JOURNAL OF BACTERIOLOGY, Apr. 1975, p. 199-205 Copyright © 1975 American Society for Microbiology
Vol. 122, No. 1 Printed in U.SA.
Tryptophan Photoproduct(s): Sensitized Induction of Strand Breaks (or Alkali-Labile Bonds) in Bacterial Deoxyribonucleic Acid During Near-Ultraviolet Irradiation GEORGE H. YOAKUM'
Division of Biological and Medical Research, Argonne National Laboratory, Argonne, Illinois
Received for publication 26 September 1974
Long-wavelength ultraviolet light (300 to 400 nm) converts L-tryptophan to a photoproduct that is toxic for bacterial cells in dark conditions. We now report that similar photoproducts of L-tryptophan sensitize bacterial deoxyribonucleic acid to 365-nm radiation, increasing the yield of deoxyribonucleic acid strand breaks (or alkali-labile bonds) by approximately 11.5-fold. Evidence is also presented which indicates that these sensitized deoxyribonucleic acid lesions contribute to lethality for Escherichia coli irradiated with 365-nm ultraviolet light in suspensions of tryptophan photoproducts. Exposure of L-tryptophan to near-ultraviolet (UV) light (320 to 400 nm), in the presence of oxygen, produces photoproducts that are toxic for bacteria (19) and mammalian cells in tissue culture (15). We have reported previously that near-UV photoproducts of L-tryptophan (TP) are especially toxic to rec and exr (X-ray sensitive) strains of Salmonella typhimurium (19). To determine the reason for the genetic specificity of TP toxicity, we conducted experiments to test the effects of TP on deoxyribonucleic (DNA) metabolism. These studies demonstrated that TP blocks a step(s) required for the iapid closure of replication gaps (21) which occur during discontinuous semiconservate DNA replication (7). In addition, we found that TP inhibits one class of X-ray repair, without apparent blockage of other X-ray repair processes.
Wild-type E. coli cells have at least two discrete mechanisms that function to repair X-ray-damaged DNA by rejoining single-strand (SS) breaks. Type II repair of X-ray-induced SS breaks requires functional polymerizing activity of the polA gene product, and can be completed in buffer at room temperature (9). Type III repair requires functional rec gene products and full medium conditions at 37 C (10). TP inhibits type III repair of X-ray-induced SS breaks without blocking type II repair of DNA SS breaks (20). Since TP blocks processes of DNA metabolism involved in the rejoining of 'Present address: University of Georgia, Department of Biochemistry, Boyd Graduate Studies Research Center, Athens, Ga. 30602.
DNA intermediates of repair and replication, we concluded that the dark toxicity of L-tryptophan photoproduct is probably a biological expression of the effect of TP on DNA metabolism (21). Many workers have reported that when intact cells are exposed to near-UV wavelengths DNA metabolism is affected. These reports suggest that endogenous photoproducts, with properties similar to those we observed for TP, may be produced in cells during near-UV irradiation of intact cells. In addition, both the production of toxic near-UV photoproducts from L-tryptophan (19) and near-UV lethality for E. coli (16) require the presence of oxygen. These correlations intimate that TP might also sensitize biological systems to near-UV wavelengths. Therefore, we tested this photoproduct for the sensitized induction of DNA strand breaks during near-UV irradiation. We now report that irradiation of cells in solutions of TP increases the yield of 365-nm DNA SS breaks (or alkali-labile bonds) by 11.5-fold and enhances the lethal effectiveness of 365-nm irradiation. MATERIALS AND METHODS Bacterial strains. The following strains were used in the experiments described below: W3110 wild type (thy) and P3478 (polAl, thy). These strains were originally derived from E. coli K-12, and their detailed genotypes and phenotypes have been described previously (21). Growth and media. Exponentially growing cells were obtained for labeling from cultures that had not exceeded 2 x 108 cells per ml. Minimal medium was buffered with M9 salts supplemented as follows
