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Lack of repair of ultraviolet light damage in Mycoplasma gallisepticum
J. Mol. BioZ. (1977) 116, 337-344
Lack of Repair of Ultraviolet Light Damage in Mycoplasma gallisepticum We have studied the repair capabilities of Mycoplasma gallisepticum, one of the smallest free-living cells, having a genome size of only 4.6 x lOa daltons. The data show that these cells have neither dark repair nor photoreactivation mechanisms for the repair of ultraviolet induced DNA damage. No other prokaryote has been reported to lack both kinds of repair systems. Studies of irradiated cell DNA indicate that the u.v.-induced pyrimidine dimers cannot be excised and block further replication. The photoreactivation experiments also showed that 32. gallisepticum is photoinactivated.
of an organism to repair damage in its DNA is essential for it to be able to maintain the structural integrity of its genome and to survive under adverse environmental conditions. The efficiencies of such DNA repair systems are themselves subject to evolutionary pressures and the rate of evolution of a species may be coupled to the efficiency and discrimination of its repair processes (Hanawalt, 1975). The studies reported here concern the repair capabilities of the smallest free-living cells, the mycoplasmas, in order to examine the ubiquity of DNA repair processes and to determine which repair processes are utilized by cells with such small genomes. The mycoplasmas are a group of small prokaryotes which do not have cell walls : each cell is bounded by a single lipoprotein “unit” membrane (reviewed by Maniloff & Morowitz, 1972). Mycoplasma isolates have been classified into five genera. The genome size for three of the genera (Acholeplasma, Spiroplasm and Thermoplasma) is 1.0 x lo9 daltons and for the other two (Mycoplusma and Ureaplasma) is 4.6 x 10s daltons. Mycoplasma, Uveaplasma and Spiroplasma require sterol for growth; the ot,her two genera do not. Only limited radiobiological data on mycoplasmas are available and all reported studies involved Acholeplusma laidluu~ii strain B. These cells can repair ultravioletinduced DNA damage by both photoreactivation and dark (excision) repair mechanisms (Folsome, 1968; Smith & Hanawalt, 1969; Das et al., 1972). Recently, A. Zuidlawii strain JAl cells have also been found to repair u.v.-irradiated mycoplasma viruses by host cell and ultraviolet reactivation (Das et aZ., 1977): the former mode involves repair of viral DNA by the cells’ excision repair system and the latter suggests the presence of a u.v.-inducible repair system in these cells. There are no reports on the repair capabilities of the other mycoplasma genera. We report here on studies of the repair of DNA damage in u.v.-irradiated Mycoplasma gallisepticum. These cells are unique biological systems. Each cell has specialized polar subcellular organelles, presumably functioning as a primitive mitotic apparatus (Maniloff & Quinlan, 1974). This is the only prokaryote whose growth has been found to be inhibited by cytochalasin B (Ghosh & Maniloff, unpublished data), a drug that inhibits eukaryotes. M. gallisepticum is one of those mycoplasmas which have the smallest genome size reported for free-living cells. Finally, studies of the cells’ ribosomal RNA have shown that M. gallisepticum is significantly different from y:! 337
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other prokaryotes : the cells lack some of the ribosomal RNA oligonucleotide sequences that are found in all ot’her prokaryotes (Woese & Maniloff, unpublished data). The mycoplasmas used in this st,udy were &I. gallise~ticum strains A5969 (described by Tourtellotte & Jacobs, 1960) and 15302 (obt’a’ined from Dr G. Kenny, University of Washington), and A. laidlawii strain JAl (described by Liss & Maniloff, 1973). A. laidlawii cells were grown in tryptose broth and assayed as colony-forming units (c.f.u.) on tryptose agar plates, as described previously (Quinlan et al., 1972). M. gaZZisepticum cells were grown in MBB medium: 21 g mycoplasma broth base (BBL, Cockeysville, Md), 50 ml yeast extract (Microbiological Associates, Bethesda, Md), 10 g dextrose and 100 ml horse serum (Flow Laboratory, Rockville, Md) per liter, adjusted to pH 7.5. They were assayed as c.f.u. on MBB agar plates containing lT/, agar. A. laidlawii plate counts were done after three to four days incubation at 37°C and M. gallisepticum after six to seven days. The survival of M. gallisepticum ,45969 following U.V. irradiation is shown in Figure l(a). For comparison, the survival of u.v.