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Changes in the rate of DNA replication fork movement during S phase in mammalian cells
J. Ilrlol. Biol. (1975) 94, 1’73-181
Changes in the Rate of DNA Replication Fork Movement During S Phase in Mammalian Cells A. HTJBERMAN
DAVID HOUSMANANDJOEL
Department of Biokgy Mamachueetts Inetitute of Technology Cam.bridge, Maes. 02139, U.X.A. (Received 25 July 1974) DNA fiber autoradiography was used to measure the rate of replication fork movement and the size of replication units as a function of time during the S phase of synchronized Chinese hamster ovary cells. The rate of fork movement increased by about threefold from early S to later S phase, with the most dramatic change occurring in the f%st hour of S phase. On the other hand, the size of replication units did not vary signif?cantly during S phase.
1. Introduction Each DNA molecule in a eukaryotic chromosome is replicated by a large number of separate replication forks. These forks generally occur in pairs; each pair starts at a common origin and moves outward bidirectionally, forming a bubble-like loop of replicated DNA (Huberman & Riggs, 1968; Huberman & Tsai, 1973; Blumenthal et al., 1973). The stretch of DNA replicated by a single pair of replication forks has been called a “replication unit” (Huberman & Riggs, 1968). The size of replication units and the rate of replication fork movement have been measured in a number of different eukaryotic species (Huberman t Riggs, 1968; Painter & Schaefer, 1969; Callan, 1972; McFarlane & Callan, 1973; Hand & Tamm, 1974). Within unsynchronized populations of mammalian cells, both replication unit size and rate of fork movement are quite heterogeneous. For instance, in Chinese hamster cells, Huberman & Riggs (1968) found replication units ranging in size from less than 10 pm (of extended DNA; 1 pm is approximately equivalent to M, = 2 x 10s) to greater than 100 pm, with a modal value of about 30 pm, and they found rates of fork movement ranging from O-5 to 1.2 pm/mm. The heterogeneity of replication unit size and fork movement rate within unsynchronized populations suggests the possibility that these parameters might be more homogeneous in synchronized populations and might vary during the cell cycle. Indeed, both Huberman & Riggs (1968) and Painter & Schaefer (1971) have attempted to measure fork movement rate at different times during S phase. Huberman & Riggs (1968) used Chinese hamster cells partially synchronized with FdUrdt and could detect no significant difference in the rate of fork movement between cells t Abbreviations used: FdUrd, 5-fluorodeoxyuridine; 173
dNTP, deoxyribonucleoside triphospbste.
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pulse-labeled at 1,3*5 or 7 hours after release of the FdUrd block. On the other hand, when Painter t Schaefer (1971) synchronized HeLa cells by mitotic selection or excess thymidine double block, they did detect a two- to threefold increase in fork movement rate during S phase. Our curiosity about this question was again aroused during the course of our recent DNA fiber autoradiographic experiments on the direction of DNA replication in mammalian cells (Huberman & Tsai, 1973). For these experiments we adopted a new and simpler method of lysing the cells and spreading the DNA for autoradiography (Lark et al., 1971). In this method the cells are lysed on the surface of a glass microscope slide, and their released DNA is spread over the surface of the slide, then autoradiographed. In scanning the slides after autoradiography we noticed that frequently the labeled regions in one small area of the slide were more uniform in length than on the slide as a whole. Occasionally two such small areas of uniform labeled lengths, each containing a different length, were found very close to each other, as in Plate I(a). We guessed that each such uniform region might be due to labeled DNA molecules originating from a single cell. The presence of these relatively uniform labeled regions emphasized the possibility that within single cells all of the replication forks might move at a much more uniform speed than in the unsynchronized cell population as a whole. If so, then we reasoned that the heterogeneity of fork movement rate seen for the total unsynchronized population might be due to differences in fork movement rate at different times during the S phase. In order to test this hypothesis, as well as to distinguish between the previous conclusions of Huberman & Riggs (1968) and those of Painter & Schaefer (1971), we used DNA autoradiography to measure the rate of replication fork movement at different times during S phase in carefully synchronized cells. We felt that it was important in this study to use a method of cell synchronization that does not interfere with or alter the pattern of DNA synthesis. Selection of cells in mitosis is such & method. In this paper we report our studies on fork movement rate and replication unit size in Chinese hamster ovary cells synchronized by mitotic selection. In agreement with the previous conclusion of Painter & Schaefer (1971), we detected a significant increase (at least threefold) in fork movement rate during S phase. In contrast to the dramatic change in fork movement rate, however, we found no sign&ant change in the size of replication units. We discuss the significance of these findings in relation to the overall control of DNA synthesis.
