163
Mutation Research, 37 (1976) 163--172 © Elsevier/North-Holland Biomedical Press
LETHAL EFFECTS OF PYRIMIDINE DIMERS INDUCED AT 365 nm IN STRAINS OF E. COL! DIFFERING IN REPAIR CAPABILITY *
R O B E R T B. WEBB, M I C K E Y S. B R O W N a n d R E X M. T Y R R E L L l
Division o f Biological and Medical Research, Argonne National Laboratory, Argonne, Illinois 60439 (U,S.A.) and I Dzstituto de Biofisica (Bloco G), Centro de Ciencias da Saude, Universidade federal do Rio de Janeiro, Ilha De Fundao, Rio de Janeiro, (Brasil) ( R e c e i v e d D e c e m b e r 23rd, 1 9 7 5 ) (Revision received May 2 5 t h , 1 9 7 6 ) ( A c c e p t e d J u n e 15th, 1 9 7 6 )
Summary Photoreactivation (PR) after 365-nm inactivation was measured in four strains of Escherichia coli differing in repair capability. Photoreactivation was observed in the recA strains K12 AB2480 and K12 AB2463 indicating a significant role of pyrimidine dimers in the lethal action of 365-nm radiation in these strains. Significant PR was not observed in the uvrA strain, K12 AB1886, or in the repair proficient strain, K12 ABl157, after 365-nm inactivation. Biological evidence indicated that stationary phase cells had not lost the capacity for photo-cnzymatic repair after fluences of 365-nm radiation of 2 X 106 J/m -2 or less. It is proposed that pyrimidine dimers, although induced, are not significant 365-nm lethal lesions in uvrA and wild-type strains because of their efficient dark repair.
Introduction Photoreactivation (PR) of damage induced by 365-nm radiation has been demonstrated in Escherichia coli K12 AB2480, a strain deficient in both excision repair (uvrA) and recombination repair (recA) [3]. This biological evidence for the induction of pyrimidine dinaers by 365-nm radiation was confirmed by chemical assay [11]. The ratio of sensitivity of strain K12 AB2480 at 254 nm and 365 nm was 106, a ratio close to that reported by Tyrrell [11] for dimer induction (7 X 10s). Although dimers clearly cause most of the inactivation at 365 nm (at high * This work supported by the U.S. Energy Research and Development Administration.
164 fluence rates and 0°C) in the UV sensitive E. coli strains K12 AB2480 (uvrA, recA) and Bs_ l (hcr, exr, ill) [3,16], their contribution to 365-nm lethal damage in more resistant strains has not been established. The photoreactivation enzyme is partially destroyed in both in vivo (E. coli) and in vitro (yeast) by the higher 365-nm fluences required to inactivate more resistant strains [16]. In this study we have attempted to assess the role of pyrimidine dimers in the lethal action of 365-nm radiation. We tested for photoreactivation after 365-nm inactivation in four isogenic strains of E. coli K12 that differ in repair capability. The presence of PR enzyme activity after 365-nm inactivation was determined by measuring photoreactivation after 365-nm inactivation and the introduction of additional dimers by 254-nm irradiation. Stationary phase cells were used because they apparently contain a greater number of active PR enzyme molecules [14]. Materials and methods
Uacterial strains Strains of E. coli differing in repair capability used in this study are listed in Table I. The strains were tested for their basic 254-nm sensitivity at the start of the study. Stocks were maintained on nutrient agar incubated at 37°C. For experiments, surface growth on nutrient agar plates incubated for 48 h was suspended in M9 salts buffer, pH 7 and washed twice by centrifugation before resuspending at approx, l 0 s cells per ml for 365-nm irradiations or at approx. 107 cells per ml for 254-nm irradiations. Cell suspensions were irradiated in quartz (254 nm) or pyrex (365 nm) vessels 1 cm in diameter with a capillary tube affixed to the b o t t o m to provide for aeration and stirring by means of a stream of moist air. A jacket of the same material surrounded the vessel to provide the circulation of a thermostated cooling solution to maintain the cell suspension at 0 to I°C during all irradiations. After irradiation procedures 0.2 ml of appropriately diluted suspensions were spread on the surface of nutrient agar plates in quadruplicate and incubated at 37°C for 48 h before counting surface colonies as a measure of the cells surviving the treatment. Radiation sources The 254-nm source was a 2 inch Penray low pressure mercury lamp with integral filter (G-275) to reduce near UV and visible radiations (SC-11 Ultra-Vio-
TABLE I S t r a i n s o f E. coli e m p l o y e d Strain
E. E. E. E.
coli coli coli coli
K12 K12 K12 K12
AB2480 AB2463 AB18S6 ABl157
a The isolate of strain AB2480 loci.
