INT . J . RADIAT . BIOL .,
1979,
VOL .
36,
NO .
6, 6 7 1 -676
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Lethality by 365 nm radiation in Escherichia coli K12 AB2480 in the presence and absence of maximal photoreactivation under aerobic and anaerobic conditionsf ROBERT B . WEBB$ and MICKEY S . BROWN Division of Biological and Medical Research, Argonne National Laboratory, Argonne, Illinois 60439, U .S .A . (Received 1 November 1978 ; accepted 18 June 1979)
1.
Introduction The absence of oxygen dependence for the inactivation of the double repairdeficient strain, Escherichia coli K12 AB2480 (uvrA recA) at the near-ultraviolet (near-UV) wavelength 365 nm, contrasts strikingly with the large oxygen dependence of single repair-deficient and repair-proficient strains for inactivation at the same wavelength (Tyrrell 1976, Webb and Brown 1979 a, b) . The basis of the strong oxygen enhancement for lethality obtained for near-UV radiation (320-400 nm) has not been established (Tyrrell 1976, Webb 1977, Webb and Brown 1979 b) . Although cyclobutyl pyrimidine dimers (Tyrrell 1973) and DNA single-strand breaks (Tyrrell, Ley and Webb 1974, Ley, Sedita and Boye 1978) are produced in biologically significant yields, the role of these and other DNA lesions has not been determined for oxygen-dependent lethality in wild-type and single repair-deficient strains (Tyrrell 1976, Webb, Brown and Tyrrell 1976) . Oxygen enhancement for the formation of pyrimidine dimers is not expected at 365 nm, since such dimers are not produced by an oxygen-dependent mechanism at 254 nm (discussed by Webb (1977)) . In contrast, the production of DNA single-strand breaks at 365 nm is completely oxygen dependent (Tyrrell, Ley and Webb 1974), suggesting that DNA breaks may be more important lethal lesions than dimers in oxygen-dependent 365 nm lethality . In this study, we measured the oxygen dependence of 365 and 254 nm inactivation in the presence and absence of maximal photoreactivation (PR) in E . coli K12 AB2480 to determine the interrelationship between oxygen-dependent and oxygen-independent lesions in lethality at 365 nm . 2.
Materials and methods Stationary-phase cells of E . coli K12 AB2480 (recA 13 uvrA 6) (described by Howard-Flanders and Boyce (1966)), 2 to 3 days old, incubated at 37°C, were suspended in M9 buffer (Anderson 1946) at pH 7, washed twice by centrifugation before adjusting the cell concentration to -2x 10' cells/ml for 254 nm irradiation and -I x 10 8 cells/ml for 365 nm irradiation . At these concentrations no correction is necessary for shielding . - Copyright U .S . Government . t Person to whom correspondence should be addressed .
Correspondence
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67 2
Inactivation and photoreactivation procedures were exactly as described previously (Brown and Webb 1972, Webb and Brown 1976, Webb et al. 1976) . Stringent anoxia was obtained by a method previously reported in detail (Webb and Brown 1979 a) . Cell suspensions were maintained at 0°C (liquid) or 25°C during irradiation at 365 nm and 25 ° C during irradiation at 254 rim . Irradiation with 365 nm radiation at high fluence rates at 0 ° C effectively prevents concomitant monomerization of pyrimidine dimers since the activity of the PR enzyme is extremely low at 0°C (Tyrrell 1973, Brown and Webb 1972) . For inactivation assays, appropriately diluted samples were plated on nutrient agar (Difco) and colonies were counted after 2 days incubation at 37 ° C . All procedures were conducted in a laboratory illuminated with gold fluorescent lamps (General Electric F40/GO) . 3.