-irradiated A. laidlawii JAl was also examined (Fig. l(b)). The JAl strain used in this stud-y has different U.V. inactivation parameters than the B strain used in all previous U.V. repair studies: the slope of the linear part of the survival curve was 77 m2/kJ for strain JAl (from Fig. l(b)) versus 200 m2/kJ for strain B (Das et al., 1972) and the zero-dose intercept of the linear part of the curve was about 3 for strain JAl versus 8 to 10 for strain B. In contrast to the A. laidluwii inactivation curve, the M. gallisepticum A5969 U.V. survival curve is almost exponential (Fig. l(a)). Th e zero-dose intercept of the M. gallisepticum U.V. survival curve is 1.53, which is experimentally indistinguishable from the value of 1.44 expected for exponentially growing cells in which each cell contains only a single genome copy. The final slope of the survival curve is 75 m2/kJ. Identical results were obtained with M. gallisepticum strain 15302. The absence of a shoulder in the M. gallisepticum U.V. survival curve suggested that these cells might not be able to repair u.v.-induced DNA damage. Therefore, experiments were carried out to investigate both dark and light repair in M. gallisepticum JAl was also studied, since this A5969. For comparative purposes, A. laidlawii species is known t,o have bot,h da,rk and light repair mechanisms (reviewed above). To measure dark repair, u.v.-irradiated cells were examined to determine whether cell survival increased if the irradiated cells in buffer were held in the dark before plating. For these experiments, at each U.V. dose in Figure 1, a sample was held for one hour at 37°C before plating. Holding resulted in an increase in survival of u.v.irradiated A. laidlawii, with a dose reduction factor of 1.45 (Fig. l(b)). However, holding u.v.-irradiated M. gallisepticum cells in the dark produced no change in the survival curve (Fig. l(a)). To examine the dark-repair kinetics, u.v.-irradiated cells were held in the dark and the number of viable cells was measured after different holding times (Fig. 2). During holding, the survival of A. hidbwii JAl cells increased exponentially and the maximum amount of dark recovery was reached in about 45 minutes (Fig. 2(b)). This is similar to the dark-recovery kinetics reported for A. laidlawii B cells (Das et al., 1972). However at each U.Y. inactivating dose, no dark recovery was seen for irradiated M. gallisepticum cells (Fig. 2(a)). For photoreactivation studies, cells were irradiated with different U.V. doses and duplicate samples were immediately plated. One set of plates was incubated in the dark and other sets were illuminated with fluorescent room light; each set being illuminated for a different length of time. The results are shown in Figure 3. Efficient
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FIQ. 1. Survival and liquid-holding (dark) recovery of u.v.-irradiated mycoplasmas. (a) M. guZZtiep~icum A6969; (b) A. Zaidlawii JAl. Exponentially growing cells were irradiated with 267 nm light, as previously described (Das et al., 1972). For t,hese studies cells in media were diluted lo-fold into /l-buffer (0.06 M-Tris, 0.166 M-N&I, 0.01 M-p-mercaptoethanol, pH 7.4) to avoid centrifugation (which causes damage to M. guZZise+xm; Maniloff & Morowitz, 1972), that would be necessary to harvest the cells for resuspension in buffer for irradiation. At each U.V. dose 2 samples were removed. One was immediately plated to determine the u.v. survival curve ( 0). The other sample was held in the dark to allow liquid-holding recovery ( A). This latter sample was diluted loo-fold into /l-buffer and held for 1 h at 37°C before plating. In this graph each point is the average of 3 to 6 experiments and a least-squares data analysis (described by Das et al., 1972) was used to calculate the inactivation parameters (slope and zero-dose intercept) and draw the curves.
photoreactivation was observed for A. laidlawii JAl cells (Fig. 3(b)), similar to that reported for A. laidlawii B (Das et al., 1972). However, at each U.V. dose, no photoreactivation was found for M. gallise@icum (Fig. 3(a)). In fact a loss of cell titer occurred while the u.v.-irradiated M. gallise$icum plates were held in the light. Unirradiated M. gallise@icum cells exposed to the fluorescent light also showed a loss of cell titer (Fig. 3(a)). The slopes of the photoinactivation curves of unirradiated cells and cells irradiated with different doses of U.V. light are about the same. This eliminates the possibility of a synergistic effect between U.V. and fluorescent light, which might have been responsible for photoinactivation. U.V. irradiation has very different effects on DNA synthesis in cells with or without
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FIG. 2. Dark-repair kinetics of u.v.-irradiated M. gallisepticum A5969 (a) and A. Zaidlawii JAI (b), inactivated to 2 different survival levels. The titer of unirradiated control cells held in buffer during the period of liquid-holding recovery remained constant (data not shown).
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Pm. 3. Photoreactivation kinetics of u.v.-irradiated M. gallisepticuna A6969 (a) and A. laidlawii JAl (b) cells, inactivated to various survival levels. After u.v. irradiation and plating of the cells, the plates were illuminated by an 80 W fluorescent lamp at a distance of 60 cm. The titer of unirradiated A. Zaidlawii JAl remained constant during the 5-h photoreactivation time.
