- Email: [email protected]
The order of DNA replication in mammalian cells
480
Preliminary notes
a result of these preliminary experiments we will, in future experiments, be able to begin the isolation and characterization of the individual proteins that migrate between the nucleus and cytoplasm in conjunction with specific cell cycle events such as mitisos.
24. LeStourgeon, W M & Wray, W, Acidic proteins of the nucleus (ed I L Cameron & J R Jeter. Jr) p. 297. Academic Press, New York (1974). 25. Kolodny, G M, J mol bio178 (1973) 197. 26. Yamaizumi,, M, Uchida, T, Okada. Y, Furusawa, M & Mitsm, H, Nature 273 (1978) 782. 27. Goldstein. L & Ko, C, J cell bio188 (1981) 516. 28. Laemmli, U K, Nature 227 (1970) 680. 29. Jeter, Jr, J R, Knieriem, K M & Cameron, I L, Cytobios 15 (1976) 183.
We gratefully acknowledge the excellent technical assistance of Mrs Lia Pedroza. This work was supported in part by NIH Grants CA-5002 and CA-07175 and by CAGNO Grant no. 531360.
Received December 8, 1980 Revised version received November 10. 1981 Accepted December 28, 1981
References 1. Johnston, R T & Rao, P N, Biol rev (1971) 97. 2. Rusch, H P, Sachsenmaier, W, Behrens, K & Gruyter, V, J cell biol31 (1966) 204. 3. Gurdon, J B, Partington, G 4 &De Robertis, E M, J embryo1 exp morph01 36 (1976) 541. 4. Legname, C & Goldstein, L? Exp cell res 75 (1972) 111. 5. Jolinek, W & Goldstein, L, J cell physiol81 (1973) 181. 6. Bolund, L, Ringertz, N R & Harris, H, J cell sci 4 (1%9) 71. 7. Ringertz, N R, Carlsson, S-A, Ege, T & Bolund, L. Proc natl acad sci US 68 (1971) 3228. 8. Appels, R, Bolund, L & Ringertz, N R; J mol biol 87 (1974) 339. 9. Appels, R, Bolund, L. Goto, S & Ringertz, N R, Exp cell res 85 (1974) 182. 10. Gilmour. R S, Acidic proteins of the nucleus (ed I L Cameron & J R Jeter, Jr) p. 297. Academic Press, New York (1974). 11. Rusch, H P, Sachsenmaier, W, Behrens. K & Gruyter, V, J cell biol31 (1966) 204. 12. Sachsenmaier, W & Rusch, H P, Exp cell res 36 (1964) 124. 13. Cummins; J E, Brewer, E N & Rusch, H P1J cell bio127 (1965) 337. 14. Hosada, E & Tanaka, N, Cytologia 38 (1973) 165. 15. Daniel, J W & Baldwin. H W, Methods in cell physiology (ed D M Prescott) vol. 1, p. 9. Academic Press. New York (1964). 16. Guttes, E & G&es, S, Methods in cell physiology (ed D M Prescott) vol. 1, p. 43. Academic Press, New York (1964). 17. Brewer, E N & Rusch, H P, Exp cell res 49 (1968) 79. 18. McCormick: J J, J cell bio162 (1974) 227. 19. Mohberg, J & Rusch, H P, Exp cell res 66 (1971) 305. 20. Teng, C SI Teng, C T & Allfrey, V G, J biol them 246 (1971) 3597. 21. Jeter. Jr. J R. Pavlat. W A & Cameron. I L. Exo. cell rks 83 (1975) 79. 22. Lowrv. 0 H. Rosebrouah. N J. Farr, A L & Randall, R J. J biol them 193 (1951) 265. 23. Sadgopal, A & Bonner, J, Biochim biophys acta 207 (1970) 206. E.vp Cell Res 138 (1982)
Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved 001J-48?::8u040-180~6502.00:0
The order of DNA replication in mammalian cells. A modified density labelling procedure for isolating sequences replicated at specific times LEO
J. GRADY
and MYRNA
L. SANDERS,
Division of Laboratories and Research, New York State Department of Health, Albany, NY 12201, USA
These experiments were directed towards establishing a procedure for isolation of the DNA sequences replicated at different times in S phase for further study. Primary considerations were thus for a method that gave fairly large quantities of cells as well synchronized in S phase as possible. The use of Chinese hamster ovary (CHO) cells synchronized by isoleucine starvation followed by exposure to hydroxyurea [l] seemed to be most promising and was explored in detail. During the course of this work, a labelling scheme was developed that involved exposure to BrdUrd during the same time window in two sequential S phases. Only those regions of the genome replicated in the same interval in each S period contained BrdUrd in both strands and these were readily isolated in CsCl gradients. As applied in the present experiments, this labelling procedure showed no untoward effects on cell-cycle traverse and gave DNA distributions in density gradients that closely approximated those that would be expected for the conditions employed.
