J. Mol. Biol. (1976) lOO, 219-225
Visualization of Sister Strand Exchanges Induced by Ultraviolet Irradiation To visualize ultraviolet-induced sister strand exchanges electron microscope autoradiography was used. Escherichia cell strain AB2500 uvrA 6 Rec +, incapable of excising pyrimidine dimers in DNA was labelled with [methyl-aH]thymidine (28 Ci/mmol) during one cycle of semiconservative replication, then irradiated with ultraviolet light (dose 40 ergs/mm 2) and incubated in non-radioactive medium for 100 minutes. By means of electron microscope autoradiography the exchanged areas, mostly shorter than 1 ~m, were observed in DNA isolated from these cells. The number of visualized exchanges comprises 10 to 15% of pyrimidine dimers. In DNA from non-irradiated cells the exchanges were observed much less frequently than in DNA from ultraviolet-irradiated cells. These results confirm the suggestion that ultraviolet light induces sister exchanges in bacteria, which can be involved in post-replication repair.
I n Escherichia cell irradiated with ultraviolet light the newly synthesized D N A strands have gaps opposite the pyrimidine dimers in old strands (Howard-Flanders et al., 1968; Rupp & ttoward-Flanders, 1968). During further incubation these gaps are filled, if the RecA function is active (Howard-Flanders et al., 1968 ; Smith & Meun, 1970). I t was suggested t h a t gaps opposite dimers are filled b y a mechanism involving u.v.-induced single-strand exchanges between sister duplexes (Howard-Flanders etal., 1968; R u p p & Howard-Flanders, 1968). The exchanges were detected b y means of CsCI density gradient techniques (Rupp etal., 1971). According to the data of Cole (1973) and Howard-Flanders & Lin (1973) a similar mechanism operates during the repair of cross-linked DNA. Holliday (1971) proposed also t h a t sister exchanges ensure the repair of D N A damaged with ionizing radiation. I n this paper the results of a direct search for u.v.-induced sister exchanges by means of electron microscope autoradiography of D N A molecules are presented. E. coli strain AB2500 uvrA 6 thy-, thre-, leu-, pro-, arg-, his- B 1- unable to excise pyrimidine dimers was used (Howard-Flanders & Boyce, 1966). The experiment was carried out according to the scheme shown in Figure 1. Cells were grown to a density of l0 s cells/ml in K - m e d i u m of R u p p etal. (1971) supplemented with 5/~g thymidine/ml, washed on a Millipore filter with K-medium, resuspended in an equal volume of K - m e d i u m and incubated with stirring for ten minutes at 37~ to exhaust the intraeellular pool of thymine. Then 2 ~g [methyl-aH]thymidine/ ml (28 Ci/mmol) were added, and incubation was continued for 30 minutes, i.e. for about one cycle of semiconservative replication. Then thymidine was added in excess (50/~g/ml) for five minutes, the cells were thoroughly washed with K - m e d i u m supplied with thymidine (20 ~g/ml) and resuspended at 2 • l0 s cells/ml in K - m e d i u m deprived of Casamino acids and thymidine. H a f t of the culture was irradiated with a dose of 40 ergs/mm 2 of u.v. light (254 nm) to produce about 200 pyrimidine dimers per genome (Rupp & Howard-Flanders, 1968; Boyle & Setlow, 1970; Friedberg & Clayton, 1972), and both portions were incubated in K - m e d i n m supplemented with 219
M. I. MOSEVITSKY
220
91 l
[3H]thymidine for 50rain ID
/~
1
Washing, u.v.- irradiation
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1
Replication
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Sister exchanges
T Y or
Version TT ~91. . . . .
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Fig. l. Diagrams showing the procedure of the experiment and possible models of u.v.-induced sister strand exchanges. (. ), Unlabelled old D N A strands; (. ), 3H-labelled old D N A strands; ( .), unlabelled D N A strands synthesized during post-irradiational replication; ( . . . . . . . . . . . ), sites of repair synthesis.