(micrograms per milliliter): glucose, 400; L-tryptophan, 20; L-proline, 10; Casamino Acids (vitamin free), 200; niacin, 1; thiamine, 1; and thymine, 10. The M9 salts (pH 6.8) contained the following (grams per liter): NH4Cl, 1.0: Na2HPO4 7H2O, 6.0; KH2PO4, 3.0; MgSO4 7H20, 0.2; and NaCl, 0.5. This medium was used for growth in all experiments in which log-phase cells were used. Stationary-phase cells were obtained from surface colonies on nutrient agar (Difco) incubated at 37 C for 48 h. Photoproduct preparation and cell irradiation. L-Tryptophan (5 mg/ml) was irradiated with 290-nm light from a Hanovia 2.5-kW xenon mercury arc lamp in a Bausch & Lomb 500-mm monochromator to an approximate absorbed dose of 106 J/m2 as described previously (21). The photoproduct (TP) was buffered and diluted with M9 buffer to an arbitrary concentration with 20% transmittance at 365 nm for a 1-cm path, as measured with a Gilford spectrophotometer. This concentration of TP was used in all experiments to facilitate accurate dosimetry. Since absorption is negligible in control samples (no TP), cells are exposed to an incident fluence rate of 365-nm radiation which is essentially equivalent to the irradiance at the surface of the sample (Io). However, L-tryptophan photoproducts (20% transmittance at 365 nm) absorb 80% of the energy incident on the surface of the sample. This absorption of 365-nm energy by TP effectively reduces the radiant energy incident on cells that are exposed to 365-nm energy in the presence of TP. To accurately determine the radiant energy incident on cells irradiated in solutions of TP an average incident fluence rate (ey) was calculated by the following formula: t = (Io - I)/ln(Io/I), where Io equals the fluence rate at the front of the sample, I equals the fluence rate after passing thru 1 cm of sample, and -y equals the average incident fluence rate in the sample. The application of this formula requires the following assumptions: (i) the important photochemical events in the TP solution, which were ultimately measured as physical or biological endpoints, occurred within the cells; (ii) absorption of photons by molecules in the suspension outside of the cells did not produce reactive intermediates with a half-life which would facilitate diffusion from outside the cell to the target molecule within the cell (i.e., DNA). Photochemical mechanisms generally require molecular contact when photon absorption occurs, and if TP sensitization operates by one of these mechanisms the corrected dosimetry is an accurate comparison of the events measured. However, if a mechanism is involved which does not require photon absorption within the cell, comparisons based on incident quanta do not apply. In this case, calculation of events based on the assumption that all quanta (absorbed and incident) in the TP solution are involved would reduce the effective yield of TP-sensitized events by approximately 50% since this is the amount of correction applied by the calculated dosimetry. Irradiation of cell suspensions was conducted at 365 nm with the monochromatic light source described previously. Control suspensions were exposed to monochromatic 365-nm radiation at a fluence rate of 500 J/m2 per s. This fluence rate was selected to match
the average incident fluence rate (y = 500 J/m2 per s) for cells irradiated in solutions of TP. A Corning Glass band-pass filter (no. 7-51) was interposed to reduce scattered-light effects. Cell suspensions were cooled on ice prior to irradiation, and the temperature of irradiation was 0 to 1 C. During irradiation cell suspensions were placed in a 1-cm2 Pyrex cuvette with a capillary bubbling tube attached to the bottom and a cooling jacket to maintain temperature. When survival was determined, stationary-phase cells were suspended in M9 buffer or TP buffered with M9 salts (TP-M9) to give a final concentration of ca. 107 cells per ml. After irradiation to various doses, samples were removed, immediately diluted, and plated in quadruplicate on nutrient agar. Survival was measured as the ability to form colonies after 48 h of incubation at 37 C. Analysis of DNA. The size of DNA after 365-nm irradiation of cells in the presence or absence of TP was determined by sedimentation through alkaline sucrose gradients by a modification of the procedure of McGrath and Williams (6). Exponentially growing W3110 cells were collected on membrane filters (washed; 0.22-Am pore size; Millipore Corp.) and suspended (ca. 5 x 107 cells per ml) in M9 medium with JCi 10 of [3H]thymidine (final specific activity, 1.5 Ci/mmol) per ml. After 90 to 120 min of incubation at 37 C, the cells were collected on membrane filters, washed with M9 buffer, and suspended