Summary.
Methods of inducing cell synchrony always run the risk that experimental results will be biased by the manipulations required to achieve synchrony. Such methods, however, are the only ones that yield large quantities of cells for biochemical analysis Printsd
m Sweden
Prelimirirary
liOieS
481
XXXI
xxxxxxxxxxr
-l-----y
and of those that have been described, the fewest drawbacks seem to be associated with the procedure developed for CHO cells by Tobey and his co-workers [ 11. The basic procedure consists of subjecting a suspension culture of log-phase cells to a period of isoleucine deprivation of sufficient length for all of the cells to become arrested in the Gl stage of the cell cycle. Upon transfer to fresh, complete medium the whole population of cells begins to traverse the cell cycle with the same kinetics as mitotically selected cells. It was later found that if hydroxyurea (HU) was added at the time of transfer to fresh medium, the cells would proceed through Gl and become blocked at the beginning of S phase. -Although it was at first thought that the block occurred at the Gl-S interface, Walters et al. [2] later showed that it was actually in early S and that the cells probably synthesized about 4% of their DNA. In any event, this is still a much better situation than has been reported for other inhibitors of DNA synthesis [4, 51. Of par-
xxxxxxxxxxl
Fig. 1. CHO cells were synchronized near the beginning of S phase by the isoleucine starvation-hydroxyurea procedure. After release from the HU block, DNA was labeiled with (‘“CjTdR(---)orBrdUrd(xxx) as shown above. Protccois [abelled A-F correspond to . .. _.
titular importance is the fact that within 1 h of release from the HU block, 90% of the cells were synthesizing DINA as measured by autoradiography [lj. This latter observation suggests that the celIs are well synchronized in the S phase. Hamlin & Pardee [6] have also shown :hat this synchrony procedure is applicable to monolayer cultures of CHO cells. As the major features of this system appeared so favorable, the main thrust of the present experiments was to determine its suitability for studying the temporal order of DNA synthesis with the ultimate goal of being abie to isolate newly replicated sequences for further characterization. Materials and Methods CHO cells were kindly provided by Dr R. A. Tobey of the Los Alamos Scientific Laboratories. Cells were grown and synchronized as described by Tobey il] using a 36 h period of isoleucine deprivation. In oblr laboratory. CHO cells exhibited a doubling time of about 18 h and the celt cycle parameters determined by the method of Volpe & Eremenko r7} compared favorably with those reported by Tobey [I]. When cells were labelled with 0.03 ,&l/ml [Y]TdR (New England Nuclear, sp. act.: 56.5 mCi!mM:
482
Preliminary notes
0
0
4
6
12 16 20 24
FRACTION
2S 32 36
40 44
NUMBER
46
52
56
BOTTOM
Fig. 2. Density distribution in CsCl gradients of DNA from synchronized cultures A, B, which were labelled with [W]TdR either early (fop) or late (borrom) in the first S phase and then with BrdUrd during corresponding times in the second S phase. Denstty increases from left to right. O---O, AzsO; 3---O, 14C cpm.
Fig. 3. Density distribution in CsCl gradients of DNA from synchronized CHO cells labelled as follows: Culture C (top), [‘“C]TdR early in first S and BrdUrd late in second S; culture D (bottom). [“C]TdR late in first S and BrdUrd early in second S. Density increases from left to right. O---O, AT6”; C---O, ‘T cpm.
effective sp. act., 8.6 mCi/mM taking into account the 2.9 PM concentration of thymidine in FIO medium) either early (O-2 h) or late (4.5-6.0 h) in S phase, the rate at which the cells entered mitosis and the fraction of the population that divided were indistinguishable from unlabelled cells. The minimal concentration of BrdUrd (Sigma) that allowed good separation in CsCl between newly replicated and unreplicated DNA without effecting the time when cells entered mitosis or the proportion of the population that divided was 6 pg/ml. The density difference in CsCl indicated that 27% of the TdR had been replaced by BrdUrd [8]. Since only one strand of newly replicated DNA contains BrdUrd and is responsible for the shift in density, in this strand about 54% of the TdR is substituted by BrdUrd. To isolate DNA, cells were suspended in buffer (0.1 M NaCl, 0.05 EDTA, 0.05 M Tris, pH 8.4) lysed with 0.4% SDS, proteinase K (Beckman) added to 200 pg/ ml, incubated at 37°C overnight, then extracted at room temperature with an equal volume of buffersaturated phenol. DNA was precipitated with cold ethanol, wrapped on a glass rod and resuspended in 0.01 M NaCl, 0.0015 M sodium citrate, pH 7.0 and incubated for 1 h at 37°C with 50 pg/ml heat-treated RNase. Then proteinase K was added to 50 pg/ml for 30 min at 37”C, followed by phenol extraction, precipitation and resuspension as above. Based on cell counts. DNA recovery was about 80% using the DNA/ cell data of Swartzendruber [9]. Sufficient CsCl (Henly) was added to the DNA solutions to give ~=1.715 g/cc (final volume of each gradient was 10 ml). Centrifugation was in a Beckman
60 Ti rotor at 36000 rpm for 73 h at 25°C. Gradients were collected from the top with a Buchler DensiFlow II apparatus. Fractions were diluted to 0.5 ml and the AZGO determined. Each fraction was then added to 10 ml of Aquasol (New England Nuclear) and its radioactivity measured.