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repair capabilities (Setlow et al., 1963; Swenson & Seblow, 1966). Therefore, after U.V. irradiation, the synthesis of M. gallisepticum and A. Zaidlawii DNA was followed by measuring the incorporation of labeled deoxythymidine into trichloroacetic acidprecipitable material. For these experiments, logarithmically growing cells were irradiated with U.V. doses to give 35 to 45% survival and then resuspended in grouth medium containing [3H]deoxythymidine. After various incubation times in the dark, trichloroacetic acid-precipitable radioactivity was measured. DNA synthesis in u.v.irradiated M. gallisepticum was reduced compared to unirradiated control cells (Fig. 4(a)). On the other hand, DNA synthesis in u.v.-irradiated A. Zaidlawii JAl cells was almost completely inhibited for about one hour, which is about the time required for maximum dark repair (Fig. 2(b)), and then resumed at the same rate as in unirradiated control cells (Fig. 4(b)). This can be explained if A. ZaidZawii JAl cells can circumvent or repair damage in their DNA, while M. gallisepticum A5969 cells cannot.
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4. DNA synthesis following ultraviolet irradiation. (a) M. gdisepticum A6969; (b) A. Zoidlowii JAl. (0) Unirradiated control; (A) u.v.-irradiated cells. Irradiated cells (to 35 to 46% survivctl) were resuspended in medium containing 60 PCi [3H]deoxythymidine/m1 (New England Nuclear, Boston, Mass.) and incubated 8t 37°C in the dark. At various times, SO-p1 semples were removed and added to equ81 volumes of cold 10% trichloroacetic acid. The precipitates were collected on filters, washed, and assayed for radioactivity in toluene/Omnifluor (New England Nuclertr, Boston, Mctss.). FIQ.
The state of the DNA in irradiated cells during liquid-holding recovery was examined by analyzing cell lysates by velocity sedimentation in alkaline 5% to 20% sucrose gradients. Figure 5 shows the sedimentation patterns of DNA from unirradiated and u.v.-irradiated M. gaZZisepticum (Fig. 5(a) to (d)) and A. Zuidlawii JAl (Fig. 5(e) to (g)) cells at different holding times. During holding, the average sedimentation rate of DNA from irradiated A. Zaidlawii first decreased and Iater increased. The weight average molecular weight of the single-stranded DNA from unirradiated JAl cells was 4.6 x 10s. After a U.V. dose of 30 J/m2 and 10 minutes of liquid-holding, the DNA molecular weight was reduced to 0.6 x lo*, and increased to 3.2 x 10s after 60 minutes of holding. This is consistent with an excision repair mechanism and similar results have been reported for A. ZaidZawii strain B (Das et al., 1972). In contrast, the sedimentation rate of M. gallisepticum DNA did not change after irradiation or during holding (Fig. 5(a) to (d)). Th e weight average molecular weight of the unirradiated single-stranded DNA was 8% x 107. The size remained unchanged
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FIG. 5. Alkaline sucrose gradient sedimentation of DNA after u.v.-irradiation and during liquidholding recovery. Ceils were grown to logarithmic phase in media containing 16 &i [3H]deoxythymidine/ml and irradiated to give a survival of 1 to 5%, as described in the legend to Fig. 1. Samples (0.1 ml) from unirradiated or u.v.-irradiated cells after different holding times were layered on top of 4.5 ml of a 5% to 20% (w / v ) sucrose gradient (adjusted to pH 12.0 with NaOH). Cells were mixed with 0.1 ml 1 N-NaOH, which had been previously layered onto the gradient. After 30 to 60 min, to allow complete digestion, the gradients were centrifuged at 28,000 revs/mm for 2 h at 20°C in a Beckman SW60.1 rotor. Fractions were collected and trichloroacetic acidprecipitable radioactivity measured as described for Fig. 4. Molecular weights were calculated, relative to marker T4 DNA which was added to each tube and shown by an arrow in each gradient as described previously (Das et al., 1972). M. gallisepticum A5969 DNA from (a) unirradiated cells, and irradiated cells after dark holding for (b) 2 min, (c) 10 min, and (d) 60 min. A. Zaidlawii JAl DNA from (e) unirradiated cells, and irradiated cells after dark-holding for (f) 10 min, and (g) 60 min.