2. Materials and Methods Chinese hamster ovary cells (obtained from D. Baltimore)
were grown on Petri plates
at 37°C in a 6% CO2 atmosphere in Joklik-modified minimal essential medium (Grand Island Biological Co.) supplemented with non-essential amino acids (Grand Island Biological Co.) and 7% fetal calf serum. Cells were synchronized by a modification of the Colcemid reversal procedure of Stubblefield (1968). The growth medium was removed after gentle swirling of the plates to dislodge dead or loosely attached cells, then replaced with medium (2 parts fresh medium and 1 part conditioned medium) containing Colcemid (Cibs) at a final concentration of 60 rig/ml. Incubation was continued at 37°C for 3 h. The Colcemid-containing medium was then removed and replaced by ELsolution of 0.06% trypsin in Tris-buffered isotonic saline (TD buffer; Huberman & Riggs, 1966) at 20°C. The plates were gently agitated for 3 to 4 minutes at 20°C and the supernatant wms carefully
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decanted. The cells removed in this way had a mitotic index of > 99% in both experiments described in this paper. The oells were then concentrated by centrifugation andmmmpended in 2 parts fresh medium plus 1 part conditioned medium at a concentration of 2 X lOs/ml. The cell suspension was distributed in l%ml portions into 35 mm x 10 mm plastic Petri plates (Falcon). The oells attached firmly to these plates within 1 h. Labeling of the cells was sooomplished by the introduction of [aH]thymidine et the specific aotivity indicated in the Figure legends to a final concentmtion of 2.6 &ml. Increasee in speoi& activity of [3H]thymidine were carried out by removing the medium containing [3H]thymidine at low specific activity, washing the cells once with TD buffer at 37°C and then adding back medium conteining QH]thymidine at high specifl~~ a&iv&y. Labeling was terminated by removing the [sH]thymidine-oont&ing medium from the plate and weshing 3 times with ioe-oold TD buffer. Cells were removed from the plate into TD buffer, either by scraping with a rubber policeman or by trypsinization. The DNA from 500 to 1000 cells was spread on glass microscope slides and autorediographed ae described previously (Huberman t Tsai, 1973). Analysis of automdiogrtuns was carried out as described in the text and Figure legends.
3. Results Although the population of Chinese ha;mster ovary cells we selected for stsrting our synchronous cultures was >99% in mitosis, their degree of synchrony diminished with progress through the cell cycle. Thus, entry of cells into S phase occurred over a span of more than four hours (Fig. l), and departure from S phase took place over a spsn of more than ten hours (Rig. 1). Nevertheless, there was sufficient synchrony in the population to allow detection of significant changes in the rate of fork movement.
0 FIG. 1. Passage of synchronized Chinese hamster ovary aells through S phase. Cells were 3yn. ohronized as deaoribed in Materials and Methods. At 2-h intervals after release from mitosis the
oells were pulse-l&&d for 10 min with [3H]thymidine (20 Ci/nunol; 17 &i/ml). The cells were then wsshed twioe with cold isotonic ssline, oolleoted by trypsinimtion, &owed to swell in hypotonic medium for 10 min, pelleted, fixed with methenol/aoetio soid (3: l), spread onto g&tin. coated glass slides, and allowed to dry. The slides were costed with Kodak AR-10 autoradiographic stripping film and exposed for 7 days. After development the slides were stsined with Uiemss stein, end at least 312 oells were sosnned to determine the peroentsge of cells lsbeled at each time point.