Esp Cell Res 138 11982)
Results and Discussion In the synchrony experiments discussed, after release from the initial HU block the cells were resuspended in fresh medium and sub-divided into as many cultures as necessary for the particular experiment. The various subcultures were designated A-F and each was labelled with [14C]TdR and BrdUrd (fig. 1). Fig. 2 shows that DNA labelled either early or late in one S phase is preferentially replicated at the same time in the next S phase. However, it is also apparent that about 50% of the DNA fails to replicate at the same time in two successive cell cycles. These data are very similar to *those reported earlier by other laboratories .[lO: Ill. Cultures C and D
Preliminary notes
6 4 2 "0 x
a I
E 10 2 b”,
8u
06
6'
0.4
4a
v 02
2
0
0 0 4 TOP
8
12 16 20 24 26
FRACTION
32 36 40 44
46
52 56 BOTTOM
NUMBER
iQ. 4. Density profiles in CsCl of DNA from svnchionized cells labelled as follows: Culture E (t&j. PClTdR earlv in first S and BrdUrd throughout second S; c&-e F (bottom), [“C]TdR late icfirst S and BrdUrd throughout second S. Density increases from left to right. e---e. Apse; O---O; “C cpm.
(fig. 3) revealed that only about 10% of the DNA that replicated early (or late) in one S phase was synthesized at the opposite end of the next S. Furthermore, cultures E and F (fig. 4) show that radiolabelled and unlabelled DNA do not differ in the extent to which they are duplicated and that 85-90% of the DNA replicates during the second S in the presence of BrdUrd. Taken together, the data in figs 3 and 4 strongly suggest that no significant fraction of DNA is sufficiently devoid of temporal control that it replicates randomly and that replication of those sequences radiolabelled in the first S phase is not impaired during the succeeding S phase. This leads to the conclusion that asynchrony in the culture is responsible for the apparent failure of 50% of the DNA to replicate at the same time in successive S phases (fig. 2). Isolation of DNA replicated at a particular time in S phase To try to reduce the variability arising from asynchrony, an experiment was carried out
453
in which BrdUrd was added during the same timespan in both the first and second S phases. The DNA replicated in the presence of BrdUrd during the first S w-iU EORtain only one strand in which BrdUrd has replaced thymidine. During the second S phase, those DNA sequences which are duplicated at the same time as in the first S will now incorporate BrdUrd into the opposite strand and will contain the analogue in both strands. (Strictly speaking, only 50% of the DNA replicating at the same time in both S periods will contain BrdUrd in both strands. The unsubstituted partner from the first S will be a hybrid with oni-y one BrdUrd containing strand.) The DNA containing BrdUrd in both strands is easily separated in a CsCl gradient from molecules which contain BrdUrd in only one strand or which lack the analogue altogether. To follow the amount of DNA duplicated during the first S phase part of the cultures labelfed both early and late were harvested at 6.5 h and the DNA extracted and examined in a CsCl gradient (data not shown). The proportion of the DNA replicated in the presence of BrdUrd was 34 and 33%, respectively. From the diagram of this experiment (fig. 5) it can be seen that ideally only 25% of the DNA replicated early in both the first and second S phases could be recovered in the heavy DNA fraction (BrdUrd in both strands). Since the earliest experiments (fig. 2) showed that only 50% of the DNA synthesized in the first S was duplicated during the same interval of the second S, the actual expected recovery of early replicating DNA would be 12.5%. A similar calculation for late replicating DNA gives an idea1 recovery of 16.6% and an expected recovery of 8.3%. The actual results of this experiment are presented in fig. 6. The small peak at the extreme right-hand side of fig. 6
484
Preliminary
notes of
Early
replicating DR.4
Maximm 251: of the DEL4 Et time of :Isrvest 'r BrdlJ :r seccnd 8 contains both strar.as
xxx-1 I
I
I
DPW. Aistritiutioo About or.+third 0" ErdU in cne strsnd
the
4
a:'ter first 5. gencme contains
-xxx
>
mxxx late
replicating DNA
IMaximun of 16.62 of the CNA at time of :larvest in secon3 S contains 3rdU in bath strands
-xxx I
I
I
I
l-lx
1 DNA disti%bution About one-shird cf BrdU in one strand
V
J
efter firs* S. she genome contaiLs
xx
=z DL4 distributiou at time z,f harvest in seccnd S
Fig. 5. Expected distribution of BrdUrd in either early (fop) or late (boftom) replicating DNA when synchronized cultures are exposed to the analogue during the same time window in two successive S
phases. Projections are for the time windows referred to in the text. The situation at the end of the first S is based on actual observations.