Results The effects of exposing cells of E . coli K12 AB2480 to maximal PR after inactivation at 365 nm (0°C) under aerobic and anaerobic conditions are shown in figure 1 . Oxygen dependence was not significant at 365 nm with strain AB2480 in the absence of photoreactivation . This result supports previous reports of the absence of oxygen dependence for lethality at 365 nm with this strain (Tyrrell 1976, Webb and Brown 1979b) . However, there was a strong oxygen enhancement of 365 nm lethality when the irradiated cells were subjected to maximal PR (figure 1) . The oxygen-enhancement ratio (OER) after maximal PR was 2 . 3 (see table) . Other interrelationships including PR and oxygen dependence are also shown in the table . For example, the PR ratio after aerobic 365 nm irradiation was 4 . 7, while the PR ratio after anaerobic irradiation was 10 . 3, a value twice as large as the result when irradiation was given under aerobic conditions . Results were also obtained in which
z0 F-U Q 0 Z J
0N
Figure 1 . Lethality of stationary-phase cells of E . coli K12 AB2480 by 365 nm radiation at 0 ° C in the presence (triangles : Q, A) and absence (circles : 0, S) of maximal PR (25 ° C) under aerobic (open symbols : A, 0) and -2 anaerobic (closed symbols : A, 0) conditions . The 365 nm fluence rate was 600 Wm . The PR (380-430) nm fluence rate was 20 \\'m 2 . The plots are composites of three independent experiments .
673
Correspondence
Wavelength (nm)
Component of sensitivity
Sensitivity component constant
PR-events plus kmax l
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Non-PR events 365
254
Non-PR events
kmint
PR-events
kPR$
PR-events
k PR§
PR-ratio
kmax/kmin
PR-events plus Non-PR events Non-PR events PR-events PR-ratio
k max
k mint k PR $ kmax/kmin
Value for aerobic irradiation 3 . 93 x 10-I M -2j-t
Value for anaerobic irradiation
Ratio of aerobic to anaerobic values
3 . 84 X 10-I M -2j-1
1 . 02
0 . 837 x 10 -5 m -2 J -1 0 . 370 X 10 - I M - 2j - 1 3 . 09 x 10-I M -2j-t 3 . 47 X 10 -5 m -2J -1 3 . 04 x 10-5M-2j _' 3 . 52 x 10-5m-21 -t 4.7 10 . 3 46 •l m -2 J -1 10 . 1m -2 J -t 36 .Om -2J -1 4.6
45-8m -2j-1 10 . 2m -2 J -t 35 . 2m -2 J -1 4.5
2. 3 0. 89 0 . 86§ 0 . 46 1 . 01 0 . 99 1 .02 1 . 02
t Sensitivity is expressed as the final slope (k) of the survival curve . Sensitivity in the absence of PR and in the presence of maximal PR is k mjn . $ The photoreactivable component (PR events) is expressed as k PR which is calculated from the equation : kPR = kmax - kmin • § Value of kpR assuming no oxygen dependence in the absence of PR . Sensitivity- and sensitivity relationships of E . coli K12 AB2480 in the presence and absence of maximal photoreactivation (PR) under aerobic and anaerobic conditions at 365 and 254 nm . is kmax
the cells were maintained at 25°C during the 365 nm irradiation . At the high fluence rates employed (600 Wm -2 ), similar responses were obtained at 0 ° C (figure 1) and 25°C (data not shown) . Thus we conclude that with high fluence rates the short irradiations required for inactivation of this radiation-sensitive strain do not allow time for significant concomitant PR to occur . At lower 365 nm fluence rates at 25 ° C, concomitant PR can be readily demonstrated with strain AB2480 (Brown and Webb 1972) . Recently, Harm (1978) presented data that also are consistent with concomitant PR during irradiation at 365 nm in E . coli AB2480 . However, Harm's conclusion that the photoreactivable DNA lesions produced at 365 nm are not cyclobutyl pyrimidine dimers is convincingly contradicted by previous chemical assays of pyrimidine dimers produced at 365 nm (Tyrrell 1973) . A set of survival curves at 254nm parallel to the 365 nm curves (figure 1) is depicted for AB2480 in figure 2 . The absence of oxygen dependence for lethality by 254 nm radiation is in accord with previous reports using other strains of microorganisms (Zetterberg 1964, Webb and Lorenz 1970) . Also distinct from the response at 365 rim, there was no oxygen dependence at 254 nm after maximal PR (figure 2) .
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674
Correspondence
Figure 2 . Lethality of stationary-phase cells of E . coli K12 AB2480 by 254nm radiation at 25°C in the presence (triangles : Q, A) and absence (circles : Q, •) of maximal PR (25°C) under aerobic (open symbols : 0, ~) and anaerobic (closed symbols : •, A) conditions . The 254nm fluence rate was 0 . 065 Wm -2 . The PR (380-430 nm fluence rate was 20 Wm -2 . The plots are composites of two independent experiments .
4.