in M9 buffer or TP-M9. The cell suspensions were then placed on ice. Samples were removed during irradiation, placed in a solution of KCN, tris(hydroxymethyl)aminomethane, and ethylenediaminetetraacetate (pH 8.0) (21), and held on ice. To avoid breakage of the DNA, 50 ,gl of cells (ca. 2 x 106) was added to 0.2 ml of 0.5% sodium dodecyl sulfate solution in 0.5 NaOH and layered on top of a 5 to 20% alkaline sucrose gradient (21). After allowing 30 min for lysis at room temperature, the gradients were centrifuged by using an SW56Ti rotor in a Beckman L-2 65B ultracentrifuge at 35,000 rpm for 95 min at 20 C. Eight-drop fractions were collected from the bottom of the gradients onto Whatman 17 paper strips by the method of Carrier and Setlow (2). The strips were washed once with 5% trichloroacetic acid and twice with 95% ethanol and air dried. The dried strips were cut and placed in vials with about 5 ml of Packard Permablend 1 (5.5 g/liter) in toluene and counted in a Packard Tri-Carb liquid scintillation counter. During some of these experiments, samples were removed to determine the loss of DNA as a result of nucleolytic activity during the irradiation treatment. To measure the loss of DNA during irradiation, 20-gl aliquots of cells were placed on Whatman 542 filter paper disks saturated with 5% trichloroacetic acid. The samples were then washed twice in 5% trichloroacetic acid and once in 95% ethanol in an ice bath to remove trichloroacetic acid-soluble radioactivity. The filter paper disks were then dried, placed in vials, and counted as above.
RESULTS Effect of TP on 365-nm induction of SS breaks in bacterial DNA. In Fig. 1 alkaline
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FIG. 1. Alkaline sucrose gradient profiles for DNA extracted from E. coli W3110 cells treated as follows: (0) sedimentation profile of DNA from untreated control cells held on ice in M9 buffer; (0) sedimentation profile of DNA from cells after 365-nm irradiation to 40 x 105 J/m2 in M9 buffer; (0) sedimentation of DNA from cells held on ice in TP-M9 solutions; and (*) sedimentation profile of DNA from cells 365-nm irradiation in TP-M9 solution (20%o transmittance at 365 nm). The number average molecular weights were calculated as described previously (5). If the dose of 365-nm radiation in the TP solution is not corrected for absorption, then the dose in the *profile is 8 x 105 J/m2. Calculation of the number SS breaks in DNA, using the uncorrected dosimetry, yields a rate of 8.7 x 10-6 SS breaks per 108 daltons m2/J in the presence of TP (*). Since 365-nm radiation with no TP present induces SS breaks at a rate of 1.4 x 10-6 SS breaks per 108 daltons m2/J (0), the uncorrected dosimetry yields an approximate sixfold increase in the rate of SS break induction in cells irradiated in solutions of TP. However, when TP absorption is accounted for, the corrected dose is 4 x 105 X J/m2, yielding a rate of 17.5 x 10-6 SS breaks per 108 daltons m2/J in TP solutions.
gradient sedimentation profiles are phage DNA as a marker as described previously shown for DNA released from E. coli W3110 (5). From the data in Fig. 1 we may calculate cells treated with 365-nm radiation in the pres- the following: (i) 40 x 108 J/m2 of 365-nm radiaence and absence of TP. Since this DNA is tion produces 5.49 SS breaks per 108 daltons sedimented in highly alkaline conditions the 365-nm radiation ), 132 x DNA lesions induced by either or both of these (16 x1 treatments may be alkali-labile bonds rather produces SS breaks in DNA at a rate of 1.47 x than actual strand breaks in the DNA molecule. 10- 6/108 daltons m 2/J; (ii) 4 x 108 J/m2 of 365In fact, recent measurements with phage DNA nm radiation in the presence of TP prosuggest that 365-nm radiation (no TP) induces duces 6.86 SS breaks per 108 daltons of DNA, primarily alkali-labile bonds in DNA (R. Ley, or TP (at 20% transmittance) + 365-nm rapersonal communication). For convenience, diation produces SS breaks in DNA at a rate these lesions will be referred to as DNA SS of 17.5 x 10-6/108 daltons m2/J. Also, experbreaks. The number of breaks induced was iments were conducted with a solution of calculated from the following formula: 0.01 M tris(hydroxymethyl)aminomethane and 0.05 M ethylenediaminetetraacetate (pH 8.0) SS breaks/108 daltons of DNA in the irradiation suspension, and no increase in the rate of SS breaks induced by 365-nm radiation with or without TP present was 108( Mn, - Mno) observed. The results in Fig. 1 are typical of data from a number of experiments over a where Mnt equals the number average molecu- dose range of 365-nm radiation both in the preslar weight of the treated DNA and Mn0 equals ence and absence of TP which were used to calthe number average molecular weight of un- culate the data shown in Fig. 2. The untreated treated control DNA (9). The number average Mn0, determined for DNA from cells held on molecular weight (Mn) was calculated with T4 ice in the presence or absence of TP (Fig. 1),