after irradiation and up to the 60-minute holding time studied. This observation, together with the decrease in DNA synthesis following irradiation, indicates that u.v.-induced pyrimidine dimers may not be excised in M. gallisepticum and act as blocks to further DNA synthesis. To investigate this explicitly, Micrococcus luteus u.v.-endonuclease (obtained from Dr L. Prakash, University of Rochester) was used to treat M. gallisepticum cells, which had been u.v.-irradiated and held for 20 minutes in the dark, and the cell DNA was analyzed in alkaline 5% to 20% sucrose gradients. This enzyme has endonuclease activity toward u.v.-irradiated DNA, is inactive on u&radiated DNA, and makes
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single-stranded breaks at pyrimidime dimers, but does not excise the dimers (Carmer & Setlow, 1970). In agreement with this, it was found that unirradiated M. gallisepticum A5969 DNA had the same sedimentation profile whether it was untreated (Fig. 6(a)) or treated with the endonuclease (Fig. 6(b)). As described above (Fig. 5(a) to (d)), no change was observed in the sedimentation pattern of unirradiated M. gallisepticum DNA (Fig. 6(a)) and DNA from cells irradiated and then held for 20 minutes (Fig. 6(c)). However, endonuclease treatment of the u.v.-irradiated DNA caused a reduction in the single-stranded DNA molecular weight (Fig. 6(d)), with no measurable loss of trichloroacetic acid-precipitable material. Therefore, u-V.-irradiated M. gallisepticum DNA must contain pyrimidime dimers which the cell is unable to repair. The cell must lack a u.v.-endonuclease specific for ppimidine dimers.
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FIQ. 6. Alkaline sucrose-gradient sedimentation of unirradiated and u.v.-irradiated M. gallise&cum DNA after u.v.-endonuclease t,reatment. Unirradiated cells were (a) untreated or (b) treated with endonucleese. u.v.-irradiated cells (to a survival of 1 to 5%) were held for 20 min in the derk (as described for Fig. 1) and eit,her (c) untreated or (cl) treated with endonuclease. In these experiments the holding buffer was 0.05 M-Tris, 0.01 M-EDTA (pH 8.1), and all cells were frozen and thawed 3 times before enzyme treatments and alkaline sucrose-gradient sedimentation (as in Fig. 5). The freeze-thaw cycles were necessary to make the cells permeable to the added endonuclease and had no effect on the sedimentation behavior of the cell DNA (compare Figs 5(a) and S(a)). For endonucleese treatment, the reaction mixture was 0.05 ml M. luteus u.v.endonuclease extract (12 mg protein/ml) end 1 ml of cells in the holding buffer. The mixture was incubated for 10 min at 37°C and the reaction was terminated by layering 0.1 ml on an alkaline sucrose gradient.
In summary, the results presented here show that wild-type M. have neither excision repair nor photoreactivation mechanisms u.v.-induced DNA damage. To our knowledge, no other prokaryote of repair systems. Therefore, M. gallisepticum may be unique with repair capabilities.
cells for the repair of lacks both kinds regard to cellular
gallisepticum
We thank David Gerling for his technical assistance, Dr L. Prakash for the gift of the endonuclease, and Dr C. Lawrence for helpful discussions. This investigation was supported in part by grant AI-07939 from the United Stat*es Public Health Service, National Institute of Allergy and Infections Diseases. One of us (J. D.) is on leave of absence from Calcutta University, Department of Physics, Calcutta, India.
344 Department University Rochester, Received
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REFERENCES Carrier, W. L. & Setlow, R. B. (1970). J. Bacterial. 102, 178-186. Das, J., Maniloff, J. & Bhattacharjee, S. B. (1972). Biochim Biophys. Acta, 259, 189- 197. Das, J., Nowak, J. & Maniloff, J. (1977). J. Bacterial. 129, 1424- 1427. Folsome, C. E. (1968). J. Gen. Microbial. 50, 43-53. Hanawalt, P. C. (1975). Genetics, 79, 179-197. Liss, A. & Maniloff, J. (1973). Virology, 55, 118-126. Maniloff, J. & Morowitz, H. J. (1972). Bacterial. Rev. 36, 263-290. Maniloff, J. & Quinlan, D. C. (1974). J. Bacterial. 120, 495-501. Quinlan, D. C., Liss, A. & Maniloff, J. ( 1972). Microbios, 6, 179-185. Setlow, R. B., Swenson, P. A. & Carrier, W. L. (1963). science, 142, 1464-1466. Smith, D. W. & Hanawalt, P. C. (1969). J. Mol. Biol. 46, 57-72. Swenson, P. A. & Setlow, R. B. (1966). J. Mol. Biol. 15, 201-219. Tourtellotte, M. E. &I Jacobs, R. E. (1960). Ann. LV.Y. Acad. Sci. 79, 521- 530.