Casamino acids and 20/zg thymidine/ml for 100 minutes. In irradiated cells, DNA strands with gaps opposite dimers were synthesized. Many of these gaps could be filled in during the time allowed (Howard-Flanders et al., 1968; Smith & Meun, 1970; Rupp et al., 1971). Both portions of cells (irradiated and non-irradiated) were washed with 0.02 •-Tris, 0-005 M-EDTA buffer, pH 7-5, resuspended in this buffer at 5 • 108 cells/ml and lysed by incubation with lysozyme (50/zg/ml) at 37~ for ten minutes followed by addition of sodium dodecyl sulphate to a final concentration of 2~ . Lysed cells were extracted with an equal volume of water-saturated phenol, pH 8. The DNA-containing aqueous phase was dialysed at 15~ against 0.1 m-ammonium acetate, pH 6"8. DNA samples for electron microscopy were prepared by sliding carbon-coated mica plates through the surface of the DNA solution. DNA molecules in the preparation were stretched predominantly in the direction of the plate movement. DNA was shadowed by evaporation of platinum-palladium alloy (8 : 2) in vacuo at an angle of about 7 ~ For the one-directional shadowing the mica plates were orientated so t h a t the direction of evaporation was normal to the DNA strands. Some DNA samples were shadowed on a rotating table. To prepare the autoradiographs shadowed DNA samples were covered with a layer of nuclear photo-emulsion (Ilford L4) by the loop method of Caro and Van Tubergen
L E T T E R S TO T H E E D I T O R
221
(Caro & Van Tubergen, 1962; Caro, 1969) with the following modification: the final dilution of stock emulsion (v/v) was 1:1.5 instead of 1:2. Therefore, the layer of emulsion was thicker in our autoradiographs. The observations of non-developed autoradiographs showed that silver halide grains are very tightly packed and even overlap. Thanks to this the efficiency (i.e. the percentage of 3H decays which give rise to developed silver grains), was raised (see also Vrensen, 1970). The shortcoming of this modification is poorer transparency of the developed autoradiographs due to the presence of more gelatin. The autoradiographs were placed in metal boxes with dry silica gel and exposed in a lead shielding at 4~ for 8 to 12 months. After the exposure, autoradiographs were developed with metol-hydroquinone developer D19 at 18~ for one-half to one minute, fixed for ten minutes, washed in distilled water and immersed in 0.05 M-Na0H at 18~ for partial solution of the gelatin. The complete removal of gelatin would favour the quality of DNA image in electron mierographs, but developed silver grains would be loosened, displaced, or even desorbed from the autoradiograph. Therefore, the autoradiographs were treated with alkali only until DI~A strands and developed grains become distinct when observed in the electron microscope. For observation in the electron microscope carbon films with preparations were transferred from mica onto a water surface and placed on electron microscope grids. For a more efficient detection of fl-particles arising at 3H decays, "sandwich" autoradiographs were also prepared (Fig. 2). In this case the collodion-coated grid was
--2 --3 - - 4
Fro. 2. Scheme of undeveloped sandwich autoradiograph. 1, layers of photo-emulsion; 2, carbon film with 3H-containing sample prepared for electron microscopy; 3, collodion film; 4, grid.
covered with the first layer of emulsion. Then the carbon film carrying the shadowed DNA sample was transferred from the mica plate to a grid and covered with the second layer of emulsion. Thanks to the permeability of collodion film, the first layer of emulsion was developed after the exposure as good as the second one. Autoradiographs were examined in a JEM-5G electron microscope. Figure 1 shows that in the conditions of our experiments u.v.-induced sister-strand exchanges might form labelled insertions in unlabelled DNA molecules and unlabelled insertions in labelled molecules. But in the latter case the exchanges could also be caused by some kind of repair synthesis occurring after the transfer of cells to unlabelled medium. I t would also be difficult to distinguish between short unlabelled insertions and their imitations caused b y irregular distribution of developed grains along a uniformly labelled strand. Taking this into account, we concentrated on looking for the labelled segments in unlabelled DNA molecules. Only compact groups of three or more silver grains placed along the DNA strand were considered as an indication that the corresponding DNA segment is labelled. The error in estimating the labelled segment length is about 0.3/~m, this being due to the
222
