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Repair of damage induced by ultraviolet light in DNA polymerase-defective Escherichia coli cells
J. Mol. Biol. (1971) 58, 623-630
Repair of Damage induced by Ultraviolet Light in DNA Polymerase-defective Escherichia coli Cells MAFCILYN MONK, MICHAEL PEACEY AND JULUN D. GROSS Medical Research Council Molecular Genetics Unit Department of Molecular Biology University of Edinburgh, Edinburgh, Scotland (Received 1 December 197’0) Cells of the Escherichia coli mutant polA1, which lack DKA polymerase activity in u&-o, are four times as sensitive as wild-type to ultraviolet irradiation. Cells of the mutant uvrA6, which are unable to excise dimers, are 12 times as sensitive as wild-type. We have shown that the double mutant poZA1 uvrA6 is only slightly more sensitive to U.V. than the uvrA6 singlemutantand conclude, therefore, that the U.V. sensitivity associated with the defect in DNA polymerase is primarily the result of a reduction in the efficiency of the excision-repair pathway. Observations on the effect of U.V. irradiation on the ability ofpolA1 cells to support the growth of phage h suggest that the post-u.v. repair function of polymerase is subsequent to the action of the uvr + gene products. Evidence is presented that the recA repair system is involved in excision-repair in po2Al cells, and we propose that it can substitute for DNA polymerase in repairing the gaps produced by dimer excision. This would account for the relatively slight effect of the poZA1
mutation on u.v. sensitivity.
1. Introduction De Lucia & Cairns (1969) have isolated a mutant of Escherichia coli designated polA1, which has little or no DNA polymerase activity in ext’racts. The mutant is more sensitive than the parent to ultraviolet irradiation and to methyl methane sulphonate, and genetic analysis has shown that a single recessive amber mutation is responsible for the absence of DNA polymerase activity and the increased radiationsensitivity (Gross & Gross, 1969). The existence of such a mutant strain suggests, though it does not prove, that DNA polymerase plays a role in DNA repair but not in DNA replication. There are believed to be two “pathways” of dark repair of U.V. damage in E. coli: one, excision repair, requires the ability to excise pyrimidine dimers and is blocked in uvr- mutants; the other, recA-mediated repair, is independent of uvr functions, but depends inter alia on the product of the recA gene which is also required for genetic recombination (Howard-Flanders, 1968). Each repair system alone is able to handle a large proportion of the dimers introduced by U.V. irradiation; if both systems are inactive, in a recA- uvr- double mutant, cells are killed by the introduction of one or two dimers per genome (Howard-Flanders, 1968). We have tried to determine which of these two repair pathways is affected inpolA1 cells, and the nature of the defect, by studying the properties of a polA1 uvr- double mutant. 623
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2. Materials and Methods (a) Bacterial strains The four isogenic strains uvrA6 polA1, uvrA6 pal+ , uvr + polA1, and uvr + pal + were constructed by Mm J. Gnmstein, making use of the transductional linkage of uvrA with m&B, and of polA with m&E (Taylor, 1970). Strain W3110 thy metE rmlB &r-r was transduced to wdB+ with Pl grown on AB1886 uvrA6 (Boyce & Howard-Flanders, 1964) and transductants examined for U.V. sensitivity. A uvrA6 and a uvr+ transductant were purified and each transduced to metE + with Pl grown on polA1 (Gross & Gross, 1969). The transductants were tested for methyl methane sulphonate sensitivity, and a polA1 and poZ+ derivative of each purified, and checked for polymerase activity by the method of De Lucia & Cairns (1969). The use of methyl methane sulphonate permitted detection of the uwrA6 polA1 double mutant, since uvr mutations do not confer sensitivity to methyl methane sulphonate (Howard-Flanders, 1968) whereas poZA1 does. All strains were checked to ensure that they were not lysogenic for P 1. The double mutant uvrA6 polA1 did not survive well if kept at 4°C. Consequently, log-phase cultures of all four strains were stored at - 6O”C, with dimethyl sulphoxide added to a concentration of 7%, and samples of these frozen stock cultures were diluted into fresh medium for use each day. (b) Media Cultures were grown in nutrient broth (Oxoid No. 2, 26g/l.) supplemented with 20 pg thymine/ml. Viable counts and phage X assays were on nutrient agar plates (Oxoid no. 2 nutrient broth 25g, Davis N. Z. agar 12*5g, distilled water to 1 1.) supplemented with 20 pg thyminelml. (0) Ultraviolet irradiation A Hanovia bactericidal ultraviolet unit was used. Depending on the dose-range required, the lamp was set to deliver 1, 3, 10 or 20 ergs mm-s set-l. Dose rates were measured with a Latarjet dosimeter (Latarjet, Morenne & Berger, 1957). Bacterial suspensions were irradiated at less than 1 mm thickness at a concentration of about 10s cells/ml. in buffer. (d) Preparation of lysogew Wild-type X was spotted on to a lawn of bacteria, and growth in streaked for single colonies (the double mutant uvrA6 polA1 gave in the phage spot). These were tested for lysogeny by cross-streaking Lysogens were made A-resistant by plating 0.1 ml. of an overnight particles and purifying resistant colonies.