Discussion The oxygen dependence of non-PR events (k m i n ) (figure 1, table) may be the consequence of the same oxygen-dependent damage that produces the large oxygen enhancement of 365 nm inactivation of uvrA rec+ , uvr+ recA, and uvr + rec + (wild type) strains . After maximal PR, the number of DNA single-strand breaks is 12 per cell per `lethal event' (per F 37 ) ( Tyrrell et al . 1974, Ley et al. 1978, Webb 1977) . Thus, although some repair of DNA single-strand breaks must occur in this uvrA recA strain, these DNA breaks are likely candidates for the oxygen-dependent lethal lesions that remain after maximal PR . The greater PR ratio after 365 nm irradiation under anaerobic conditions (PR ratio =10. 3) than under aerobic conditions (PR ratio =4 . 7) is a provocative result (see table) . If only the damage subject to photoreactivation (kma.-kmin=kPR) is considered, the rate of production of PR events (k PR ) by 365 nm radiation under -1 ) anaerobic conditions (3 . 47 x 10 -5 m -2J is significantly larger than the rate of production of PR events under aerobic conditions (3 . 09 x 10-IM-2j-1) (see table) . Furthermore, if it is assumed that in the absence of PR the survival curves at 365 nm under aerobic and anaerobic conditions are identical for strain AB2480, as repeated experiments have indicated (data not shown), the difference in the rate of production of PR events under aerobic and anaerobic conditions is greater than that shown in the table . Since enzymatic photoreactivation is specific for cyclobutyl pyrimidine dimers (Setlow 1967), PR events represent the dimer component of lethality . If .the correspondence between PR events and pyrimidine dieters at 365 nm is the same for aerobic and anaerobic irradiation, results shown in figure 1 and the table indicate that more pyrimidine dimers are produced at a given fluence of 365 nm radiation under
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Correspondence
675
anaerobic than aerobic conditions . This result suggests that the excited states leading to the production of pyrimidine dimers at 365 nm may be partially quenched by oxygen . The induction of pyrimidine dimers mediated by triplet sensitizers such as acetophenone is also partially inhibited by oxygen (Rahn 1973, Rahn et al . 1974). Thus the greater yield of PR events under anaerobic conditions than under aerobic conditions (see table) is consistent with the endogenously sensitized production of pyrimidine dimers by 365 nm radiation . Strikingly similar strain differences have been obtained for both oxygen dependence and PR with gamma and 365 nm radiation . Recently, photoreactivation was demonstrated after gamma-radiation inactivation in E . coli strains Bs-1 (uvrB lexA Ion) and K12 AB2480 (uvrA recA) (Myasnik and Morozov 1977, Redpath and Tortorello 1977, Vinicombe, Moss and Davies 1978) . Furthermore, the production of cyclobutyl pyrimidine dimers by gamma radiation was demonstrated by chemical assay (Wang and Smith 1978) . A strong oxygen enhancement has been obtained for lethal effects with both 365 nm (Tyrrell 1976, Webb and Brown 1979 a, b) and gamma radiation (Vinicombe et al . 1978) in repair-proficient, uvrA rec + , uvr + recA and polA strains . However, little or no oxygen dependence has been observed with the uvrA recA strains for either 365 nm (figure 1) (Tyrrell 1976, Webb and Brown 1979 b) or gamma radiation (Vinicombe et al . 1978). In addition, PR of lethality can be readily demonstrated in uvrA recA and uvrB lexA strains for both 365 nm (Brown and Webb 1972, Tyrrell et al . 1973, Webb et al . 1976) and gamma radiation (Myasnik and Morazov 1977, Vinicombe et al . 1977, Wang and Smith 1978) . However, differences in the PR responses have been observed for 365 nm and gamma radiation with more radiation-resistant strains . For 365 nm lethality, a small but significant amount of PR was obtained with recA strains ; PR was not obtained with uvrA and wild-type strains (Webb et al . 1976). For gamma-radiation lethality, no PR was demonstrated for recA, uvrA or wild-type strains (Myasnik and Morozov 1977, Vinicombe et al . 1977) . The absence of significant PR in strains capable of excision and/or recombination repair can be accounted for with both 365 nm and gamma radiation by postulating a high efficiency of repair of dimers when they are produced by either of these two radiations . In contrast, pyrimidine dimers produced at 254 nm may be less efficiently repaired by dark repair processes, possibly because of the production of lesions that interfere with the dark repair of dimers . Although pyrimidine dimers account for 75 to 80% of the lethal damage at both 254 and 365 nm in strain AB2480 (Tyrrell 1973, Brown and Webb 1972), results presented here indicate that the mechanism of induction of cyclobutyl pyrimidine dimers is different at the two wavelengths . Acknowledgment This work was supported by the U .S . Department of Energy . References ANDERSON, E . H ., 1946, P roc . natn . Acad. Sci . U .S . A ., 32, 120 . BROWN, M . S ., and WEBB, R . B ., 1972, Mutation . Res ., 15, 348 . HARM, W ., 1978, Mutation Res ., 51, 301 . HOWARD-FLANDERS, P ., and BOYCE, R . P ., 1966, Radiat . Res . Suppl ., 6, 156 . LEY, R . D ., SEDITA, A ., and BOYE, E ., 1978, Photochem . Photobiol ., 27, 323 . MYASNIK, M . N ., and MoROZOV, I . I ., 1977, Int . J . Radiat . Biol., 31, 95 .