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MOSEVITSKY
size of developed grains (0-1 to 0.15 ~m) and to the range of fl-particles paths in emulsion (see Salpeter et al., 1969). The length of DNA molecules was mostly 5 to 8 ~m (10 to 16 • 106 daltons). The total length of DNA inspected in autoradiograph preparations from u.v.-irradiated cells was about 2000/~m. The same total length of DNA was inspected in the case of unirradiated (control) cells. 34 labelled inner segments were found in DNA molecules of u.v.-irradiated cells (Figs 3 and 4). The size distribution of these insertions was as follows: 23 were shorter than I ~m (at least 15 of them ranged from 0-5 to 1/zm, six ranged from 1 ~m to 2 ~m, two from 2 ~m to 3 ~m and three from 3 to 4 ~m). We did not include four molecules with label in a terminal segment because this type of labelling could be caused by presence of the replication point in the corresponding area of the chromosome at the moment of transfer from unlabelled to labelled medium, or vice versa. In the case of DNA isolated from control cells only two DNA molecules with labelled insertions (0-5 and 1-5 ~m) and three molecules with label in a terminal segment were observed. These results confirm the suggestion that u.v.-irradiation stimulates sister strand exchanges in bacteria. However, they do not exclude the less frequent spontaneous exchanges. We see that u.v.-stimulated exchanges are mostly short. This is in accord with hypothesis of Howard-Flanders et al. (Rupp et al., 1971; Howard-Fianders, 1973) that the induced sister exchanges participate in post-replication repair (see Fig. 11 in Rupp et al., 1971 and Fig. 1. in this paper). The dose of irradiation (40 ergs/mm 2) was determined to within 10%, and the estimate of the number of dimers (about 200 per E. coli genome, or 300 per 2000 ~m) was made using data of different authors, which are in good agreement with each other (Rupp & Howard-Flanders, 1968; Boyle & Setlow, 1970; Friedberg & Clayton, 1972). Therefore, the number of labelled insertions observed in DNA of u.v.-irradiated cells (34 insertions/2000 ~m) comprises not more than 10 to 15% of the expected number, if sister strand recombinations were the only mode of the post-replication repair. Several interpretations of this fact might be considered, which do not exclude one another. (1) Some (mostly short) labelled insertions might be missed because they have induced a low number (less than 3) of developed grains in emulsion. (2) Dimers arise by a co-operative mechanism in pyrimidine tracts, and several dimers form a tight group in a polynucleotide chain (Bruak, 1973). The corresponding post-replication defects can be repaired b y a single sister exchange. (3) Not all post-replication defects have been repaired in our experiments. (4) Besides sister exchanges other mechanisms using DNA synthesis for the filling of gaps opposite dimers are involved in the post-replication repair (Witkin & George, 1973; Buhl et al., 1974). In our experiments we could not observe the patches of new DNA, because after u.v.irradiation the cells were incubated in unlabelled medium. I t is difficult at present to state the relative contributions of these possibilities. The experimental results presented here, and also our previous work (Bresler et al., 1970,1972) indicate that the electron microscope autoradiography of nucleic acids permits the measurements of the lengths of 3H-labelled segments, if the exposure is sufficiently long. The method can be highly useful for those scientists who intend to observe the recombination areas in DNA and DNA segments newly synthesized during replicative or repair synthesis.
LETTERS
TO T H E E D I T O R
223
Fie. 3. Electron microscope autoradiographs of the DNA strands containing ~ segments exchanged during u.v.-induced sister recombination. Exposure for 10 months. (a) Onesided autoradiograph. Shadowing by rotation. Magnification 40,000 • (b) Sandwich autoradiograph. One-directional shadowing. Magnification 50,000 x . (c) One-sided autoradiograph. Shadowing by rotation. Magrffieation 50,000 •
224
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MOSEVITSKY
i
FIo. 4. The same as Fig. 3. (a) Sandwich autoradiograph. One-directional shadowing. Magnification 25,000 x . (b) Sandwich autoradiograph. One-directional shadowing. Magnification 40,000 •
LETTERS
TO T H E E D I T O R
225
I a m grateful to Professor S. E. Bresler for discussions and to Dr L. K. K o r o v i n for help in experiments. L a b o r a t o r y of Biopolymers I n s t i t u t e of Nuclear Physics A c a d e m y of Sciences of U.S.S.R. Leningrad, Gatchina 188350, U.S.S.R.