represents the heavy-heavy DNA. Since this material represents such a small proportion of the total DNA, fractions 44-52 of each gradient were pooled and rebanded, to assure that the heavy DNA contained therein banded true and to estimate the degree of contamination with intermediate density DNA (data not shown). Based on refractive index, in both the first and second CsCl gradients the heavy DNA banded at 1.76 g/cc for early replicating DNA and 1.765 g/cc for late replicating DNA. Heavy DNA comprised 63 and 78% respectively of the pooled fractions. Using this information in conjunction with the data in fig. 6, it was calculated that about 9% of the DNA synthesized early in S was recovered in the heavy fraction (12.5 % best expected) and for late DNA the pro-
portion was 12% recovered (8.3 % best expected). Allowing for the error inherent in these kinds of calculations, it was concluded that pulsing with BrdUrd for the same interval in two successive S phases is a valid method for isolating the DNA sequences replicated during a particular portion of the S phase for further characterization. At the same time, it must be recognized that the low yield of DNA puts limits on how narrow a window can be used to examine DNA synthesis during S phase. Under the conditions described, a 7.50 ml culture yields about 120 pg of either early or late replicated DNA. Returning to fig. 6, the replication scheme illustrated in fig. 5 predicts that for early replicating DNA, the distribution should be roughly 38% having normal density, 50%
E.rp Cell Res 138 11982)
Preliininar~l notes 0.4
8. Luk, D C &r Bick, M D. Anal biochem 77 (1977) 9. Swartzendruber D E, 3 cell physioi 90 (1977) 445. 10. May, M S & Belle, L J: Exp cell res 83 (1974) 79. 11. Mueller; G L & Kajiwara, K; Biochim biophys
i :I: i
485
I,14c19,
,,.
Revised version received 1981 December 2, 1981 Re=i;:;e=i;;;;;:8*
;;
111~IIIIIIIIIIIIIIIIIIIII 0
4
8
Top
I2
16 20
FRACTION
24
28 32
36
40
44
48 52 BOTTOM
NUMBER
Fig. 6. CsCl density gradient profile of DNA from synchronized cells labelled with BrdUrd either (A) early or (B) late during two sequential S phases. Density increases from left to right. O---O, AZ6”.
having hybrid density and 12% having BrdUrd in both strands. Calculated from fig. 6A, the actual distribution was 41, 50 and 9%: respectively. Similarly, for late replicating DNA the prediction would be 58 % with normal density, 33 % in hybrids and 8% with BrdUrd in both strands. The observed distribution (fig. 6B) was 60, 29 and 12%. In both cases the close agreement between the expected and observed DNA distributions is very important as it provides evidence that the process of DNA synthesis was not unduly perturbed by introduction of the density label.
Copyright @ 1982 bl- Academic Press. inc. AII rights of reproduction in any form reserved M)l4~827;8?!040485-0~$0~.~~0
Correlation of increased nuclease activity with enhanced virus reactivation Y. NISHIY4MA,l K. MAENO’ and S. YOSHIDA,’ ‘Department of Microbiology, Germfree Li$ Research Institute, Xagova G’niverrir)’ Schooi Gf Medicine, Nagoya, 464 and ?Departmen? qf Biochemistry, Institute for Developmental Research, Al&i Prefecture Colony, Kasugai;480-03. Japat? Summary. An increase in nuclease activity, which degraded both unit-radiated and ultraviolet (UV)-irradiated DN4, was observed in the extract of monkey Vero cells after irradiation with an appropriate amount of UVI In contrast, no increase was observed with mouse L cells. Neither DNA polymerases nor uraciDNA glycosylase was enhanced but rather suppressed by UV irradiation in both cell lines. Cytological studies showed that, in Vero cells, the reactivation of UV-irradiated herpes simplex virus was markedly enhanced by irradiating cells with UV before infection. However, no enhancement was observed with L cells. These results suggest that an increase in nuclease activity may be one of underlying mechanisms for the enhanced reactivation of DNA viruses.
Inducible DNA repair systems in bacteria Tobey, R A, Methods in cell biology (ed D M have been well characterized. Treatment of Prescott) vol. 6, p. 67. Academic Press, New York bacterial cells with UV, mitomycin C or (1973). acid induces the error-prone Walters, R Al Tobey, R A & Hildebrand, C E, nalidixic B&hem biophys res commun 64 (1976) 212. “SOS” repair pathway which depends on Studzinski, G P & Lambert. W C, J cell physiol73 the function of the recA and 1exA genes (1969) 109. Bostock, C J, Prescott, D M & Hatch, F T, Exp [l], but the treatment with a low concencell res 68 (1971) 163. of N-methyl-W-nitrosoguanidine Amaldi, F, Leoni, L & Mariotti. D, Exp cell res 74 tration (1972) 367. (MNNG) induces another type of repair Hamlin, J L & Pardee, AB, Exp cell res 100(1976) which is error-free [2}. In mammalian cells: 265. Volpe, P & Eremenko, T, Methods in cell biology the existence of inducible repair systems (ed D M Prescott) vol. 6, p. 113. Academic Press, has been suggested to explain the enhanced New York (1973).