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676
Correspondence
RAHN, R . 0 ., 1973, Photophysiology, Edited by A . C . Giese, Vol . 8 (New York : Academic Press) pp . 231-255 . RAHN, R . 0 ., LANDRY, L . C ., and CARRIER, W . L ., 1974, Photochem . Photobiol ., 19, 75 . REDPATH, J . L ., and TORTORELLO, M . L ., 1977, Int . J . Radiat . Biol., 32, 505 . SETLOW, J . K ., 1967, Comprehensive Biochemistry, Photobiology, Ionizing Radiations edited by M . Florkin and E . H . Stotz (New York : Elsevier Publishing Co .), .pp . 157-203 . TYRRELL, R. M ., 1973, Photochem . Photobiol ., 17, 69 ; 1976, Ibid., 23, 13 . TYRRELL, R. M ., and DAVIES, D . J . G ., 1974, Mutation Res ., 23, 151 . TYRRELL, R . M ., LEY, R . D ., and WEBB, R . B ., 1974, Photochem . Photobiol., 20, 395 . VINICOMBE, D . A ., Moss, S . H ., and DAVIES, D . J . G ., 1978, Int . J . Radiat . Biol ., 23, 483 . WANG, T . V ., and SMITH, K . C ., 1978, Radiat . Res ., 76, 540 . WEBB, R. B ., 1977, Photochemical and Photobiological Reviews, edited by K . C . Smith, Vol . 2 (New York : Plenum Press) pp . 169-261 . WEBB, R . B ., and BROWN, M . S ., 1976, Photochem . Photobiol ., 24,425 ; 1979 a, Ibid ., 29, 407 ; 1979b, Radiat . Res . (submitted) . WEBB, R . B ., BROWN, M . S ., and TYRRELL, R . M ., 1976, Mutation Res ., 37, 163 . WEBB, R . B . and LORENZ, J . R ., 1970, Photochem . Photobiol ., 12, 283 . ZETTERBERG, G ., 1964, Photophysiology, edited by A . C . Giese, Vol . 3 pp . 247-281, (New York : Academic Press) .
1979,
VOL .
36,
NO .
6, 6 7 1 -676
Int J Radiat Biol Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/02/14 For personal use only.
Lethality by 365 nm radiation in Escherichia coli K12 AB2480 in the presence and absence of maximal photoreactivation under aerobic and anaerobic conditionsf ROBERT B . WEBB$ and MICKEY S . BROWN Division of Biological and Medical Research, Argonne National Laboratory, Argonne, Illinois 60439, U .S .A . (Received 1 November 1978 ; accepted 18 June 1979)
1.