MARK I. MOSEVITSKY
Received 28 May 1975, and in revised form 19 October 1975 REFERENCES Boyle, J. & Setlow, R. (1970). J. Mol. Biol. 51, 131-144. Bresler, S. E., D a d i v a n j a n , L. P. & Mosevitsky, M. I. (1970). Biochim. Biophys. Acta, 224, 249-252. Bresler, S. E., Dadivanjan, L. P. & Mosevitsky, M. I. (1972). Mol. Biol. U.S.S.R. 6, 226-230. Brunk, C. F. (1973). Nature New Biol. 241, 74-76. Buhl, S. N., Setlow, R. B. & Regan, J. D. (1974). Biophys. J. 14, 791-804. Caro, L. G. (1969). J. Cell Biol. 41,918-919. Caro, L. G. & Van Tubergen, R. P. (1962). J. Cell Biol. 15, 173-188. Cole, R. S. (1973). Proc. Nat. Aead. Sci., U.S.A. 70, 1064-1069. Friedberg, E. & Clayton, D. (1972). Nature (London), 237, 99-100. Holliday, R. (1971). Nature New Biol. 232, 233-236. Howard-Flanders, P. (1973). Brit. Med. Bull. 29, 226-235. Howard-Flanders, P. & Boyee, R. P. (1966). Rad. Res. Suppl. @, 156-184. Howard-Flanders, P. & Lin, P.-F. (1973). Genetics 73, 85-90. Howard-Flanders, P., R u p p , W. D., Wilkins, B. M. & Cole, R. S. (1968). Cold Spring Harbor Symp. Quant. Biol. 33, 195-205. R u p p , W. D. & Howard-Flanders, P. (1968). J. Mol. Biol. 31, 291-304. R u p p , W. D., Wilde, C. E., Reno, D. L. & Howard-Flanders, P. (1971). J. Mol. Biol. 61, 25-44. Salpeter, M. M., Baehmarm, L. & Salpeter, E. E. (1969). J. Cell Biol. 41, 1-20. Smith, K. C. & Meun, D. H. C. (1970). J. Mol. Biol. 51,459-472. Vrensen, G. F. J. M. (1970). J. Histochem. Cytochem. 18, 278-290. Witkin, E. M. & George, D. L. (1973). Genetics 73, 91-108.
Visualization of Sister Strand Exchanges Induced by Ultraviolet Irradiation To visualize ultraviolet-induced sister strand exchanges electron microscope autoradiography was used. Escherichia cell strain AB2500 uvrA 6 Rec +, incapable of excising pyrimidine dimers in DNA was labelled with [methyl-aH]thymidine (28 Ci/mmol) during one cycle of semiconservative replication, then irradiated with ultraviolet light (dose 40 ergs/mm 2) and incubated in non-radioactive medium for 100 minutes. By means of electron microscope autoradiography the exchanged areas, mostly shorter than 1 ~m, were observed in DNA isolated from these cells. The number of visualized exchanges comprises 10 to 15% of pyrimidine dimers. In DNA from non-irradiated cells the exchanges were observed much less frequently than in DNA from ultraviolet-irradiated cells. These results confirm the suggestion that ultraviolet light induces sister exchanges in bacteria, which can be involved in post-replication repair.