Repair genotype
Nutritional requirements
Source
uvrA6, recA13 cecAl3 uvrA6 wild-type
Pro, his, his, arg,
Howard-Flanders ttoward-Flanders Howard-Flanders Howard-Flanders
thi arg, arg, his,
a p r o , thr, leu, thi p r o , thr, leu, thi p r o , thr, leu, thi
u s e d i n t h i s s t u d y w a s f o u n d t o h a v e r e v e r t e d a t t h e his, arg, t h r a n d l e u
165 let Products, Inc.). Exposures were determined with a shutter controlled by an electronic timer. Fluence rates (approx. 0.1 W/m 2 for strain AB2480 and 0.8 W/m 2 for the other strains) were measured with a calibrated General Electric Germicidal meter. The 365-nm source was a Hanovia 2.5-kW Hg/Xe arc lamp operating in a Schoeffel housing with a predispersion prism. The 365-nm band was isolated by a Bausch and Lomb 500-mm monochromator. Short wavelength scattered light was eliminated with a Corning 7-51 filter. Fluence rates of 1000 to 1500 W/ m 2 were obtained with this source. Fluence rates were measured with a calibrated radiometer (Yellow Springs Instrument Co., l~ettering Model 65). Photoreactivation Cell suspensions containing between 106 and 107 cells per ml were irradiated in 1-cm square pyrex cuvettes by a 500 W quartz-iodine lamp in a Sawyer 707Q projector operating at 135 V to increase the near UV and blue parts of the spectrum. The beam was filtered through a bath containing CuSO4 and CoSo4 resulting in a band from 380 to 440 with a peak at 400 nm. An additional lens was added to provide a focused field of 10 cm square. Fluence rate was 20 W/m 2 for the radiation sensitive strains and 50 W/m z for the radiation resistant strains. PR exposures were 30 min. No inactivation was observed from the PR treatment alone. In some experiments 405-nm radiation from the 2.5-kW Hg/ Xe arc lamp used for the 365-nm source was also used for photoreactivation. Shorter wavelength scattered light was reduced by an optics technology LP400 filter (1% transmittance at 370 nm). All procedures were carried out in a laboratory illuminated with yellow fluorescent lamps (Champion F40/G0). Results Photorepair after 365-nm inactivation in the far-UV sensitive double m u t a n t E. coli K12 AB2480 is shown in Fig. 1A. The PR sector ((k o --keR)/ko where ko and kpR are rate constants for inactivation in the absence and presence of photoreactivation respectively) of 0.8 compares favorably with that after 254nm inactivation (Fig. 2A) [3,16]. The tail on the 365 nm survival curve previously reported for this strain [3,16], attributed to concomitant PR, was not observed at the greater fluence rates used in these studies. The demonstration by chemical assay of 365-nm induced dimers by Tyrrell [11], together with the observation of a large PR sector clearly establish a predominant role of pyrimidine dimers in the lethal action of this wavelength on strain AB2480. E. coli K12 AB2463 (recA) is much more resistant than AB2480 to 365-nm radiation, The photoreactivable sector of AB2463, although significant in the low dose range of inactivation, was small (Fig.lC). The inactivation fluence of 365-nm radiation at which PR was no longer obtainable was 2.0 × 106 J/m 2. The much greater photoreactivation of AB2463 after 254-nm inactivation is shown in Fig. 2C. E. coli K12 AB1886 (uvrA) showed little, if any, photoreactivation at any inactivation fluence level of 365-nm radiation (Fig.lB). Strain A B l 1 5 7 (wildtype) also showed no photoreactivation after 365-nm inactivation (Fig.lD). With this strain cells inactivated at 365 nm showed a small additional reduc-
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KI2 A B 2 4 6 5 rec~
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?'ig. 1. I n a c t i v a t i o n a t 3 6 5 n m (O°C) in t h e p r e s e n c e a n d a b s e n c e o f p h o t o r e a c t i v a t i o n ( 2 5 ° C ) o f f o u r s t r a i n s o f E. coli K 1 2 i n s t a t i o n a r y p h a s e . T h e i n f l u e n c e r a t e s ~t 3 6 5 n m w e r e 1 , 0 - - 1 . 2 X 1 0 3 W / m 2. F l u e n c e r a t e s o f P R i l l u m i n a t i o n (380--430nm) wcre 20 W/m 2 for AB2480,100 W/m 2 for AB1886 and AB2463, and 150 ~V/m 2 f o r A B l 1 5 7 .