the area of lysis was barely visible growth against Xc1 and ,I&. culture with log htir
(e) Measurements of viable count, optical density, infectious cerztrer, and free phqe Viable counts were measured by plating in soft agar. Optical density was followed in a Klett-Summerson calorimeter. Infectioue centrm. Phage h was adsorbed to cells suspended in 0.01 M-MgSO., for 16 min at 37°C. A sample of 0.1 ml. of an appropriate dilution of infectious centres was inoculated into 2.5 ml. of soft agar seeded with 0.3 ml. of C600 h-s indicator and poured on to a nutrient agar plate. To measure unadsorbed phage the adsorption mixtures were chloroformed before assay. Free phage. After 140 min at 37°C to allow for lysis, samples were chloroformed, diluted and Oal-ml. samples plated with A-.9indicator as above.
3. Results The colonies formed by cells of the uvrA6 polA1 double mutant after overnight incubation on agar plates are smaller than those formed by wild-type cells or by the single mutants polA1 or uvrA6. We have followed the growth of these strains in exponential broth culture, measuring both optical density and numbers of viable
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FIG. 1. Growth curves of wild-type, uvrA6, polA1 and uvrA6 polA1 cells. Log-phase cultures were diluted in nutrient broth and incubated with aeration at 37°C. Optical density (-•-a-) end viable counts (-O-O--) were measured a~ described in M&xi& and Methods.
cells. The results in Figure 1 show that the ratio of viable counts to optical density is lower for the polA1 cells than for either wild-type or uvr- cells and that this effect is accentuated in the double mutant, which also has a longer generation time. Microscopic examination of cells from exponential cultures (Plate I) indicates that the discrepancy between optical density and viable count may be accounted for by increased average cell sizes. polA1 cultures contain cells of up to five times normal length, while half the cells in uvrA6 polA1 cultures are filaments which can reach 50 cell lengths. Hertman (1969) has noted similar slow growth and filament&ion of a, uvr- recA - double mutant. Figure 2 presents U.V. survival curves of the same four strains. polA1 cells sre approximately four times as sensitive as wild-type cells. uvrA6 cells, which are believed to be completely unable to excise dimers (Boyce &Howard-Flanders, 1964), are about 12 times as sensitive. The uvr- pal- double mutant is only slightly more sensitive than uvrA6 itself; in the experiment shown in Figure 2 the double mutant was about 30% more sensitive than uvrA6, and in another experiment, not shown, it was only 10% more sensitive. This result is in marked contrast with the strict additivity of t,he effects of uvr- and recA- mutations on the exponential rate of U.V. killing (Howard-Flanders, 1968; Hertman, 1969). It indicates that the U.V. sensitivity associated withpolA1 is primarily the result of a reduction in the eillciency of the excision-repair pathway r&her than of recA-mediated repair. Some indication of the step in excision repair that is defective in pot cells has 41
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FIU. 2. Ultraviolet survival curves of wild-type, uvrA6, polA1 and uvrA6 plA1 cells. Logphase oultures at lo* cells/ml. were centrifuged and resuspended in ohilled buffer. Volumes of 7 ml. were irradiated. Samples of 0.1 ml. were removed, diluted and plated in soft agar on nutrient agar plates. The plates were incubated at 37% for 48 hr. The survival curve of wild-type cells showed e shoulder of up to about 400 ergs mmWa; only the final slope is indicated in the Figure.