I n Escherichia cell irradiated with ultraviolet light the newly synthesized D N A strands have gaps opposite the pyrimidine dimers in old strands (Howard-Flanders et al., 1968; Rupp & ttoward-Flanders, 1968). During further incubation these gaps are filled, if the RecA function is active (Howard-Flanders et al., 1968 ; Smith & Meun, 1970). I t was suggested t h a t gaps opposite dimers are filled b y a mechanism involving u.v.-induced single-strand exchanges between sister duplexes (Howard-Flanders etal., 1968; R u p p & Howard-Flanders, 1968). The exchanges were detected b y means of CsCI density gradient techniques (Rupp etal., 1971). According to the data of Cole (1973) and Howard-Flanders & Lin (1973) a similar mechanism operates during the repair of cross-linked DNA. Holliday (1971) proposed also t h a t sister exchanges ensure the repair of D N A damaged with ionizing radiation. I n this paper the results of a direct search for u.v.-induced sister exchanges by means of electron microscope autoradiography of D N A molecules are presented. E. coli strain AB2500 uvrA 6 thy-, thre-, leu-, pro-, arg-, his- B 1- unable to excise pyrimidine dimers was used (Howard-Flanders & Boyce, 1966). The experiment was carried out according to the scheme shown in Figure 1. Cells were grown to a density of l0 s cells/ml in K - m e d i u m of R u p p etal. (1971) supplemented with 5/~g thymidine/ml, washed on a Millipore filter with K-medium, resuspended in an equal volume of K - m e d i u m and incubated with stirring for ten minutes at 37~ to exhaust the intraeellular pool of thymine. Then 2 ~g [methyl-aH]thymidine/ ml (28 Ci/mmol) were added, and incubation was continued for 30 minutes, i.e. for about one cycle of semiconservative replication. Then thymidine was added in excess (50/~g/ml) for five minutes, the cells were thoroughly washed with K - m e d i u m supplied with thymidine (20 ~g/ml) and resuspended at 2 • l0 s cells/ml in K - m e d i u m deprived of Casamino acids and thymidine. H a f t of the culture was irradiated with a dose of 40 ergs/mm 2 of u.v. light (254 nm) to produce about 200 pyrimidine dimers per genome (Rupp & Howard-Flanders, 1968; Boyle & Setlow, 1970; Friedberg & Clayton, 1972), and both portions were incubated in K - m e d i n m supplemented with 219
M. I. MOSEVITSKY
220
91 l
[3H]thymidine for 50rain ID
/~
1
Washing, u.v.- irradiation
/~
1
Replication
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Sister exchanges
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Version TT ~91. . . . .
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Fig. l. Diagrams showing the procedure of the experiment and possible models of u.v.-induced sister strand exchanges. (. ), Unlabelled old D N A strands; (. ), 3H-labelled old D N A strands; ( .), unlabelled D N A strands synthesized during post-irradiational replication; ( . . . . . . . . . . . ), sites of repair synthesis.
Casamino acids and 20/zg thymidine/ml for 100 minutes. In irradiated cells, DNA strands with gaps opposite dimers were synthesized. Many of these gaps could be filled in during the time allowed (Howard-Flanders et al., 1968; Smith & Meun, 1970; Rupp et al., 1971). Both portions of cells (irradiated and non-irradiated) were washed with 0.02 •-Tris, 0-005 M-EDTA buffer, pH 7-5, resuspended in this buffer at 5 • 108 cells/ml and lysed by incubation with lysozyme (50/zg/ml) at 37~ for ten minutes followed by addition of sodium dodecyl sulphate to a final concentration of 2~ . Lysed cells were extracted with an equal volume of water-saturated phenol, pH 8. The DNA-containing aqueous phase was dialysed at 15~ against 0.1 m-ammonium acetate, pH 6"8. DNA samples for electron microscopy were prepared by sliding carbon-coated mica plates through the surface of the DNA solution. DNA molecules in the preparation were stretched predominantly in the direction of the plate movement. DNA was shadowed by evaporation of platinum-palladium alloy (8 : 2) in vacuo at an angle of about 7 ~ For the one-directional shadowing the mica plates were orientated so t h a t the direction of evaporation was normal to the DNA strands. Some DNA samples were shadowed on a rotating table. To prepare the autoradiographs shadowed DNA samples were covered with a layer of nuclear photo-emulsion (Ilford L4) by the loop method of Caro and Van Tubergen
L E T T E R S TO T H E E D I T O R
221
(Caro & Van Tubergen, 1962; Caro, 1969) with the following modification: the final dilution of stock emulsion (v/v) was 1:1.5 instead of 1:2. Therefore, the layer of emulsion was thicker in our autoradiographs. The observations of non-developed autoradiographs showed that silver halide grains are very tightly packed and even overlap. Thanks to this the efficiency (i.e. the percentage of 3H decays which give rise to developed silver grains), was raised (see also Vrensen, 1970). The shortcoming of this modification is poorer transparency of the developed autoradiographs due to the presence of more gelatin. The autoradiographs were placed in metal boxes with dry silica gel and exposed in a lead shielding at 4~ for 8 to 12 months. After the exposure, autoradiographs were developed with metol-hydroquinone developer D19 at 18~ for one-half to one minute, fixed for ten minutes, washed in distilled water and immersed in 0.05 M-Na0H at 18~ for partial solution of the gelatin. The complete removal of gelatin would favour the quality of DNA image in electron mierographs, but developed silver grains would be loosened, displaced, or even desorbed from the autoradiograph. Therefore, the autoradiographs were treated with alkali only until DI~A strands and developed grains become distinct when observed in the electron microscope. For observation in the electron microscope carbon films with preparations were transferred from mica onto a water surface and placed on electron microscope grids. For a more efficient detection of fl-particles arising at 3H decays, "sandwich" autoradiographs were also prepared (Fig. 2). In this case the collodion-coated grid was
--2 --3 - - 4
Fro. 2. Scheme of undeveloped sandwich autoradiograph. 1, layers of photo-emulsion; 2, carbon film with 3H-containing sample prepared for electron microscopy; 3, collodion film; 4, grid.