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Mutation Research, 37 (1976) 163--172 © Elsevier/North-Holland Biomedical Press
LETHAL EFFECTS OF PYRIMIDINE DIMERS INDUCED AT 365 nm IN STRAINS OF E. COL! DIFFERING IN REPAIR CAPABILITY *
R O B E R T B. WEBB, M I C K E Y S. B R O W N a n d R E X M. T Y R R E L L l
Division o f Biological and Medical Research, Argonne National Laboratory, Argonne, Illinois 60439 (U,S.A.) and I Dzstituto de Biofisica (Bloco G), Centro de Ciencias da Saude, Universidade federal do Rio de Janeiro, Ilha De Fundao, Rio de Janeiro, (Brasil) ( R e c e i v e d D e c e m b e r 23rd, 1 9 7 5 ) (Revision received May 2 5 t h , 1 9 7 6 ) ( A c c e p t e d J u n e 15th, 1 9 7 6 )
Summary Photoreactivation (PR) after 365-nm inactivation was measured in four strains of Escherichia coli differing in repair capability. Photoreactivation was observed in the recA strains K12 AB2480 and K12 AB2463 indicating a significant role of pyrimidine dimers in the lethal action of 365-nm radiation in these strains. Significant PR was not observed in the uvrA strain, K12 AB1886, or in the repair proficient strain, K12 ABl157, after 365-nm inactivation. Biological evidence indicated that stationary phase cells had not lost the capacity for photo-cnzymatic repair after fluences of 365-nm radiation of 2 X 106 J/m -2 or less. It is proposed that pyrimidine dimers, although induced, are not significant 365-nm lethal lesions in uvrA and wild-type strains because of their efficient dark repair.
Introduction Photoreactivation (PR) of damage induced by 365-nm radiation has been demonstrated in Escherichia coli K12 AB2480, a strain deficient in both excision repair (uvrA) and recombination repair (recA) [3]. This biological evidence for the induction of pyrimidine dinaers by 365-nm radiation was confirmed by chemical assay [11]. The ratio of sensitivity of strain K12 AB2480 at 254 nm and 365 nm was 106, a ratio close to that reported by Tyrrell [11] for dimer induction (7 X 10s). Although dimers clearly cause most of the inactivation at 365 nm (at high * This work supported by the U.S. Energy Research and Development Administration.
164 fluence rates and 0°C) in the UV sensitive E. coli strains K12 AB2480 (uvrA, recA) and Bs_ l (hcr, exr, ill) [3,16], their contribution to 365-nm lethal damage in more resistant strains has not been established. The photoreactivation enzyme is partially destroyed in both in vivo (E. coli) and in vitro (yeast) by the higher 365-nm fluences required to inactivate more resistant strains [16]. In this study we have attempted to assess the role of pyrimidine dimers in the lethal action of 365-nm radiation. We tested for photoreactivation after 365-nm inactivation in four isogenic strains of E. coli K12 that differ in repair capability. The presence of PR enzyme activity after 365-nm inactivation was determined by measuring photoreactivation after 365-nm inactivation and the introduction of additional dimers by 254-nm irradiation. Stationary phase cells were used because they apparently contain a greater number of active PR enzyme molecules [14]. Materials and methods
Uacterial strains Strains of E. coli differing in repair capability used in this study are listed in Table I. The strains were tested for their basic 254-nm sensitivity at the start of the study. Stocks were maintained on nutrient agar incubated at 37°C. For experiments, surface growth on nutrient agar plates incubated for 48 h was suspended in M9 salts buffer, pH 7 and washed twice by centrifugation before resuspending at approx, l 0 s cells per ml for 365-nm irradiations or at approx. 107 cells per ml for 254-nm irradiations. Cell suspensions were irradiated in quartz (254 nm) or pyrex (365 nm) vessels 1 cm in diameter with a capillary tube affixed to the b o t t o m to provide for aeration and stirring by means of a stream of moist air. A jacket of the same material surrounded the vessel to provide the circulation of a thermostated cooling solution to maintain the cell suspension at 0 to I°C during all irradiations. After irradiation procedures 0.2 ml of appropriately diluted suspensions were spread on the surface of nutrient agar plates in quadruplicate and incubated at 37°C for 48 h before counting surface colonies as a measure of the cells surviving the treatment. Radiation sources The 254-nm source was a 2 inch Penray low pressure mercury lamp with integral filter (G-275) to reduce near UV and visible radiations (SC-11 Ultra-Vio-