been obtained by examining their “capacity” (Benzer & Jacob, 1963), that is, the effect of U.V. irradiation on their ability to support the growth of infecting phage h. Devoret & Coquerelle (1966) and Young & Sinsheimer (1967) have shown previously that the capacity of UVT+ cells is significantly decreased by doses of U.V. above 2000 ergs rnrnw3 whereas the same dose has little effect on the capacity of UVP- cells. We have also found the capacity of uvr- cells more resistant at higher doses of U.V. (see Fig. 3). This may be because extensive excision-repair in the wild-type cells at high U.V. doses may pre-empt an enzyme or enzymes required for multiplication of the phage. Figure 3 shows that the pal- mutation has a much more pronounced effect on capacity than the uvr- mutation and that the double mutant UVT- pal- has the same high capacity as the uvv single mutant. Therefore, the defect in pal- cells which results in low capacity is not expressed when the cells are unable to excise dimers; it must, therefore, be at a step subsequent to the action of the UVT gene products. If one role of DNA polymerase in repair is to illl in gaps after excision, and if the enzyme is absent in polA1 cells, these cells should be extremely U.V. sensitive. Since they are not, it is conceivable that some other enzyme or repair process can substitute for the missing DNA polymerase. We have considered the possibility that the recA repair pathway can act in place of DNA polymerase after excision. One diagnostic feature of the extent of involvement of this system appears to be the sensitivity of X lysogens to prophage induction (see Discussion). Therefore, we have examined the sensitivity of lysogenic derivatives of the four strains to induction by U.V. Figure 4 shows the results of these experiments. The interpretation of the induction curves is complicated by the low capacity of polA1 cells, and the reduced ability of uvr- cells
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Fra. 3. Capacity curves for wild-type, uwAG, polA1 and uwAG polA1 cells. Log-phase cultures at 10s cells/ml. were centrifuged and resuspended in chilled O-01 ar-MgSO+ Volumes of 8 ml. were irradiated. Samples of 1 ml. were removed to small tubes, phage h added to each at a multiplicity of infection of 0,06, and adsorption allowed to take place for 16 min at 37°C. Samples were gently diluted and assayed for infectious centres on a C600 h-8 indicator. Two tubes for each series were chloroformed and assayed for unedsorbed phage. Only the infectious centres for poZA1 cells receiving 600 ergs mm-s required correction for un~dsorbed phage. The time from addition of the phage to the final plating for infectious centres WES less than 46 min. In all c.sses the plaques arising from infectious oentres became less clear at the highest WV. doses. In the case of the wildtype cells the plaques faded to extinction and points on the dashed part of the curve are lower estimates.
to repair dimers in the prophage genome. However, the responses at low doses show clearly that polA1 cells are considerably more sensitive to induction than wild-type cells and that they have approximately the same sensitivity as uvr- and UVT- palcells. The low absolute yield of phage from the double mutant is presumably related to its abnormal growth properties, described above.
4. Discussion We have found that the U.V. sensitivity of the poEA uvrA6 double mutant is only very slightly greater than the sensitivity of the wvrA6 single mutant. This result is in striking contrast to that obtained with uvr- recA- double mutant’s whose sensitivity is the product of the sensitivities of strains carrying the component single mutations (Howard-Flanders, 1968; Hertman, 1969). It indicates that the pal- mutation confers P.V. sensitivity by reducing the eiEciency of the excision-repair pathway and does not significantly affect the ability to overcome the lethal effect of unexcised
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FIG. 4. Ultraviolet induction ourves for cells. Log-phase cultures at 10s cells/ml. Volumes of 7 ml. were irradiated. Samples large test tubes and incubated 140 min at for free phage.
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h lysogens of wild-type, uvrA6, polA1 and uvrA6 polA1 were oentrifuged and resuspended in chilled buffer. of 0.1 ml. were removed to 0.5 ml. of nutrient broth in 37°C. Samples were chloroformed, diluted and assayed
