SYNTHESIS AND REPAIR OF DNA*

Abstract— Photoprotecting effects of near UV radiations (300–400 nm, maximum at 360 nm) against far UV radiations (primarily 254 nm) have been studied in Escherichia coli B/r cells in minimal medium with glycerol as a carbon source. Near UV light (10⁵ Jm^-2) has a negligible effect on survival, but causes transitory inhibition of respiration, growth, DNA, RNA, and protein syntheses and cell division. Far UV (52 J m^-2) reduces survival to about 0.5 per cent; respiration, growth and RNA and protein syntheses proceed for about 60 min, after which they nearly cease for several hours. Near UV given before this fluence of far UV increases survival 10-fold and the above processes resume at times and with kinetics characteristic of those produced by lower fluences of far UV. Single-strand breaks appear in the DNA of both unprotected and photoprotected cells; repair of the breaks is essentially complete in protected but not unprotected cells. The viability kinetics for far-UV-irradiated cells with and without photoprotecting treatment are identical except that the curve for the latter is displaced upward about 1 log; exponential increases (cell division) begin at 120 min in each case. The data suggest that, in B/r cells grown under our particular conditions, namely in minimal medium with glycerol, photoprotection is not the result of growth or division delays, but reflects an increased repair capability due to continued respiration.     

EXCITED-STATE PROPERTIES OF DNA METHYLATED AT THE NV7 POSITION OF GUANINE AND ITS FREE FLUOROPHORE AT ROOM TEMPERATURE

The room-temperature optical properties of calf thymus DNA, with about 75% of its guanine residues methylated at position N-7, are compared with those of 7-methyl GMP which has the same fluorophore. The fluorescence spectrum of the methylated guanine residues depends strongly on the excitation wavelength, shifting to the blue as the wavelength increases. The fluorescence quantum yield, corrected for the contribution to absorption by the other virtually nonfluorescent residues, exhibits a pronounced drop at long excitation wavelengths relative to that for excitation at 265 nm. The degree of fluorescence polarization exhibits a weak dependence on the excitation and emission wavelengths. For 7-methyl GMP, the fluorescence spectrum is very weakly dependent on the excitation wavelength and its fluorescence quantum yield shows a moderate increase at long wavelengths. The degree of fluorescence polarization increases with increasing excitation wavelength particularly when monitoring the emission in the short wavelength region of the fluorescence spectrum. A pronounced drop of unknown origin is observed when exciting at 265 nm, which is not observed for methylated DNA. The methylated DNA data are interpreted in terms of a combination of (i) a heterogeneous environment of the methylated guanine residues, which results from sequence-dependent stacking interactions, and (ii) transfer of excitation energy from the other residues to the fluorescing methylated guanine residues. From the values of the quantum yields and those of the decay times, which we have recently reported (Georghiou et al., 1985), the following values are obtained for the radiative, k_t, and the sum of the nonradiative, σk₁, rate constants for deexcitation of the excited states of methylated DNA and its free fluorophore: 1.6 × 10⁸ s^-1 7 × 10⁷ s^-1 and 5 × 10¹⁰ s^-lvs 6 × 10⁹ s^-1. Because of energy transfer from the other residues. the k_f value for the methylated guanine residues is overestimated but their σk₁, value is not affected significantly and is by about an order of magnitude larger than that for 7-methyl GMP, apparently because of stacking interactions.     

NONRECIPROCAL SYNERGISTIC LETHAL INTERACTION BETWEEN 365-nm and 405-nm RADIATION IN WILD TYPE and uvrA STRAINS OF ESCHERICHIA COLI*

Abstract— Lethality by 405-nm radiation in three repair-proficient and two uvrA strains of Escherichia coli that belong to two isogenic series was greatly enhanced by prior exposures to 365-nm radiation at fluences greater than 1 times 10₆Jm_-2. Fluences at 365 nm that yielded a surviving fraction of 0.10 (>1 times 10₆ Jm_-2) in the 5 strains tested resulted in the following 405-nm fluence enhancement factors (FEF, ratio of the 405-nm F₃₇ in the absence of a prior 365-nm irradiation to that in the presence): strain K.12 AB1157 (wild type), 8.7; strain B/r (wild type), 52; strain WP2 (wild type), 25; strain WP2s (uvrA), 13; strain K.12 AB1886 (uvrA), 15. The maximal 405-nm FEF value obtained after a prior 365-nm irradiation at greater fluences was 83 in the wild-type strain B/r. Enhancement of anoxic 405-nm radiation after a prior aerobic 365-nm exposure was not detectable, suggesting that prior aerobic irradiation at 365-nm increased the effects of damage produced at 405 nm by means of an oxygen-dependent process. Single-strand breaks (or alkali-labile bonds) were produced by 405-nm radiation at 3.0 times 10_-5 breaks per 2.5 times 10₉ daltons per Jm_-2 in the polA strain P3478; pyrimidine dimers were not detected by biological assay (photoreactivation) at 405 nm. Although the introduction of different DNA lesions produced by 365- and 405-nm radiations cannot be ruled out, we propose that the strong synergistic effect of 365-nm irradiation on 405-nm lethality is the consequence of pronounced inhibition by 365-nm radiation of components of the DNA-repair systems that can mend or bypass damage produced by 405-nm radiation.     

The Fluorescence of native DNA at room temperature

The fluorescence spectra (300–500nm) quantum yields (φ_f) and excitation spectra (240–285 nm) are reported for neutral aqueous sloutions of purified native DNA from calf thymus, E. coli bacterium, and hen erythrocyte near 20°C. The same properties were also measured for a reference solution of mononucleotides, and direct comparisons were made. Whether purified or not, the DNA spectra all closely resemble that from the monomer mixture between 300 and 360 nm but shows a broad, low level shoulder at λ_max ≈ 450nm which is absent in the monomer spectrum. The φ_f for the purified DNA is (4 = 1) x 10^-5, about half that of the monomer reference solution and unpurified DNA. The excitation spectrum is slightly red-shifted from the absorption for both the DNA and the monomer mixture, but not for the individual monomers. The fluorescence exhibits abrupt changes associated with the denaturation of DNA at ≈ 80°C and pII) <4 or > 11.