covered with the first layer of emulsion. Then the carbon film carrying the shadowed DNA sample was transferred from the mica plate to a grid and covered with the second layer of emulsion. Thanks to the permeability of collodion film, the first layer of emulsion was developed after the exposure as good as the second one. Autoradiographs were examined in a JEM-5G electron microscope. Figure 1 shows that in the conditions of our experiments u.v.-induced sister-strand exchanges might form labelled insertions in unlabelled DNA molecules and unlabelled insertions in labelled molecules. But in the latter case the exchanges could also be caused by some kind of repair synthesis occurring after the transfer of cells to unlabelled medium. I t would also be difficult to distinguish between short unlabelled insertions and their imitations caused b y irregular distribution of developed grains along a uniformly labelled strand. Taking this into account, we concentrated on looking for the labelled segments in unlabelled DNA molecules. Only compact groups of three or more silver grains placed along the DNA strand were considered as an indication that the corresponding DNA segment is labelled. The error in estimating the labelled segment length is about 0.3/~m, this being due to the
222
M.I.
MOSEVITSKY
size of developed grains (0-1 to 0.15 ~m) and to the range of fl-particles paths in emulsion (see Salpeter et al., 1969). The length of DNA molecules was mostly 5 to 8 ~m (10 to 16 • 106 daltons). The total length of DNA inspected in autoradiograph preparations from u.v.-irradiated cells was about 2000/~m. The same total length of DNA was inspected in the case of unirradiated (control) cells. 34 labelled inner segments were found in DNA molecules of u.v.-irradiated cells (Figs 3 and 4). The size distribution of these insertions was as follows: 23 were shorter than I ~m (at least 15 of them ranged from 0-5 to 1/zm, six ranged from 1 ~m to 2 ~m, two from 2 ~m to 3 ~m and three from 3 to 4 ~m). We did not include four molecules with label in a terminal segment because this type of labelling could be caused by presence of the replication point in the corresponding area of the chromosome at the moment of transfer from unlabelled to labelled medium, or vice versa. In the case of DNA isolated from control cells only two DNA molecules with labelled insertions (0-5 and 1-5 ~m) and three molecules with label in a terminal segment were observed. These results confirm the suggestion that u.v.-irradiation stimulates sister strand exchanges in bacteria. However, they do not exclude the less frequent spontaneous exchanges. We see that u.v.-stimulated exchanges are mostly short. This is in accord with hypothesis of Howard-Flanders et al. (Rupp et al., 1971; Howard-Fianders, 1973) that the induced sister exchanges participate in post-replication repair (see Fig. 11 in Rupp et al., 1971 and Fig. 1. in this paper). The dose of irradiation (40 ergs/mm 2) was determined to within 10%, and the estimate of the number of dimers (about 200 per E. coli genome, or 300 per 2000 ~m) was made using data of different authors, which are in good agreement with each other (Rupp & Howard-Flanders, 1968; Boyle & Setlow, 1970; Friedberg & Clayton, 1972). Therefore, the number of labelled insertions observed in DNA of u.v.-irradiated cells (34 insertions/2000 ~m) comprises not more than 10 to 15% of the expected number, if sister strand recombinations were the only mode of the post-replication repair. Several interpretations of this fact might be considered, which do not exclude one another. (1) Some (mostly short) labelled insertions might be missed because they have induced a low number (less than 3) of developed grains in emulsion. (2) Dimers arise by a co-operative mechanism in pyrimidine tracts, and several dimers form a tight group in a polynucleotide chain (Bruak, 1973). The corresponding post-replication defects can be repaired b y a single sister exchange. (3) Not all post-replication defects have been repaired in our experiments. (4) Besides sister exchanges other mechanisms using DNA synthesis for the filling of gaps opposite dimers are involved in the post-replication repair (Witkin & George, 1973; Buhl et al., 1974). In our experiments we could not observe the patches of new DNA, because after u.v.irradiation the cells were incubated in unlabelled medium. I t is difficult at present to state the relative contributions of these possibilities. The experimental results presented here, and also our previous work (Bresler et al., 1970,1972) indicate that the electron microscope autoradiography of nucleic acids permits the measurements of the lengths of 3H-labelled segments, if the exposure is sufficiently long. The method can be highly useful for those scientists who intend to observe the recombination areas in DNA and DNA segments newly synthesized during replicative or repair synthesis.