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Lack of Repair of Ultraviolet Light Damage in Mycoplasma gallisepticum We have studied the repair capabilities of Mycoplasma gallisepticum, one of the smallest free-living cells, having a genome size of only 4.6 x lOa daltons. The data show that these cells have neither dark repair nor photoreactivation mechanisms for the repair of ultraviolet induced DNA damage. No other prokaryote has been reported to lack both kinds of repair systems. Studies of irradiated cell DNA indicate that the u.v.-induced pyrimidine dimers cannot be excised and block further replication. The photoreactivation experiments also showed that 32. gallisepticum is photoinactivated.
of an organism to repair damage in its DNA is essential for it to be able to maintain the structural integrity of its genome and to survive under adverse environmental conditions. The efficiencies of such DNA repair systems are themselves subject to evolutionary pressures and the rate of evolution of a species may be coupled to the efficiency and discrimination of its repair processes (Hanawalt, 1975). The studies reported here concern the repair capabilities of the smallest free-living cells, the mycoplasmas, in order to examine the ubiquity of DNA repair processes and to determine which repair processes are utilized by cells with such small genomes. The mycoplasmas are a group of small prokaryotes which do not have cell walls : each cell is bounded by a single lipoprotein “unit” membrane (reviewed by Maniloff & Morowitz, 1972). Mycoplasma isolates have been classified into five genera. The genome size for three of the genera (Acholeplasma, Spiroplasm and Thermoplasma) is 1.0 x lo9 daltons and for the other two (Mycoplusma and Ureaplasma) is 4.6 x 10s daltons. Mycoplasma, Uveaplasma and Spiroplasma require sterol for growth; the ot,her two genera do not. Only limited radiobiological data on mycoplasmas are available and all reported studies involved Acholeplusma laidluu~ii strain B. These cells can repair ultravioletinduced DNA damage by both photoreactivation and dark (excision) repair mechanisms (Folsome, 1968; Smith & Hanawalt, 1969; Das et al., 1972). Recently, A. Zuidlawii strain JAl cells have also been found to repair u.v.-irradiated mycoplasma viruses by host cell and ultraviolet reactivation (Das et aZ., 1977): the former mode involves repair of viral DNA by the cells’ excision repair system and the latter suggests the presence of a u.v.-inducible repair system in these cells. There are no reports on the repair capabilities of the other mycoplasma genera. We report here on studies of the repair of DNA damage in u.v.-irradiated Mycoplasma gallisepticum. These cells are unique biological systems. Each cell has specialized polar subcellular organelles, presumably functioning as a primitive mitotic apparatus (Maniloff & Quinlan, 1974). This is the only prokaryote whose growth has been found to be inhibited by cytochalasin B (Ghosh & Maniloff, unpublished data), a drug that inhibits eukaryotes. M. gallisepticum is one of those mycoplasmas which have the smallest genome size reported for free-living cells. Finally, studies of the cells’ ribosomal RNA have shown that M. gallisepticum is significantly different from y:! 337
The ability
338
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J.
Ij.1f-S ANI)
.I. MASILOPh
other prokaryotes : the cells lack some of the ribosomal RNA oligonucleotide sequences that are found in all ot’her prokaryotes (Woese & Maniloff, unpublished data). The mycoplasmas used in this st,udy were &I. gallise~ticum strains A5969 (described by Tourtellotte & Jacobs, 1960) and 15302 (obt’a’ined from Dr G. Kenny, University of Washington), and A. laidlawii strain JAl (described by Liss & Maniloff, 1973). A. laidlawii cells were grown in tryptose broth and assayed as colony-forming units (c.f.u.) on tryptose agar plates, as described previously (Quinlan et al., 1972). M. gaZZisepticum cells were grown in MBB medium: 21 g mycoplasma broth base (BBL, Cockeysville, Md), 50 ml yeast extract (Microbiological Associates, Bethesda, Md), 10 g dextrose and 100 ml horse serum (Flow Laboratory, Rockville, Md) per liter, adjusted to pH 7.5. They were assayed as c.f.u. on MBB agar plates containing lT/, agar. A. laidlawii plate counts were done after three to four days incubation at 37°C and M. gallisepticum after six to seven days. The survival of M. gallisepticum ,45969 following U.V. irradiation is shown in Figure l(a). For comparison, the survival of u.v.