References 1. 2. 3. 4. 5. 6. 7.
Printed
in Sweden
E.rp Ceil Res i38 (f?Q)
Preliminary notes
a result of these preliminary experiments we will, in future experiments, be able to begin the isolation and characterization of the individual proteins that migrate between the nucleus and cytoplasm in conjunction with specific cell cycle events such as mitisos.
24. LeStourgeon, W M & Wray, W, Acidic proteins of the nucleus (ed I L Cameron & J R Jeter. Jr) p. 297. Academic Press, New York (1974). 25. Kolodny, G M, J mol bio178 (1973) 197. 26. Yamaizumi,, M, Uchida, T, Okada. Y, Furusawa, M & Mitsm, H, Nature 273 (1978) 782. 27. Goldstein. L & Ko, C, J cell bio188 (1981) 516. 28. Laemmli, U K, Nature 227 (1970) 680. 29. Jeter, Jr, J R, Knieriem, K M & Cameron, I L, Cytobios 15 (1976) 183.
We gratefully acknowledge the excellent technical assistance of Mrs Lia Pedroza. This work was supported in part by NIH Grants CA-5002 and CA-07175 and by CAGNO Grant no. 531360.
Received December 8, 1980 Revised version received November 10. 1981 Accepted December 28, 1981
References 1. Johnston, R T & Rao, P N, Biol rev (1971) 97. 2. Rusch, H P, Sachsenmaier, W, Behrens, K & Gruyter, V, J cell biol31 (1966) 204. 3. Gurdon, J B, Partington, G 4 &De Robertis, E M, J embryo1 exp morph01 36 (1976) 541. 4. Legname, C & Goldstein, L? Exp cell res 75 (1972) 111. 5. Jolinek, W & Goldstein, L, J cell physiol81 (1973) 181. 6. Bolund, L, Ringertz, N R & Harris, H, J cell sci 4 (1%9) 71. 7. Ringertz, N R, Carlsson, S-A, Ege, T & Bolund, L. Proc natl acad sci US 68 (1971) 3228. 8. Appels, R, Bolund, L & Ringertz, N R; J mol biol 87 (1974) 339. 9. Appels, R, Bolund, L. Goto, S & Ringertz, N R, Exp cell res 85 (1974) 182. 10. Gilmour. R S, Acidic proteins of the nucleus (ed I L Cameron & J R Jeter, Jr) p. 297. Academic Press, New York (1974). 11. Rusch, H P, Sachsenmaier, W, Behrens. K & Gruyter, V, J cell biol31 (1966) 204. 12. Sachsenmaier, W & Rusch, H P, Exp cell res 36 (1964) 124. 13. Cummins; J E, Brewer, E N & Rusch, H P1J cell bio127 (1965) 337. 14. Hosada, E & Tanaka, N, Cytologia 38 (1973) 165. 15. Daniel, J W & Baldwin. H W, Methods in cell physiology (ed D M Prescott) vol. 1, p. 9. Academic Press. New York (1964). 16. Guttes, E & G&es, S, Methods in cell physiology (ed D M Prescott) vol. 1, p. 43. Academic Press, New York (1964). 17. Brewer, E N & Rusch, H P, Exp cell res 49 (1968) 79. 18. McCormick: J J, J cell bio162 (1974) 227. 19. Mohberg, J & Rusch, H P, Exp cell res 66 (1971) 305. 20. Teng, C SI Teng, C T & Allfrey, V G, J biol them 246 (1971) 3597. 21. Jeter. Jr. J R. Pavlat. W A & Cameron. I L. Exo. cell rks 83 (1975) 79. 22. Lowrv. 0 H. Rosebrouah. N J. Farr, A L & Randall, R J. J biol them 193 (1951) 265. 23. Sadgopal, A & Bonner, J, Biochim biophys acta 207 (1970) 206. E.vp Cell Res 138 (1982)
Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved 001J-48?::8u040-180~6502.00:0
The order of DNA replication in mammalian cells. A modified density labelling procedure for isolating sequences replicated at specific times LEO
J. GRADY
and MYRNA
L. SANDERS,
Division of Laboratories and Research, New York State Department of Health, Albany, NY 12201, USA
These experiments were directed towards establishing a procedure for isolation of the DNA sequences replicated at different times in S phase for further study. Primary considerations were thus for a method that gave fairly large quantities of cells as well synchronized in S phase as possible. The use of Chinese hamster ovary (CHO) cells synchronized by isoleucine starvation followed by exposure to hydroxyurea [l] seemed to be most promising and was explored in detail. During the course of this work, a labelling scheme was developed that involved exposure to BrdUrd during the same time window in two sequential S phases. Only those regions of the genome replicated in the same interval in each S period contained BrdUrd in both strands and these were readily isolated in CsCl gradients. As applied in the present experiments, this labelling procedure showed no untoward effects on cell-cycle traverse and gave DNA distributions in density gradients that closely approximated those that would be expected for the conditions employed.