dimers by means of the recA+ repair pathway. The same conclusion has been reached by Klein & Niebch (1971) and Kato $ Kondo (1970). Our observations also show that pre-irradiated pal- cells have greatly reduced ability to support the growth of phage X, whereas the pal- UVT- double mutant has the high capacity of uvr- cells. Since uvr- cells sre unable to carry out the initial incision at a dimer the defect in pal- cells must be in a step after incision. The s&me inference may be drawn from the fact that the DNA of polA1 cells that have been incubated after irradiation has a lower average molecular weight than the DNA of similarly treated poZ+ cells (Kanner & Hanawalt, 1970; Boyle, Paterson & Setlow, 1970). Boyle et ccl. (1970) have observed that the rate and final extent of dimer excision are essentially normal in polA1 cells. This finding, which is noteworthy in view of the ability of DNA polymerase to excise dimers from nicked DNA in vitro (Kelly, Atkinson, Huberman & Kornberg, 1969), indics,tes that the U.V. sensitivity of pal- cells is not due to their inability to excise tiers. It would seem plausible therefore, that the step which is defective in pal- cells is repair of the gaps resulting from excision. However, if pal- cells were completely unable to repairthese gaps, one or two dimers would be lethal; instead, 37% of irradiatedpolAI cells survive & dose of U.V. which introduces well over a hundred dimers per genome. One way of explaining this relatively high resistance would be to suppose, as Kanner & Hanawalt (1970) and Boyle et cd. (1970)
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have done, that polA1 cells retain significant DNA polymerase activity. Although this cannot be excluded, a substantial body of evidence renders it unlikely (Moses & Richardson, 1970; Knippers & Strlitling, 1970; Gross, Grunstein & Witkin, 1971), and we believe that some other explanation must be found. The greater sensitivity to prophage induction of uvr- cells compared to wild-type cells (Mattern, van Winden & Rijrsch, 1965) is thought to be due to the fact that the activity of the recA-repair pathway is implicated in induction; since no dimers are removed by excision in a uvr- cell, more of them are diverted to the recA-repair system, and so there is a higher probability of induction for a given U.V. dose. We have shown above (see also Kondo, Ichikawa, Iwo & Kato, 1971) that polA1 cells show approximately the same sensitivity to prophage induction as uvr cells. This suggests that the recA-repair system is active to the same extent after irradiation of polA1 cells as after irradiation of uvr- cells. It is possible, therefore, that polA1 cells are relatively insensitive to killing by U.V. because the recA-repair system can somehow substitute for DNA polymerase in repairing gaps resulting from excision. Since pal- cells are more sensitive than wild-type, one would assume that the substitute system is not quite as e6lcient as DNA polymerase in performing this function. Independent evidence that DNA polymerase and the recA gene product can perform a common function is presented by Gross et al. (1971). The idea that the recA system can act on strand interruptions is supported by the fact that recA- cells show increased sensitivity to 32P decay (Cairns & Davern, 1966) and to X-rays (Kapp t Smith, 1970). Since such strand interruptions would lead to chromosome fragmentation during replication, they must be repaired either before or during replication, but not after replication as has been suggested for recA-mediated repair of U.V. damage in uvr- cells (Rupp & Howard-Flanders, 1968). It is possible that the recA gene product is, or functions in association with, a DNA polymerase distinct from the Kornberg polymerase. That the presumptive recA-mediated repair of excision gaps in pal- cells is different from its action in overcoming the lethal effect of unexcised dimers is indicated by the finding that U.V. mutagenesis, which is, like prophage induction, believed to depend primarily on recA-repair (Witkin, 1969), is not increased inpol- cells, whereas it is increased in uvr- cells (Witkin, 1971; Kondo et al., 1971; Brammar, personal communication). REFERENCES Benzer, S. & Jacob, F. (1953). Ann. In&. Pasteur, 84, 186. Boyce, R. P. & Howard-Flanders, P. (1964). Proc. Nat. Acad. Sci., Wash. 51, 293. Boyle, J. M., Paterson, M. C. & Setlow, R. B. (1970). Nature, 226, 708. Cairns, J. & Davcm, C. I. (1966). J. Mol. Biol. 17, 418. De Lucia, P. & Cairns, J. (1969). Nature, 224, 1164. of Ru&osen&ivity : Mechanisms Devoret, R. & Coquerelle, T. (1966). Genetic Aqects of Repair. Vienna: International Atomic Energy Agency. Gross, J. & Gross, M. (1969). Nature, 224, 1166. Gross, J. D., Grunstem, J. & Witkin, E. M. (1971). J. Mol. Biol. 58, 631. Hertman, I. M. (1969). CIenet. Rea. 14, 291. Howard-Flanders, P. (1968). Ann. Rev. Biochem. 37, 175. Kanner, L. & Hanawalt, P. (1970). Biochem. Biophya. Res. Comm. 39, 149. Kapp, D. S. & Smith, K. C. (1970). J. Bad. 103, 49. Kato, T. & Kondo, S. (1970). J. Butt. 104, 871. Kelly, R. B., Atkinson, M. R., Huberman, J. A. & Kornberg, A. (1969). Nature, 224, 495. Klein, A. & Niebch, U. (1971). Nature, New Biology, 229, 82.
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Knippers, R. & Stratling, W. (1970). Nature, 226, 713. Kondo, S., Ichikawa, H., Iwo, K. & Kato, T. (1971). ffenetica, in the press. Latarjet, R., Morenne, P. & Berger, R. (1957). Ann. In&. Pasteur, 85, 174. Mattern, I., van Winden, M. & RGrsch, A. (1965). Mutation Rm. 2, 111. Moses, R. E. & Richardson, C. C. (1970). Proc. Nat. Acad. Sci., WC&. 67, 674. Rupp, W. D. & Howard-Flanders, P. (1908). J. Mo2. Biol. 31, 291. Taylor, A. L. (1970). Bact. Rev. 34, 166. Witkin, E. M. (1969). Ann. Rev. Microbial. 23, 487. Witkiu, E. M. (1971). Nature, New Biology, 229, 81. Young, E. T. & Sinsheimer, R. L. (1967). J. Mol. Biol. 30, 165.