Introduction The absence of oxygen dependence for the inactivation of the double repairdeficient strain, Escherichia coli K12 AB2480 (uvrA recA) at the near-ultraviolet (near-UV) wavelength 365 nm, contrasts strikingly with the large oxygen dependence of single repair-deficient and repair-proficient strains for inactivation at the same wavelength (Tyrrell 1976, Webb and Brown 1979 a, b) . The basis of the strong oxygen enhancement for lethality obtained for near-UV radiation (320-400 nm) has not been established (Tyrrell 1976, Webb 1977, Webb and Brown 1979 b) . Although cyclobutyl pyrimidine dimers (Tyrrell 1973) and DNA single-strand breaks (Tyrrell, Ley and Webb 1974, Ley, Sedita and Boye 1978) are produced in biologically significant yields, the role of these and other DNA lesions has not been determined for oxygen-dependent lethality in wild-type and single repair-deficient strains (Tyrrell 1976, Webb, Brown and Tyrrell 1976) . Oxygen enhancement for the formation of pyrimidine dimers is not expected at 365 nm, since such dimers are not produced by an oxygen-dependent mechanism at 254 nm (discussed by Webb (1977)) . In contrast, the production of DNA single-strand breaks at 365 nm is completely oxygen dependent (Tyrrell, Ley and Webb 1974), suggesting that DNA breaks may be more important lethal lesions than dimers in oxygen-dependent 365 nm lethality . In this study, we measured the oxygen dependence of 365 and 254 nm inactivation in the presence and absence of maximal photoreactivation (PR) in E . coli K12 AB2480 to determine the interrelationship between oxygen-dependent and oxygen-independent lesions in lethality at 365 nm . 2.
Materials and methods Stationary-phase cells of E . coli K12 AB2480 (recA 13 uvrA 6) (described by Howard-Flanders and Boyce (1966)), 2 to 3 days old, incubated at 37°C, were suspended in M9 buffer (Anderson 1946) at pH 7, washed twice by centrifugation before adjusting the cell concentration to -2x 10' cells/ml for 254 nm irradiation and -I x 10 8 cells/ml for 365 nm irradiation . At these concentrations no correction is necessary for shielding . - Copyright U .S . Government . t Person to whom correspondence should be addressed .
Correspondence
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67 2
Inactivation and photoreactivation procedures were exactly as described previously (Brown and Webb 1972, Webb and Brown 1976, Webb et al. 1976) . Stringent anoxia was obtained by a method previously reported in detail (Webb and Brown 1979 a) . Cell suspensions were maintained at 0°C (liquid) or 25°C during irradiation at 365 nm and 25 ° C during irradiation at 254 rim . Irradiation with 365 nm radiation at high fluence rates at 0 ° C effectively prevents concomitant monomerization of pyrimidine dimers since the activity of the PR enzyme is extremely low at 0°C (Tyrrell 1973, Brown and Webb 1972) . For inactivation assays, appropriately diluted samples were plated on nutrient agar (Difco) and colonies were counted after 2 days incubation at 37 ° C . All procedures were conducted in a laboratory illuminated with gold fluorescent lamps (General Electric F40/GO) . 3.
Results The effects of exposing cells of E . coli K12 AB2480 to maximal PR after inactivation at 365 nm (0°C) under aerobic and anaerobic conditions are shown in figure 1 . Oxygen dependence was not significant at 365 nm with strain AB2480 in the absence of photoreactivation . This result supports previous reports of the absence of oxygen dependence for lethality at 365 nm with this strain (Tyrrell 1976, Webb and Brown 1979b) . However, there was a strong oxygen enhancement of 365 nm lethality when the irradiated cells were subjected to maximal PR (figure 1) . The oxygen-enhancement ratio (OER) after maximal PR was 2 . 3 (see table) . Other interrelationships including PR and oxygen dependence are also shown in the table . For example, the PR ratio after aerobic 365 nm irradiation was 4 . 7, while the PR ratio after anaerobic irradiation was 10 . 3, a value twice as large as the result when irradiation was given under aerobic conditions . Results were also obtained in which
z0 F-U Q 0 Z J
0N
Figure 1 . Lethality of stationary-phase cells of E . coli K12 AB2480 by 365 nm radiation at 0 ° C in the presence (triangles : Q, A) and absence (circles : 0, S) of maximal PR (25 ° C) under aerobic (open symbols : A, 0) and -2 anaerobic (closed symbols : A, 0) conditions . The 365 nm fluence rate was 600 Wm . The PR (380-430) nm fluence rate was 20 \\'m 2 . The plots are composites of three independent experiments .