TABLE I S t r a i n s o f E. coli e m p l o y e d Strain
E. E. E. E.
coli coli coli coli
K12 K12 K12 K12
AB2480 AB2463 AB18S6 ABl157
a The isolate of strain AB2480 loci.
Repair genotype
Nutritional requirements
Source
uvrA6, recA13 cecAl3 uvrA6 wild-type
Pro, his, his, arg,
Howard-Flanders ttoward-Flanders Howard-Flanders Howard-Flanders
thi arg, arg, his,
a p r o , thr, leu, thi p r o , thr, leu, thi p r o , thr, leu, thi
u s e d i n t h i s s t u d y w a s f o u n d t o h a v e r e v e r t e d a t t h e his, arg, t h r a n d l e u
165 let Products, Inc.). Exposures were determined with a shutter controlled by an electronic timer. Fluence rates (approx. 0.1 W/m 2 for strain AB2480 and 0.8 W/m 2 for the other strains) were measured with a calibrated General Electric Germicidal meter. The 365-nm source was a Hanovia 2.5-kW Hg/Xe arc lamp operating in a Schoeffel housing with a predispersion prism. The 365-nm band was isolated by a Bausch and Lomb 500-mm monochromator. Short wavelength scattered light was eliminated with a Corning 7-51 filter. Fluence rates of 1000 to 1500 W/ m 2 were obtained with this source. Fluence rates were measured with a calibrated radiometer (Yellow Springs Instrument Co., l~ettering Model 65). Photoreactivation Cell suspensions containing between 106 and 107 cells per ml were irradiated in 1-cm square pyrex cuvettes by a 500 W quartz-iodine lamp in a Sawyer 707Q projector operating at 135 V to increase the near UV and blue parts of the spectrum. The beam was filtered through a bath containing CuSO4 and CoSo4 resulting in a band from 380 to 440 with a peak at 400 nm. An additional lens was added to provide a focused field of 10 cm square. Fluence rate was 20 W/m 2 for the radiation sensitive strains and 50 W/m z for the radiation resistant strains. PR exposures were 30 min. No inactivation was observed from the PR treatment alone. In some experiments 405-nm radiation from the 2.5-kW Hg/ Xe arc lamp used for the 365-nm source was also used for photoreactivation. Shorter wavelength scattered light was reduced by an optics technology LP400 filter (1% transmittance at 370 nm). All procedures were carried out in a laboratory illuminated with yellow fluorescent lamps (Champion F40/G0). Results Photorepair after 365-nm inactivation in the far-UV sensitive double m u t a n t E. coli K12 AB2480 is shown in Fig. 1A. The PR sector ((k o --keR)/ko where ko and kpR are rate constants for inactivation in the absence and presence of photoreactivation respectively) of 0.8 compares favorably with that after 254nm inactivation (Fig. 2A) [3,16]. The tail on the 365 nm survival curve previously reported for this strain [3,16], attributed to concomitant PR, was not observed at the greater fluence rates used in these studies. The demonstration by chemical assay of 365-nm induced dimers by Tyrrell [11], together with the observation of a large PR sector clearly establish a predominant role of pyrimidine dimers in the lethal action of this wavelength on strain AB2480. E. coli K12 AB2463 (recA) is much more resistant than AB2480 to 365-nm radiation, The photoreactivable sector of AB2463, although significant in the low dose range of inactivation, was small (Fig.lC). The inactivation fluence of 365-nm radiation at which PR was no longer obtainable was 2.0 × 106 J/m 2. The much greater photoreactivation of AB2463 after 254-nm inactivation is shown in Fig. 2C. E. coli K12 AB1886 (uvrA) showed little, if any, photoreactivation at any inactivation fluence level of 365-nm radiation (Fig.lB). Strain A B l 1 5 7 (wildtype) also showed no photoreactivation after 365-nm inactivation (Fig.lD). With this strain cells inactivated at 365 nm showed a small additional reduc-
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KI2 A B 2 4 6 5 rec~
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