Repair of Damage induced by Ultraviolet Light in DNA Polymerase-defective Escherichia coli Cells MAFCILYN MONK, MICHAEL PEACEY AND JULUN D. GROSS Medical Research Council Molecular Genetics Unit Department of Molecular Biology University of Edinburgh, Edinburgh, Scotland (Received 1 December 197’0) Cells of the Escherichia coli mutant polA1, which lack DKA polymerase activity in u&-o, are four times as sensitive as wild-type to ultraviolet irradiation. Cells of the mutant uvrA6, which are unable to excise dimers, are 12 times as sensitive as wild-type. We have shown that the double mutant poZA1 uvrA6 is only slightly more sensitive to U.V. than the uvrA6 singlemutantand conclude, therefore, that the U.V. sensitivity associated with the defect in DNA polymerase is primarily the result of a reduction in the efficiency of the excision-repair pathway. Observations on the effect of U.V. irradiation on the ability ofpolA1 cells to support the growth of phage h suggest that the post-u.v. repair function of polymerase is subsequent to the action of the uvr + gene products. Evidence is presented that the recA repair system is involved in excision-repair in po2Al cells, and we propose that it can substitute for DNA polymerase in repairing the gaps produced by dimer excision. This would account for the relatively slight effect of the poZA1
mutation on u.v. sensitivity.
1. Introduction De Lucia & Cairns (1969) have isolated a mutant of Escherichia coli designated polA1, which has little or no DNA polymerase activity in ext’racts. The mutant is more sensitive than the parent to ultraviolet irradiation and to methyl methane sulphonate, and genetic analysis has shown that a single recessive amber mutation is responsible for the absence of DNA polymerase activity and the increased radiationsensitivity (Gross & Gross, 1969). The existence of such a mutant strain suggests, though it does not prove, that DNA polymerase plays a role in DNA repair but not in DNA replication. There are believed to be two “pathways” of dark repair of U.V. damage in E. coli: one, excision repair, requires the ability to excise pyrimidine dimers and is blocked in uvr- mutants; the other, recA-mediated repair, is independent of uvr functions, but depends inter alia on the product of the recA gene which is also required for genetic recombination (Howard-Flanders, 1968). Each repair system alone is able to handle a large proportion of the dimers introduced by U.V. irradiation; if both systems are inactive, in a recA- uvr- double mutant, cells are killed by the introduction of one or two dimers per genome (Howard-Flanders, 1968). We have tried to determine which of these two repair pathways is affected inpolA1 cells, and the nature of the defect, by studying the properties of a polA1 uvr- double mutant. 623
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2. Materials and Methods (a) Bacterial strains The four isogenic strains uvrA6 polA1, uvrA6 pal+ , uvr + polA1, and uvr + pal + were constructed by Mm J. Gnmstein, making use of the transductional linkage of uvrA with m&B, and of polA with m&E (Taylor, 1970). Strain W3110 thy metE rmlB &r-r was transduced to wdB+ with Pl grown on AB1886 uvrA6 (Boyce & Howard-Flanders, 1964) and transductants examined for U.V. sensitivity. A uvrA6 and a uvr+ transductant were purified and each transduced to metE + with Pl grown on polA1 (Gross & Gross, 1969). The transductants were tested for methyl methane sulphonate sensitivity, and a polA1 and poZ+ derivative of each purified, and checked for polymerase activity by the method of De Lucia & Cairns (1969). The use of methyl methane sulphonate permitted detection of the uwrA6 polA1 double mutant, since uvr mutations do not confer sensitivity to methyl methane sulphonate (Howard-Flanders, 1968) whereas poZA1 does. All strains were checked to ensure that they were not lysogenic for P 1. The double mutant uvrA6 polA1 did not survive well if kept at 4°C. Consequently, log-phase cultures of all four strains were stored at - 6O”C, with dimethyl sulphoxide added to a concentration of 7%, and samples of these frozen stock cultures were diluted into fresh medium for use each day. (b) Media Cultures were grown in nutrient broth (Oxoid No. 2, 26g/l.) supplemented with 20 pg thymine/ml. Viable counts and phage X assays were on nutrient agar plates (Oxoid no. 2 nutrient broth 25g, Davis N. Z. agar 12*5g, distilled water to 1 1.) supplemented with 20 pg thyminelml. (0) Ultraviolet irradiation A Hanovia bactericidal ultraviolet unit was used. Depending on the dose-range required, the lamp was set to deliver 1, 3, 10 or 20 ergs mm-s set-l. Dose rates were measured with a Latarjet dosimeter (Latarjet, Morenne & Berger, 1957). Bacterial suspensions were irradiated at less than 1 mm thickness at a concentration of about 10s cells/ml. in buffer. (d) Preparation of lysogew Wild-type X was spotted on to a lawn of bacteria, and growth in streaked for single colonies (the double mutant uvrA6 polA1 gave in the phage spot). These were tested for lysogeny by cross-streaking Lysogens were made A-resistant by plating 0.1 ml. of an overnight particles and purifying resistant colonies.