Summary.
Methods of inducing cell synchrony always run the risk that experimental results will be biased by the manipulations required to achieve synchrony. Such methods, however, are the only ones that yield large quantities of cells for biochemical analysis Printsd
m Sweden
Prelimirirary
liOieS
481
XXXI
xxxxxxxxxxr
-l-----y
and of those that have been described, the fewest drawbacks seem to be associated with the procedure developed for CHO cells by Tobey and his co-workers [ 11. The basic procedure consists of subjecting a suspension culture of log-phase cells to a period of isoleucine deprivation of sufficient length for all of the cells to become arrested in the Gl stage of the cell cycle. Upon transfer to fresh, complete medium the whole population of cells begins to traverse the cell cycle with the same kinetics as mitotically selected cells. It was later found that if hydroxyurea (HU) was added at the time of transfer to fresh medium, the cells would proceed through Gl and become blocked at the beginning of S phase. -Although it was at first thought that the block occurred at the Gl-S interface, Walters et al. [2] later showed that it was actually in early S and that the cells probably synthesized about 4% of their DNA. In any event, this is still a much better situation than has been reported for other inhibitors of DNA synthesis [4, 51. Of par-
xxxxxxxxxxl
Fig. 1. CHO cells were synchronized near the beginning of S phase by the isoleucine starvation-hydroxyurea procedure. After release from the HU block, DNA was labeiled with (‘“CjTdR(---)orBrdUrd(xxx) as shown above. Protccois [abelled A-F correspond to . .. _.
titular importance is the fact that within 1 h of release from the HU block, 90% of the cells were synthesizing DINA as measured by autoradiography [lj. This latter observation suggests that the celIs are well synchronized in the S phase. Hamlin & Pardee [6] have also shown :hat this synchrony procedure is applicable to monolayer cultures of CHO cells. As the major features of this system appeared so favorable, the main thrust of the present experiments was to determine its suitability for studying the temporal order of DNA synthesis with the ultimate goal of being abie to isolate newly replicated sequences for further characterization. Materials and Methods CHO cells were kindly provided by Dr R. A. Tobey of the Los Alamos Scientific Laboratories. Cells were grown and synchronized as described by Tobey il] using a 36 h period of isoleucine deprivation. In oblr laboratory. CHO cells exhibited a doubling time of about 18 h and the celt cycle parameters determined by the method of Volpe & Eremenko r7} compared favorably with those reported by Tobey [I]. When cells were labelled with 0.03 ,&l/ml [Y]TdR (New England Nuclear, sp. act.: 56.5 mCi!mM:
482
Preliminary notes
0
0
4
6
12 16 20 24
FRACTION
2S 32 36
40 44
NUMBER
46
52
56
BOTTOM
Fig. 2. Density distribution in CsCl gradients of DNA from synchronized cultures A, B, which were labelled with [W]TdR either early (fop) or late (borrom) in the first S phase and then with BrdUrd during corresponding times in the second S phase. Denstty increases from left to right. O---O, AzsO; 3---O, 14C cpm.
Fig. 3. Density distribution in CsCl gradients of DNA from synchronized CHO cells labelled as follows: Culture C (top), [‘“C]TdR early in first S and BrdUrd late in second S; culture D (bottom). [“C]TdR late in first S and BrdUrd early in second S. Density increases from left to right. O---O, AT6”; C---O, ‘T cpm.