-irradiated A. laidlawii JAl was also examined (Fig. l(b)). The JAl strain used in this stud-y has different U.V. inactivation parameters than the B strain used in all previous U.V. repair studies: the slope of the linear part of the survival curve was 77 m2/kJ for strain JAl (from Fig. l(b)) versus 200 m2/kJ for strain B (Das et al., 1972) and the zero-dose intercept of the linear part of the curve was about 3 for strain JAl versus 8 to 10 for strain B. In contrast to the A. laidluwii inactivation curve, the M. gallisepticum A5969 U.V. survival curve is almost exponential (Fig. l(a)). Th e zero-dose intercept of the M. gallisepticum U.V. survival curve is 1.53, which is experimentally indistinguishable from the value of 1.44 expected for exponentially growing cells in which each cell contains only a single genome copy. The final slope of the survival curve is 75 m2/kJ. Identical results were obtained with M. gallisepticum strain 15302. The absence of a shoulder in the M. gallisepticum U.V. survival curve suggested that these cells might not be able to repair u.v.-induced DNA damage. Therefore, experiments were carried out to investigate both dark and light repair in M. gallisepticum JAl was also studied, since this A5969. For comparative purposes, A. laidlawii species is known t,o have bot,h da,rk and light repair mechanisms (reviewed above). To measure dark repair, u.v.-irradiated cells were examined to determine whether cell survival increased if the irradiated cells in buffer were held in the dark before plating. For these experiments, at each U.V. dose in Figure 1, a sample was held for one hour at 37°C before plating. Holding resulted in an increase in survival of u.v.irradiated A. laidlawii, with a dose reduction factor of 1.45 (Fig. l(b)). However, holding u.v.-irradiated M. gallisepticum cells in the dark produced no change in the survival curve (Fig. l(a)). To examine the dark-repair kinetics, u.v.-irradiated cells were held in the dark and the number of viable cells was measured after different holding times (Fig. 2). During holding, the survival of A. hidbwii JAl cells increased exponentially and the maximum amount of dark recovery was reached in about 45 minutes (Fig. 2(b)). This is similar to the dark-recovery kinetics reported for A. laidlawii B cells (Das et al., 1972). However at each U.Y. inactivating dose, no dark recovery was seen for irradiated M. gallisepticum cells (Fig. 2(a)). For photoreactivation studies, cells were irradiated with different U.V. doses and duplicate samples were immediately plated. One set of plates was incubated in the dark and other sets were illuminated with fluorescent room light; each set being illuminated for a different length of time. The results are shown in Figure 3. Efficient
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FIQ. 1. Survival and liquid-holding (dark) recovery of u.v.-irradiated mycoplasmas. (a) M. guZZtiep~icum A6969; (b) A. Zaidlawii JAl. Exponentially growing cells were irradiated with 267 nm light, as previously described (Das et al., 1972). For t,hese studies cells in media were diluted lo-fold into /l-buffer (0.06 M-Tris, 0.166 M-N&I, 0.01 M-p-mercaptoethanol, pH 7.4) to avoid centrifugation (which causes damage to M. guZZise+xm; Maniloff & Morowitz, 1972), that would be necessary to harvest the cells for resuspension in buffer for irradiation. At each U.V. dose 2 samples were removed. One was immediately plated to determine the u.v. survival curve ( 0). The other sample was held in the dark to allow liquid-holding recovery ( A). This latter sample was diluted loo-fold into /l-buffer and held for 1 h at 37°C before plating. In this graph each point is the average of 3 to 6 experiments and a least-squares data analysis (described by Das et al., 1972) was used to calculate the inactivation parameters (slope and zero-dose intercept) and draw the curves.
photoreactivation was observed for A. laidlawii JAl cells (Fig. 3(b)), similar to that reported for A. laidlawii B (Das et al., 1972). However, at each U.V. dose, no photoreactivation was found for M. gallise@icum (Fig. 3(a)). In fact a loss of cell titer occurred while the u.v.-irradiated M. gallise$icum plates were held in the light. Unirradiated M. gallise@icum cells exposed to the fluorescent light also showed a loss of cell titer (Fig. 3(a)). The slopes of the photoinactivation curves of unirradiated cells and cells irradiated with different doses of U.V. light are about the same. This eliminates the possibility of a synergistic effect between U.V. and fluorescent light, which might have been responsible for photoinactivation. U.V. irradiation has very different effects on DNA synthesis in cells with or without
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FIG. 2. Dark-repair kinetics of u.v.-irradiated M. gallisepticum A5969 (a) and A. Zaidlawii JAI (b), inactivated to 2 different survival levels. The titer of unirradiated control cells held in buffer during the period of liquid-holding recovery remained constant (data not shown).
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Pm. 3. Photoreactivation kinetics of u.v.-irradiated M. gallisepticuna A6969 (a) and A. laidlawii JAl (b) cells, inactivated to various survival levels. After u.v. irradiation and plating of the cells, the plates were illuminated by an 80 W fluorescent lamp at a distance of 60 cm. The titer of unirradiated A. Zaidlawii JAl remained constant during the 5-h photoreactivation time.