the area of lysis was barely visible growth against Xc1 and ,I&. culture with log htir
(e) Measurements of viable count, optical density, infectious cerztrer, and free phqe Viable counts were measured by plating in soft agar. Optical density was followed in a Klett-Summerson calorimeter. Infectioue centrm. Phage h was adsorbed to cells suspended in 0.01 M-MgSO., for 16 min at 37°C. A sample of 0.1 ml. of an appropriate dilution of infectious centres was inoculated into 2.5 ml. of soft agar seeded with 0.3 ml. of C600 h-s indicator and poured on to a nutrient agar plate. To measure unadsorbed phage the adsorption mixtures were chloroformed before assay. Free phage. After 140 min at 37°C to allow for lysis, samples were chloroformed, diluted and Oal-ml. samples plated with A-.9indicator as above.
3. Results The colonies formed by cells of the uvrA6 polA1 double mutant after overnight incubation on agar plates are smaller than those formed by wild-type cells or by the single mutants polA1 or uvrA6. We have followed the growth of these strains in exponential broth culture, measuring both optical density and numbers of viable
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FIG. 1. Growth curves of wild-type, uvrA6, polA1 and uvrA6 polA1 cells. Log-phase cultures were diluted in nutrient broth and incubated with aeration at 37°C. Optical density (-•-a-) end viable counts (-O-O--) were measured a~ described in M&xi& and Methods.
cells. The results in Figure 1 show that the ratio of viable counts to optical density is lower for the polA1 cells than for either wild-type or uvr- cells and that this effect is accentuated in the double mutant, which also has a longer generation time. Microscopic examination of cells from exponential cultures (Plate I) indicates that the discrepancy between optical density and viable count may be accounted for by increased average cell sizes. polA1 cultures contain cells of up to five times normal length, while half the cells in uvrA6 polA1 cultures are filaments which can reach 50 cell lengths. Hertman (1969) has noted similar slow growth and filament&ion of a, uvr- recA - double mutant. Figure 2 presents U.V. survival curves of the same four strains. polA1 cells sre approximately four times as sensitive as wild-type cells. uvrA6 cells, which are believed to be completely unable to excise dimers (Boyce &Howard-Flanders, 1964), are about 12 times as sensitive. The uvr- pal- double mutant is only slightly more sensitive than uvrA6 itself; in the experiment shown in Figure 2 the double mutant was about 30% more sensitive than uvrA6, and in another experiment, not shown, it was only 10% more sensitive. This result is in marked contrast with the strict additivity of t,he effects of uvr- and recA- mutations on the exponential rate of U.V. killing (Howard-Flanders, 1968; Hertman, 1969). It indicates that the U.V. sensitivity associated withpolA1 is primarily the result of a reduction in the eillciency of the excision-repair pathway r&her than of recA-mediated repair. Some indication of the step in excision repair that is defective in pot cells has 41
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FIU. 2. Ultraviolet survival curves of wild-type, uvrA6, polA1 and uvrA6 plA1 cells. Logphase oultures at lo* cells/ml. were centrifuged and resuspended in ohilled buffer. Volumes of 7 ml. were irradiated. Samples of 0.1 ml. were removed, diluted and plated in soft agar on nutrient agar plates. The plates were incubated at 37% for 48 hr. The survival curve of wild-type cells showed e shoulder of up to about 400 ergs mmWa; only the final slope is indicated in the Figure.