673
Correspondence
Wavelength (nm)
Component of sensitivity
Sensitivity component constant
PR-events plus kmax l
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Non-PR events 365
254
Non-PR events
kmint
PR-events
kPR$
PR-events
k PR§
PR-ratio
kmax/kmin
PR-events plus Non-PR events Non-PR events PR-events PR-ratio
k max
k mint k PR $ kmax/kmin
Value for aerobic irradiation 3 . 93 x 10-I M -2j-t
Value for anaerobic irradiation
Ratio of aerobic to anaerobic values
3 . 84 X 10-I M -2j-1
1 . 02
0 . 837 x 10 -5 m -2 J -1 0 . 370 X 10 - I M - 2j - 1 3 . 09 x 10-I M -2j-t 3 . 47 X 10 -5 m -2J -1 3 . 04 x 10-5M-2j _' 3 . 52 x 10-5m-21 -t 4.7 10 . 3 46 •l m -2 J -1 10 . 1m -2 J -t 36 .Om -2J -1 4.6
45-8m -2j-1 10 . 2m -2 J -t 35 . 2m -2 J -1 4.5
2. 3 0. 89 0 . 86§ 0 . 46 1 . 01 0 . 99 1 .02 1 . 02
t Sensitivity is expressed as the final slope (k) of the survival curve . Sensitivity in the absence of PR and in the presence of maximal PR is k mjn . $ The photoreactivable component (PR events) is expressed as k PR which is calculated from the equation : kPR = kmax - kmin • § Value of kpR assuming no oxygen dependence in the absence of PR . Sensitivity- and sensitivity relationships of E . coli K12 AB2480 in the presence and absence of maximal photoreactivation (PR) under aerobic and anaerobic conditions at 365 and 254 nm . is kmax
the cells were maintained at 25°C during the 365 nm irradiation . At the high fluence rates employed (600 Wm -2 ), similar responses were obtained at 0 ° C (figure 1) and 25°C (data not shown) . Thus we conclude that with high fluence rates the short irradiations required for inactivation of this radiation-sensitive strain do not allow time for significant concomitant PR to occur . At lower 365 nm fluence rates at 25 ° C, concomitant PR can be readily demonstrated with strain AB2480 (Brown and Webb 1972) . Recently, Harm (1978) presented data that also are consistent with concomitant PR during irradiation at 365 nm in E . coli AB2480 . However, Harm's conclusion that the photoreactivable DNA lesions produced at 365 nm are not cyclobutyl pyrimidine dimers is convincingly contradicted by previous chemical assays of pyrimidine dimers produced at 365 nm (Tyrrell 1973) . A set of survival curves at 254nm parallel to the 365 nm curves (figure 1) is depicted for AB2480 in figure 2 . The absence of oxygen dependence for lethality by 254 nm radiation is in accord with previous reports using other strains of microorganisms (Zetterberg 1964, Webb and Lorenz 1970) . Also distinct from the response at 365 rim, there was no oxygen dependence at 254 nm after maximal PR (figure 2) .
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674
Correspondence
Figure 2 . Lethality of stationary-phase cells of E . coli K12 AB2480 by 254nm radiation at 25°C in the presence (triangles : Q, A) and absence (circles : Q, •) of maximal PR (25°C) under aerobic (open symbols : 0, ~) and anaerobic (closed symbols : •, A) conditions . The 254nm fluence rate was 0 . 065 Wm -2 . The PR (380-430 nm fluence rate was 20 Wm -2 . The plots are composites of two independent experiments .
4.