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repair capabilities (Setlow et al., 1963; Swenson & Seblow, 1966). Therefore, after U.V. irradiation, the synthesis of M. gallisepticum and A. Zaidlawii DNA was followed by measuring the incorporation of labeled deoxythymidine into trichloroacetic acidprecipitable material. For these experiments, logarithmically growing cells were irradiated with U.V. doses to give 35 to 45% survival and then resuspended in grouth medium containing [3H]deoxythymidine. After various incubation times in the dark, trichloroacetic acid-precipitable radioactivity was measured. DNA synthesis in u.v.irradiated M. gallisepticum was reduced compared to unirradiated control cells (Fig. 4(a)). On the other hand, DNA synthesis in u.v.-irradiated A. Zaidlawii JAl cells was almost completely inhibited for about one hour, which is about the time required for maximum dark repair (Fig. 2(b)), and then resumed at the same rate as in unirradiated control cells (Fig. 4(b)). This can be explained if A. ZaidZawii JAl cells can circumvent or repair damage in their DNA, while M. gallisepticum A5969 cells cannot.
: :)
(b)
4. DNA synthesis following ultraviolet irradiation. (a) M. gdisepticum A6969; (b) A. Zoidlowii JAl. (0) Unirradiated control; (A) u.v.-irradiated cells. Irradiated cells (to 35 to 46% survivctl) were resuspended in medium containing 60 PCi [3H]deoxythymidine/m1 (New England Nuclear, Boston, Mass.) and incubated 8t 37°C in the dark. At various times, SO-p1 semples were removed and added to equ81 volumes of cold 10% trichloroacetic acid. The precipitates were collected on filters, washed, and assayed for radioactivity in toluene/Omnifluor (New England Nuclertr, Boston, Mctss.). FIQ.
The state of the DNA in irradiated cells during liquid-holding recovery was examined by analyzing cell lysates by velocity sedimentation in alkaline 5% to 20% sucrose gradients. Figure 5 shows the sedimentation patterns of DNA from unirradiated and u.v.-irradiated M. gaZZisepticum (Fig. 5(a) to (d)) and A. Zuidlawii JAl (Fig. 5(e) to (g)) cells at different holding times. During holding, the average sedimentation rate of DNA from irradiated A. Zaidlawii first decreased and Iater increased. The weight average molecular weight of the single-stranded DNA from unirradiated JAl cells was 4.6 x 10s. After a U.V. dose of 30 J/m2 and 10 minutes of liquid-holding, the DNA molecular weight was reduced to 0.6 x lo*, and increased to 3.2 x 10s after 60 minutes of holding. This is consistent with an excision repair mechanism and similar results have been reported for A. ZaidZawii strain B (Das et al., 1972). In contrast, the sedimentation rate of M. gallisepticum DNA did not change after irradiation or during holding (Fig. 5(a) to (d)). Th e weight average molecular weight of the unirradiated single-stranded DNA was 8% x 107. The size remained unchanged
342
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FIG. 5. Alkaline sucrose gradient sedimentation of DNA after u.v.-irradiation and during liquidholding recovery. Ceils were grown to logarithmic phase in media containing 16 &i [3H]deoxythymidine/ml and irradiated to give a survival of 1 to 5%, as described in the legend to Fig. 1. Samples (0.1 ml) from unirradiated or u.v.-irradiated cells after different holding times were layered on top of 4.5 ml of a 5% to 20% (w / v ) sucrose gradient (adjusted to pH 12.0 with NaOH). Cells were mixed with 0.1 ml 1 N-NaOH, which had been previously layered onto the gradient. After 30 to 60 min, to allow complete digestion, the gradients were centrifuged at 28,000 revs/mm for 2 h at 20°C in a Beckman SW60.1 rotor. Fractions were collected and trichloroacetic acidprecipitable radioactivity measured as described for Fig. 4. Molecular weights were calculated, relative to marker T4 DNA which was added to each tube and shown by an arrow in each gradient as described previously (Das et al., 1972). M. gallisepticum A5969 DNA from (a) unirradiated cells, and irradiated cells after dark holding for (b) 2 min, (c) 10 min, and (d) 60 min. A. Zaidlawii JAl DNA from (e) unirradiated cells, and irradiated cells after dark-holding for (f) 10 min, and (g) 60 min.