been obtained by examining their “capacity” (Benzer & Jacob, 1963), that is, the effect of U.V. irradiation on their ability to support the growth of infecting phage h. Devoret & Coquerelle (1966) and Young & Sinsheimer (1967) have shown previously that the capacity of UVT+ cells is significantly decreased by doses of U.V. above 2000 ergs rnrnw3 whereas the same dose has little effect on the capacity of UVP- cells. We have also found the capacity of uvr- cells more resistant at higher doses of U.V. (see Fig. 3). This may be because extensive excision-repair in the wild-type cells at high U.V. doses may pre-empt an enzyme or enzymes required for multiplication of the phage. Figure 3 shows that the pal- mutation has a much more pronounced effect on capacity than the uvr- mutation and that the double mutant UVT- pal- has the same high capacity as the uvv single mutant. Therefore, the defect in pal- cells which results in low capacity is not expressed when the cells are unable to excise dimers; it must, therefore, be at a step subsequent to the action of the UVT gene products. If one role of DNA polymerase in repair is to illl in gaps after excision, and if the enzyme is absent in polA1 cells, these cells should be extremely U.V. sensitive. Since they are not, it is conceivable that some other enzyme or repair process can substitute for the missing DNA polymerase. We have considered the possibility that the recA repair pathway can act in place of DNA polymerase after excision. One diagnostic feature of the extent of involvement of this system appears to be the sensitivity of X lysogens to prophage induction (see Discussion). Therefore, we have examined the sensitivity of lysogenic derivatives of the four strains to induction by U.V. Figure 4 shows the results of these experiments. The interpretation of the induction curves is complicated by the low capacity of polA1 cells, and the reduced ability of uvr- cells
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2000 Ultraviolet dose (ergs mm-‘)
I
3000
Fra. 3. Capacity curves for wild-type, uwAG, polA1 and uwAG polA1 cells. Log-phase cultures at 10s cells/ml. were centrifuged and resuspended in chilled O-01 ar-MgSO+ Volumes of 8 ml. were irradiated. Samples of 1 ml. were removed to small tubes, phage h added to each at a multiplicity of infection of 0,06, and adsorption allowed to take place for 16 min at 37°C. Samples were gently diluted and assayed for infectious centres on a C600 h-8 indicator. Two tubes for each series were chloroformed and assayed for unedsorbed phage. Only the infectious centres for poZA1 cells receiving 600 ergs mm-s required correction for un~dsorbed phage. The time from addition of the phage to the final plating for infectious centres WES less than 46 min. In all c.sses the plaques arising from infectious oentres became less clear at the highest WV. doses. In the case of the wildtype cells the plaques faded to extinction and points on the dashed part of the curve are lower estimates.
to repair dimers in the prophage genome. However, the responses at low doses show clearly that polA1 cells are considerably more sensitive to induction than wild-type cells and that they have approximately the same sensitivity as uvr- and UVT- palcells. The low absolute yield of phage from the double mutant is presumably related to its abnormal growth properties, described above.
4. Discussion We have found that the U.V. sensitivity of the poEA uvrA6 double mutant is only very slightly greater than the sensitivity of the wvrA6 single mutant. This result is in striking contrast to that obtained with uvr- recA- double mutant’s whose sensitivity is the product of the sensitivities of strains carrying the component single mutations (Howard-Flanders, 1968; Hertman, 1969). It indicates that the pal- mutation confers P.V. sensitivity by reducing the eiEciency of the excision-repair pathway and does not significantly affect the ability to overcome the lethal effect of unexcised
628
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105 1 0
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I 100 Ultraviolet
FIG. 4. Ultraviolet induction ourves for cells. Log-phase cultures at 10s cells/ml. Volumes of 7 ml. were irradiated. Samples large test tubes and incubated 140 min at for free phage.
AND
I
J. D.
I
I 200 dose (erp
GROSS
I 300
mm‘“)
h lysogens of wild-type, uvrA6, polA1 and uvrA6 polA1 were oentrifuged and resuspended in chilled buffer. of 0.1 ml. were removed to 0.5 ml. of nutrient broth in 37°C. Samples were chloroformed, diluted and assayed
dimers by means of the recA+ repair pathway. The same conclusion has been reached by Klein & Niebch (1971) and Kato $ Kondo (1970). Our observations also show that pre-irradiated pal- cells have greatly reduced ability to support the growth of phage X, whereas the pal- UVT- double mutant has the high capacity of uvr- cells. Since uvr- cells sre unable to carry out the initial incision at a dimer the defect in pal- cells must be in a step after incision. The s&me inference may be drawn from the fact that the DNA of polA1 cells that have been incubated after irradiation has a lower average molecular weight than the DNA of similarly treated poZ+ cells (Kanner & Hanawalt, 1970; Boyle, Paterson & Setlow, 1970). Boyle et ccl. (1970) have observed that the rate and final extent of dimer excision are essentially normal in polA1 cells. This finding, which is noteworthy in view of the ability of DNA polymerase to excise dimers from nicked DNA in vitro (Kelly, Atkinson, Huberman & Kornberg, 1969), indics,tes that the U.V. sensitivity of pal- cells is not due to their inability to excise tiers. It would seem plausible therefore, that the step which is defective in pal- cells is repair of the gaps resulting from excision. However, if pal- cells were completely unable to repairthese gaps, one or two dimers would be lethal; instead, 37% of irradiatedpolAI cells survive & dose of U.V. which introduces well over a hundred dimers per genome. One way of explaining this relatively high resistance would be to suppose, as Kanner & Hanawalt (1970) and Boyle et cd. (1970)
DNA
POLYMERASE-DEFECTIVE
E.