Discussion The oxygen dependence of non-PR events (k m i n ) (figure 1, table) may be the consequence of the same oxygen-dependent damage that produces the large oxygen enhancement of 365 nm inactivation of uvrA rec+ , uvr+ recA, and uvr + rec + (wild type) strains . After maximal PR, the number of DNA single-strand breaks is 12 per cell per `lethal event' (per F 37 ) ( Tyrrell et al . 1974, Ley et al. 1978, Webb 1977) . Thus, although some repair of DNA single-strand breaks must occur in this uvrA recA strain, these DNA breaks are likely candidates for the oxygen-dependent lethal lesions that remain after maximal PR . The greater PR ratio after 365 nm irradiation under anaerobic conditions (PR ratio =10. 3) than under aerobic conditions (PR ratio =4 . 7) is a provocative result (see table) . If only the damage subject to photoreactivation (kma.-kmin=kPR) is considered, the rate of production of PR events (k PR ) by 365 nm radiation under -1 ) anaerobic conditions (3 . 47 x 10 -5 m -2J is significantly larger than the rate of production of PR events under aerobic conditions (3 . 09 x 10-IM-2j-1) (see table) . Furthermore, if it is assumed that in the absence of PR the survival curves at 365 nm under aerobic and anaerobic conditions are identical for strain AB2480, as repeated experiments have indicated (data not shown), the difference in the rate of production of PR events under aerobic and anaerobic conditions is greater than that shown in the table . Since enzymatic photoreactivation is specific for cyclobutyl pyrimidine dimers (Setlow 1967), PR events represent the dimer component of lethality . If .the correspondence between PR events and pyrimidine dieters at 365 nm is the same for aerobic and anaerobic irradiation, results shown in figure 1 and the table indicate that more pyrimidine dimers are produced at a given fluence of 365 nm radiation under
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Correspondence
675
anaerobic than aerobic conditions . This result suggests that the excited states leading to the production of pyrimidine dimers at 365 nm may be partially quenched by oxygen . The induction of pyrimidine dimers mediated by triplet sensitizers such as acetophenone is also partially inhibited by oxygen (Rahn 1973, Rahn et al . 1974). Thus the greater yield of PR events under anaerobic conditions than under aerobic conditions (see table) is consistent with the endogenously sensitized production of pyrimidine dimers by 365 nm radiation . Strikingly similar strain differences have been obtained for both oxygen dependence and PR with gamma and 365 nm radiation . Recently, photoreactivation was demonstrated after gamma-radiation inactivation in E . coli strains Bs-1 (uvrB lexA Ion) and K12 AB2480 (uvrA recA) (Myasnik and Morozov 1977, Redpath and Tortorello 1977, Vinicombe, Moss and Davies 1978) . Furthermore, the production of cyclobutyl pyrimidine dimers by gamma radiation was demonstrated by chemical assay (Wang and Smith 1978) . A strong oxygen enhancement has been obtained for lethal effects with both 365 nm (Tyrrell 1976, Webb and Brown 1979 a, b) and gamma radiation (Vinicombe et al . 1978) in repair-proficient, uvrA rec + , uvr + recA and polA strains . However, little or no oxygen dependence has been observed with the uvrA recA strains for either 365 nm (figure 1) (Tyrrell 1976, Webb and Brown 1979 b) or gamma radiation (Vinicombe et al . 1978). In addition, PR of lethality can be readily demonstrated in uvrA recA and uvrB lexA strains for both 365 nm (Brown and Webb 1972, Tyrrell et al . 1973, Webb et al . 1976) and gamma radiation (Myasnik and Morazov 1977, Vinicombe et al . 1977, Wang and Smith 1978) . However, differences in the PR responses have been observed for 365 nm and gamma radiation with more radiation-resistant strains . For 365 nm lethality, a small but significant amount of PR was obtained with recA strains ; PR was not obtained with uvrA and wild-type strains (Webb et al . 1976). For gamma-radiation lethality, no PR was demonstrated for recA, uvrA or wild-type strains (Myasnik and Morozov 1977, Vinicombe et al . 1977) . The absence of significant PR in strains capable of excision and/or recombination repair can be accounted for with both 365 nm and gamma radiation by postulating a high efficiency of repair of dimers when they are produced by either of these two radiations . In contrast, pyrimidine dimers produced at 254 nm may be less efficiently repaired by dark repair processes, possibly because of the production of lesions that interfere with the dark repair of dimers . Although pyrimidine dimers account for 75 to 80% of the lethal damage at both 254 and 365 nm in strain AB2480 (Tyrrell 1973, Brown and Webb 1972), results presented here indicate that the mechanism of induction of cyclobutyl pyrimidine dimers is different at the two wavelengths . Acknowledgment This work was supported by the U .S . Department of Energy . References ANDERSON, E . H ., 1946, P roc . natn . Acad. Sci . U .S . A ., 32, 120 . BROWN, M . S ., and WEBB, R . B ., 1972, Mutation . Res ., 15, 348 . HARM, W ., 1978, Mutation Res ., 51, 301 . HOWARD-FLANDERS, P ., and BOYCE, R . P ., 1966, Radiat . Res . Suppl ., 6, 156 . LEY, R . D ., SEDITA, A ., and BOYE, E ., 1978, Photochem . Photobiol ., 27, 323 . MYASNIK, M . N ., and MoROZOV, I . I ., 1977, Int . J . Radiat . Biol., 31, 95 .
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676
Correspondence
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