after irradiation and up to the 60-minute holding time studied. This observation, together with the decrease in DNA synthesis following irradiation, indicates that u.v.-induced pyrimidine dimers may not be excised in M. gallisepticum and act as blocks to further DNA synthesis. To investigate this explicitly, Micrococcus luteus u.v.-endonuclease (obtained from Dr L. Prakash, University of Rochester) was used to treat M. gallisepticum cells, which had been u.v.-irradiated and held for 20 minutes in the dark, and the cell DNA was analyzed in alkaline 5% to 20% sucrose gradients. This enzyme has endonuclease activity toward u.v.-irradiated DNA, is inactive on u&radiated DNA, and makes
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single-stranded breaks at pyrimidime dimers, but does not excise the dimers (Carmer & Setlow, 1970). In agreement with this, it was found that unirradiated M. gallisepticum A5969 DNA had the same sedimentation profile whether it was untreated (Fig. 6(a)) or treated with the endonuclease (Fig. 6(b)). As described above (Fig. 5(a) to (d)), no change was observed in the sedimentation pattern of unirradiated M. gallisepticum DNA (Fig. 6(a)) and DNA from cells irradiated and then held for 20 minutes (Fig. 6(c)). However, endonuclease treatment of the u.v.-irradiated DNA caused a reduction in the single-stranded DNA molecular weight (Fig. 6(d)), with no measurable loss of trichloroacetic acid-precipitable material. Therefore, u-V.-irradiated M. gallisepticum DNA must contain pyrimidime dimers which the cell is unable to repair. The cell must lack a u.v.-endonuclease specific for ppimidine dimers.
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FIQ. 6. Alkaline sucrose-gradient sedimentation of unirradiated and u.v.-irradiated M. gallise&cum DNA after u.v.-endonuclease t,reatment. Unirradiated cells were (a) untreated or (b) treated with endonucleese. u.v.-irradiated cells (to a survival of 1 to 5%) were held for 20 min in the derk (as described for Fig. 1) and eit,her (c) untreated or (cl) treated with endonuclease. In these experiments the holding buffer was 0.05 M-Tris, 0.01 M-EDTA (pH 8.1), and all cells were frozen and thawed 3 times before enzyme treatments and alkaline sucrose-gradient sedimentation (as in Fig. 5). The freeze-thaw cycles were necessary to make the cells permeable to the added endonuclease and had no effect on the sedimentation behavior of the cell DNA (compare Figs 5(a) and S(a)). For endonucleese treatment, the reaction mixture was 0.05 ml M. luteus u.v.endonuclease extract (12 mg protein/ml) end 1 ml of cells in the holding buffer. The mixture was incubated for 10 min at 37°C and the reaction was terminated by layering 0.1 ml on an alkaline sucrose gradient.
In summary, the results presented here show that wild-type M. have neither excision repair nor photoreactivation mechanisms u.v.-induced DNA damage. To our knowledge, no other prokaryote of repair systems. Therefore, M. gallisepticum may be unique with repair capabilities.
cells for the repair of lacks both kinds regard to cellular
gallisepticum
We thank David Gerling for his technical assistance, Dr L. Prakash for the gift of the endonuclease, and Dr C. Lawrence for helpful discussions. This investigation was supported in part by grant AI-07939 from the United Stat*es Public Health Service, National Institute of Allergy and Infections Diseases. One of us (J. D.) is on leave of absence from Calcutta University, Department of Physics, Calcutta, India.
344 Department University Rochester, Received
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REFERENCES Carrier, W. L. & Setlow, R. B. (1970). J. Bacterial. 102, 178-186. Das, J., Maniloff, J. & Bhattacharjee, S. B. (1972). Biochim Biophys. Acta, 259, 189- 197. Das, J., Nowak, J. & Maniloff, J. (1977). J. Bacterial. 129, 1424- 1427. Folsome, C. E. (1968). J. Gen. Microbial. 50, 43-53. Hanawalt, P. C. (1975). Genetics, 79, 179-197. Liss, A. & Maniloff, J. (1973). Virology, 55, 118-126. Maniloff, J. & Morowitz, H. J. (1972). Bacterial. Rev. 36, 263-290. Maniloff, J. & Quinlan, D. C. (1974). J. Bacterial. 120, 495-501. Quinlan, D. C., Liss, A. & Maniloff, J. ( 1972). Microbios, 6, 179-185. Setlow, R. B., Swenson, P. A. & Carrier, W. L. (1963). science, 142, 1464-1466. Smith, D. W. & Hanawalt, P. C. (1969). J. Mol. Biol. 46, 57-72. Swenson, P. A. & Setlow, R. B. (1966). J. Mol. Biol. 15, 201-219. Tourtellotte, M. E. &I Jacobs, R. E. (1960). Ann. LV.Y. Acad. Sci. 79, 521- 530.
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requests should be addressed.