effective sp. act., 8.6 mCi/mM taking into account the 2.9 PM concentration of thymidine in FIO medium) either early (O-2 h) or late (4.5-6.0 h) in S phase, the rate at which the cells entered mitosis and the fraction of the population that divided were indistinguishable from unlabelled cells. The minimal concentration of BrdUrd (Sigma) that allowed good separation in CsCl between newly replicated and unreplicated DNA without effecting the time when cells entered mitosis or the proportion of the population that divided was 6 pg/ml. The density difference in CsCl indicated that 27% of the TdR had been replaced by BrdUrd [8]. Since only one strand of newly replicated DNA contains BrdUrd and is responsible for the shift in density, in this strand about 54% of the TdR is substituted by BrdUrd. To isolate DNA, cells were suspended in buffer (0.1 M NaCl, 0.05 EDTA, 0.05 M Tris, pH 8.4) lysed with 0.4% SDS, proteinase K (Beckman) added to 200 pg/ ml, incubated at 37°C overnight, then extracted at room temperature with an equal volume of buffersaturated phenol. DNA was precipitated with cold ethanol, wrapped on a glass rod and resuspended in 0.01 M NaCl, 0.0015 M sodium citrate, pH 7.0 and incubated for 1 h at 37°C with 50 pg/ml heat-treated RNase. Then proteinase K was added to 50 pg/ml for 30 min at 37”C, followed by phenol extraction, precipitation and resuspension as above. Based on cell counts. DNA recovery was about 80% using the DNA/ cell data of Swartzendruber [9]. Sufficient CsCl (Henly) was added to the DNA solutions to give ~=1.715 g/cc (final volume of each gradient was 10 ml). Centrifugation was in a Beckman
60 Ti rotor at 36000 rpm for 73 h at 25°C. Gradients were collected from the top with a Buchler DensiFlow II apparatus. Fractions were diluted to 0.5 ml and the AZGO determined. Each fraction was then added to 10 ml of Aquasol (New England Nuclear) and its radioactivity measured.
Esp Cell Res 138 11982)
Results and Discussion In the synchrony experiments discussed, after release from the initial HU block the cells were resuspended in fresh medium and sub-divided into as many cultures as necessary for the particular experiment. The various subcultures were designated A-F and each was labelled with [14C]TdR and BrdUrd (fig. 1). Fig. 2 shows that DNA labelled either early or late in one S phase is preferentially replicated at the same time in the next S phase. However, it is also apparent that about 50% of the DNA fails to replicate at the same time in two successive cell cycles. These data are very similar to *those reported earlier by other laboratories .[lO: Ill. Cultures C and D
Preliminary notes
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iQ. 4. Density profiles in CsCl of DNA from svnchionized cells labelled as follows: Culture E (t&j. PClTdR earlv in first S and BrdUrd throughout second S; c&-e F (bottom), [“C]TdR late icfirst S and BrdUrd throughout second S. Density increases from left to right. e---e. Apse; O---O; “C cpm.
(fig. 3) revealed that only about 10% of the DNA that replicated early (or late) in one S phase was synthesized at the opposite end of the next S. Furthermore, cultures E and F (fig. 4) show that radiolabelled and unlabelled DNA do not differ in the extent to which they are duplicated and that 85-90% of the DNA replicates during the second S in the presence of BrdUrd. Taken together, the data in figs 3 and 4 strongly suggest that no significant fraction of DNA is sufficiently devoid of temporal control that it replicates randomly and that replication of those sequences radiolabelled in the first S phase is not impaired during the succeeding S phase. This leads to the conclusion that asynchrony in the culture is responsible for the apparent failure of 50% of the DNA to replicate at the same time in successive S phases (fig. 2). Isolation of DNA replicated at a particular time in S phase To try to reduce the variability arising from asynchrony, an experiment was carried out
453
in which BrdUrd was added during the same timespan in both the first and second S phases. The DNA replicated in the presence of BrdUrd during the first S w-iU EORtain only one strand in which BrdUrd has replaced thymidine. During the second S phase, those DNA sequences which are duplicated at the same time as in the first S will now incorporate BrdUrd into the opposite strand and will contain the analogue in both strands. (Strictly speaking, only 50% of the DNA replicating at the same time in both S periods will contain BrdUrd in both strands. The unsubstituted partner from the first S will be a hybrid with oni-y one BrdUrd containing strand.) The DNA containing BrdUrd in both strands is easily separated in a CsCl gradient from molecules which contain BrdUrd in only one strand or which lack the analogue altogether. To follow the amount of DNA duplicated during the first S phase part of the cultures labelfed both early and late were harvested at 6.5 h and the DNA extracted and examined in a CsCl gradient (data not shown). The proportion of the DNA replicated in the presence of BrdUrd was 34 and 33%, respectively. From the diagram of this experiment (fig. 5) it can be seen that ideally only 25% of the DNA replicated early in both the first and second S phases could be recovered in the heavy DNA fraction (BrdUrd in both strands). Since the earliest experiments (fig. 2) showed that only 50% of the DNA synthesized in the first S was duplicated during the same interval of the second S, the actual expected recovery of early replicating DNA would be 12.5%. A similar calculation for late replicating DNA gives an idea1 recovery of 16.6% and an expected recovery of 8.3%. The actual results of this experiment are presented in fig. 6. The small peak at the extreme right-hand side of fig. 6
484
Preliminary
notes of
Early
replicating DR.4
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IMaximun of 16.62 of the CNA at time of :larvest in secon3 S contains 3rdU in bath strands
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Fig. 5. Expected distribution of BrdUrd in either early (fop) or late (boftom) replicating DNA when synchronized cultures are exposed to the analogue during the same time window in two successive S
phases. Projections are for the time windows referred to in the text. The situation at the end of the first S is based on actual observations.