YOAKUM 8 0-
B. 365Nm Irradiation
A TP + 365NM Irradiation 4 C
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FIG. 2. Induction of DNA SS breaks (or alkali-labile bonds) in E. coli W3110 wild type by 365-nm irradiation with and without TP present. The data in this figure are a compilation of data similar to those shown in Fig. 1. (A) Number of SS breaks per 108 daltons of DNA induced by 365-nm irradiation of cells suspended in TP-M9 (20% transmittance at 365 nm). (---) Induction of SS breaks in DNA from cells irradiated in M9 (no TP) plotted on the same linear dose scale. (U) Number of SS breaks per 108 daltons of DNA induced in cell irradiated with 365-nm energy in M9 (no TP) as a function of dose (J/m2). (+ +) Plot of the number of SS breaks induced in cells irradiated with 365-nm energy in the presence of TP plotted on the same dose scale. The number of SS breaks per 108 daltons of DNA was calculated as described previously (8). was the same (within experimental error) for all experiments shown in Fig. 2. The data in Fig. 2A and B indicate that, both in the presence and absence of TP, SS breaks are induced by 365-nm radiation in dosedependent quantities. The value obtained from the slope of the data plotted in Fig. 2B indicates that 365-nm radiation of E. coli W3110 cells induces SS breaks in bacterial DNA at a rate of 1.38 x 10-6 SS breaks per 108 daltons m2/J. This value is in good agreement with data for bacterial DNA reported previously by Tyrrell et al. (13). However, when bacterial cells are irradiated with 365-nm radiation in suspensions containing TP (Fig. 2A), the rate of SS break induction is 15.9 x 10-6 SS breaks per 108 daltons m2/J. Thus, L-tryptophan photoproduct(s) (20% transmittance at 365 nm) has increased the yield of SS breaks induced during 365-nm radiation by 11.5-fold. The increased rate of SS breaks in the presence of TP indicates that some component of the photoproduct is a near-UV chromophore that sensitizes the DNA to 365-nm radiation. The loss of trichloroacetic acid-precipitable counts from cellular DNA during 365-nm irradiation in the presence and absence of TP was measured. During 365-nm irradiation with TP present, approximately 8% of trichloroacetic acid-insoluble radioactivity was solubilized, and
during 365-nm irradiation without TP present approximately 9% of trichloroacetic acid-insoluble radioactivity was solubilized. However, unirradiated cells held on ice during the irradiation procedure solubilized approximately 12% of trichloroacetic acid-insoluble radioactivity. Effect of TP plus 365-nm radiation on survival. The colony-forming ability of strains W3110 and P3478 polAl after 365-nm radiation, in the presence and absence of TP, is shown in Fig. 3. The presence of TP in the suspension during 365-nm irradiation (20% transmittance at 365 nm) enhances the lethality of irradiation for both wild-type and polAl strains. The dose enhancement factor (DEF) was calculated at lie (D37) by the following formula: DEF
where cD37 is dose required to inactivate, to 37% survival, cells irradiated in M9, and I,D37 is the dose required to inactivate, to 37% survival, cells irradiated in TP-M9. These results indicate that the presence of TP gives a DEF of 3.9 for polAl and 3.5 for wild type. A comparison of the number of SS breaks per lethal event in these two conditions may be obtained from the data in Fig. 2. For E. coli P3478 polA1, the
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DOSE (X10 5J/M2) FIG. 3. Survival of stationary-phase E. coli W3110 wild type and P3478 polAl after 365-nm irradiation in the presence and absence of TP. (0) Survival response of P378 polA1 to 365-nm irradiation in M9 buffer. (A) Survival response of P3478 poLAl to 365-nm irradiation in TP-M9. (0) Survival response of W3110 wild type to 365-nm irradiation in M9 buffer. (