LETTERS
TO T H E E D I T O R
223
Fie. 3. Electron microscope autoradiographs of the DNA strands containing ~ segments exchanged during u.v.-induced sister recombination. Exposure for 10 months. (a) Onesided autoradiograph. Shadowing by rotation. Magnification 40,000 • (b) Sandwich autoradiograph. One-directional shadowing. Magnification 50,000 x . (c) One-sided autoradiograph. Shadowing by rotation. Magrffieation 50,000 •
224
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MOSEVITSKY
i
FIo. 4. The same as Fig. 3. (a) Sandwich autoradiograph. One-directional shadowing. Magnification 25,000 x . (b) Sandwich autoradiograph. One-directional shadowing. Magnification 40,000 •
LETTERS
TO T H E E D I T O R
225
I a m grateful to Professor S. E. Bresler for discussions and to Dr L. K. K o r o v i n for help in experiments. L a b o r a t o r y of Biopolymers I n s t i t u t e of Nuclear Physics A c a d e m y of Sciences of U.S.S.R. Leningrad, Gatchina 188350, U.S.S.R.
MARK I. MOSEVITSKY
Received 28 May 1975, and in revised form 19 October 1975 REFERENCES Boyle, J. & Setlow, R. (1970). J. Mol. Biol. 51, 131-144. Bresler, S. E., D a d i v a n j a n , L. P. & Mosevitsky, M. I. (1970). Biochim. Biophys. Acta, 224, 249-252. Bresler, S. E., Dadivanjan, L. P. & Mosevitsky, M. I. (1972). Mol. Biol. U.S.S.R. 6, 226-230. Brunk, C. F. (1973). Nature New Biol. 241, 74-76. Buhl, S. N., Setlow, R. B. & Regan, J. D. (1974). Biophys. J. 14, 791-804. Caro, L. G. (1969). J. Cell Biol. 41,918-919. Caro, L. G. & Van Tubergen, R. P. (1962). J. Cell Biol. 15, 173-188. Cole, R. S. (1973). Proc. Nat. Aead. Sci., U.S.A. 70, 1064-1069. Friedberg, E. & Clayton, D. (1972). Nature (London), 237, 99-100. Holliday, R. (1971). Nature New Biol. 232, 233-236. Howard-Flanders, P. (1973). Brit. Med. Bull. 29, 226-235. Howard-Flanders, P. & Boyee, R. P. (1966). Rad. Res. Suppl. @, 156-184. Howard-Flanders, P. & Lin, P.-F. (1973). Genetics 73, 85-90. Howard-Flanders, P., R u p p , W. D., Wilkins, B. M. & Cole, R. S. (1968). Cold Spring Harbor Symp. Quant. Biol. 33, 195-205. R u p p , W. D. & Howard-Flanders, P. (1968). J. Mol. Biol. 31, 291-304. R u p p , W. D., Wilde, C. E., Reno, D. L. & Howard-Flanders, P. (1971). J. Mol. Biol. 61, 25-44. Salpeter, M. M., Baehmarm, L. & Salpeter, E. E. (1969). J. Cell Biol. 41, 1-20. Smith, K. C. & Meun, D. H. C. (1970). J. Mol. Biol. 51,459-472. Vrensen, G. F. J. M. (1970). J. Histochem. Cytochem. 18, 278-290. Witkin, E. M. & George, D. L. (1973). Genetics 73, 91-108.