represents the heavy-heavy DNA. Since this material represents such a small proportion of the total DNA, fractions 44-52 of each gradient were pooled and rebanded, to assure that the heavy DNA contained therein banded true and to estimate the degree of contamination with intermediate density DNA (data not shown). Based on refractive index, in both the first and second CsCl gradients the heavy DNA banded at 1.76 g/cc for early replicating DNA and 1.765 g/cc for late replicating DNA. Heavy DNA comprised 63 and 78% respectively of the pooled fractions. Using this information in conjunction with the data in fig. 6, it was calculated that about 9% of the DNA synthesized early in S was recovered in the heavy fraction (12.5 % best expected) and for late DNA the pro-
portion was 12% recovered (8.3 % best expected). Allowing for the error inherent in these kinds of calculations, it was concluded that pulsing with BrdUrd for the same interval in two successive S phases is a valid method for isolating the DNA sequences replicated during a particular portion of the S phase for further characterization. At the same time, it must be recognized that the low yield of DNA puts limits on how narrow a window can be used to examine DNA synthesis during S phase. Under the conditions described, a 7.50 ml culture yields about 120 pg of either early or late replicated DNA. Returning to fig. 6, the replication scheme illustrated in fig. 5 predicts that for early replicating DNA, the distribution should be roughly 38% having normal density, 50%
E.rp Cell Res 138 11982)
Preliininar~l notes 0.4
8. Luk, D C &r Bick, M D. Anal biochem 77 (1977) 9. Swartzendruber D E, 3 cell physioi 90 (1977) 445. 10. May, M S & Belle, L J: Exp cell res 83 (1974) 79. 11. Mueller; G L & Kajiwara, K; Biochim biophys
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485
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Fig. 6. CsCl density gradient profile of DNA from synchronized cells labelled with BrdUrd either (A) early or (B) late during two sequential S phases. Density increases from left to right. O---O, AZ6”.
having hybrid density and 12% having BrdUrd in both strands. Calculated from fig. 6A, the actual distribution was 41, 50 and 9%: respectively. Similarly, for late replicating DNA the prediction would be 58 % with normal density, 33 % in hybrids and 8% with BrdUrd in both strands. The observed distribution (fig. 6B) was 60, 29 and 12%. In both cases the close agreement between the expected and observed DNA distributions is very important as it provides evidence that the process of DNA synthesis was not unduly perturbed by introduction of the density label.
Copyright @ 1982 bl- Academic Press. inc. AII rights of reproduction in any form reserved M)l4~827;8?!040485-0~$0~.~~0
Correlation of increased nuclease activity with enhanced virus reactivation Y. NISHIY4MA,l K. MAENO’ and S. YOSHIDA,’ ‘Department of Microbiology, Germfree Li$ Research Institute, Xagova G’niverrir)’ Schooi Gf Medicine, Nagoya, 464 and ?Departmen? qf Biochemistry, Institute for Developmental Research, Al&i Prefecture Colony, Kasugai;480-03. Japat? Summary. An increase in nuclease activity, which degraded both unit-radiated and ultraviolet (UV)-irradiated DN4, was observed in the extract of monkey Vero cells after irradiation with an appropriate amount of UVI In contrast, no increase was observed with mouse L cells. Neither DNA polymerases nor uraciDNA glycosylase was enhanced but rather suppressed by UV irradiation in both cell lines. Cytological studies showed that, in Vero cells, the reactivation of UV-irradiated herpes simplex virus was markedly enhanced by irradiating cells with UV before infection. However, no enhancement was observed with L cells. These results suggest that an increase in nuclease activity may be one of underlying mechanisms for the enhanced reactivation of DNA viruses.
Inducible DNA repair systems in bacteria Tobey, R A, Methods in cell biology (ed D M have been well characterized. Treatment of Prescott) vol. 6, p. 67. Academic Press, New York bacterial cells with UV, mitomycin C or (1973). acid induces the error-prone Walters, R Al Tobey, R A & Hildebrand, C E, nalidixic B&hem biophys res commun 64 (1976) 212. “SOS” repair pathway which depends on Studzinski, G P & Lambert. W C, J cell physiol73 the function of the recA and 1exA genes (1969) 109. Bostock, C J, Prescott, D M & Hatch, F T, Exp [l], but the treatment with a low concencell res 68 (1971) 163. of N-methyl-W-nitrosoguanidine Amaldi, F, Leoni, L & Mariotti. D, Exp cell res 74 tration (1972) 367. (MNNG) induces another type of repair Hamlin, J L & Pardee, AB, Exp cell res 100(1976) which is error-free [2}. In mammalian cells: 265. Volpe, P & Eremenko, T, Methods in cell biology the existence of inducible repair systems (ed D M Prescott) vol. 6, p. 113. Academic Press, has been suggested to explain the enhanced New York (1973).
References 1. 2. 3. 4. 5. 6. 7.
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E.rp Ceil Res i38 (f?Q)