COLI
CELLS
b’9
have done, that polA1 cells retain significant DNA polymerase activity. Although this cannot be excluded, a substantial body of evidence renders it unlikely (Moses & Richardson, 1970; Knippers & Strlitling, 1970; Gross, Grunstein & Witkin, 1971), and we believe that some other explanation must be found. The greater sensitivity to prophage induction of uvr- cells compared to wild-type cells (Mattern, van Winden & Rijrsch, 1965) is thought to be due to the fact that the activity of the recA-repair pathway is implicated in induction; since no dimers are removed by excision in a uvr- cell, more of them are diverted to the recA-repair system, and so there is a higher probability of induction for a given U.V. dose. We have shown above (see also Kondo, Ichikawa, Iwo & Kato, 1971) that polA1 cells show approximately the same sensitivity to prophage induction as uvr cells. This suggests that the recA-repair system is active to the same extent after irradiation of polA1 cells as after irradiation of uvr- cells. It is possible, therefore, that polA1 cells are relatively insensitive to killing by U.V. because the recA-repair system can somehow substitute for DNA polymerase in repairing gaps resulting from excision. Since pal- cells are more sensitive than wild-type, one would assume that the substitute system is not quite as e6lcient as DNA polymerase in performing this function. Independent evidence that DNA polymerase and the recA gene product can perform a common function is presented by Gross et al. (1971). The idea that the recA system can act on strand interruptions is supported by the fact that recA- cells show increased sensitivity to 32P decay (Cairns & Davern, 1966) and to X-rays (Kapp t Smith, 1970). Since such strand interruptions would lead to chromosome fragmentation during replication, they must be repaired either before or during replication, but not after replication as has been suggested for recA-mediated repair of U.V. damage in uvr- cells (Rupp & Howard-Flanders, 1968). It is possible that the recA gene product is, or functions in association with, a DNA polymerase distinct from the Kornberg polymerase. That the presumptive recA-mediated repair of excision gaps in pal- cells is different from its action in overcoming the lethal effect of unexcised dimers is indicated by the finding that U.V. mutagenesis, which is, like prophage induction, believed to depend primarily on recA-repair (Witkin, 1969), is not increased inpol- cells, whereas it is increased in uvr- cells (Witkin, 1971; Kondo et al., 1971; Brammar, personal communication). REFERENCES Benzer, S. & Jacob, F. (1953). Ann. In&. Pasteur, 84, 186. Boyce, R. P. & Howard-Flanders, P. (1964). Proc. Nat. Acad. Sci., Wash. 51, 293. Boyle, J. M., Paterson, M. C. & Setlow, R. B. (1970). Nature, 226, 708. Cairns, J. & Davcm, C. I. (1966). J. Mol. Biol. 17, 418. De Lucia, P. & Cairns, J. (1969). Nature, 224, 1164. of Ru&osen&ivity : Mechanisms Devoret, R. & Coquerelle, T. (1966). Genetic Aqects of Repair. Vienna: International Atomic Energy Agency. Gross, J. & Gross, M. (1969). Nature, 224, 1166. Gross, J. D., Grunstem, J. & Witkin, E. M. (1971). J. Mol. Biol. 58, 631. Hertman, I. M. (1969). CIenet. Rea. 14, 291. Howard-Flanders, P. (1968). Ann. Rev. Biochem. 37, 175. Kanner, L. & Hanawalt, P. (1970). Biochem. Biophya. Res. Comm. 39, 149. Kapp, D. S. & Smith, K. C. (1970). J. Bad. 103, 49. Kato, T. & Kondo, S. (1970). J. Butt. 104, 871. Kelly, R. B., Atkinson, M. R., Huberman, J. A. & Kornberg, A. (1969). Nature, 224, 495. Klein, A. & Niebch, U. (1971). Nature, New Biology, 229, 82.
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Knippers, R. & Stratling, W. (1970). Nature, 226, 713. Kondo, S., Ichikawa, H., Iwo, K. & Kato, T. (1971). ffenetica, in the press. Latarjet, R., Morenne, P. & Berger, R. (1957). Ann. In&. Pasteur, 85, 174. Mattern, I., van Winden, M. & RGrsch, A. (1965). Mutation Rm. 2, 111. Moses, R. E. & Richardson, C. C. (1970). Proc. Nat. Acad. Sci., WC&. 67, 674. Rupp, W. D. & Howard-Flanders, P. (1908). J. Mo2. Biol. 31, 291. Taylor, A. L. (1970). Bact. Rev. 34, 166. Witkin, E. M. (1969). Ann. Rev. Microbial. 23, 487. Witkiu, E. M. (1971). Nature, New Biology, 229, 81. Young, E. T. & Sinsheimer, R. L. (1967). J. Mol. Biol. 30, 165.