Our initial experiment (experiment 1) was designed to give a rapid estimate of the rate of replication fork movement by measuring the lengths of DNA labeled during a 20-minute pulse with [3H]thymidine. The basic procedure was to label synchronized cells for 20 minutes with [3H]thymidine at various times during S phase, subject the DNA to autoradiography, and then measure the lengths of labeled regions of DNA in
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the autoradiographs. Examples of such autoradiographs are shown in Plate I(b) and (c). A striking feature of the slides from which the examples in Plates I(b) and (c) were taken is that, on each slide, the distribution of labeled lengths appears muoh more homogeneous than would be the case for unsynchronized cells, and the average length of labeled region appears to increase during S phase. This trend is documented in the histograms to be described below. An estimate of the fork movement rate for each labeled region can be made by dividing the length of the labeled region by 20 (to give the rate in pm/n&). Histograms showing the relative frequency of different replication rates at several times
dl-I
LI
’ ‘5h
Repllcatlon
rate
(pm/mln)
FIQ. 2. Apparent replktion rates in synohronized Chinese hamster ovary aells at various times during S phase. Cells were synohronized, pulse-lebeled for 20 min at the indicated times, and autoradiographed aa desoribed in the legend to Plate I. Measurements were made ouly in the internal regions of taudem arrays. Au effort was made to ohoose areas of the slide free from aggregation, but onoe the ohoioe of area was made, all interual segments of tandem arrays in that area were measured. M easurements were made using an eyepiece micrometer at a final mqpifieation of 400 x . Measured lengths were divided by 20 to obtein replioation rates in -/min.
PLATE 1 (a) Fiber autoradiograph of HeLa cell DNA showing 2 distinct classes of replicated regions. Unsynchronized HeLa cells growing on Petri plates were pulse-labeled with [eH]thymidine at 15 Ci/mmol for 16 min and then with [aH]thymidine at 51 Ci/mmol for an additional 15 min. The cells were lysed with sodium dodecyl sulfate and then their DNA was spread onto a glass microscope slide and autoradiographed. Details of the procedure are in the Materials and Methodi section and in Huberman & Tsai, 1973. The exposure time was 7.75 months. (b) and (c) DNA fiber autoradiographs from synchronized Chinese hamster ovary cells pulselabeled at various times during S phase. Cells were grown and synchronized as described in Materials and Methods. At 2 h (b) or 7.6 h (c) after mitosis the cells were pulse-labeled for 20 min with [eH]thymidine (60 Ci/mmol). The pulse was terminated and the cells were lysed and autoradiographed as described in Materials and Methods. Autoradiographic exposure time was for 2 to 3 months. (d) and (e) DNA fiber autoradiographs from synchronized Chinese hamster ovary cells pulselabeled with a low to high specific activity shift at different stages of S phase. Cells were grown and synchronized as described in Materials and Methods. At 2.3 h (d) or 10.3 h (e) after mitosis the cells were labeled for 16 min with [3H]thymidine at 8 Ci/mmol followed by 16 min at 50 Ci/mmol. The labeling was terminated and the cells were lysed and autoradiographed as described in Materials and Methods. Exposure time was 8 months.
[facing p. 176
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S PHASE
TABLE 1
Average fork movement rates ad replication unit sizes at various times after mitosis in sy?whronized Chinese hamder ovary Gel& Time after mitosis 04 Experiment
Experiment
Average fork movement r&e b.dmW
Average replication tit size (P-4
lt 2 3 4 6 6 7-b 9 10.5
0.21 O-28 O-36 0.42 0.42 0.63 0.64 0.69
2.3 4.3 6-3 10-3
0.27 0.67 0.70 O-68
2$
t C&ml&ions $ Calculations
32 31 28
baaed on d&a from Fig. 2. based on d&a from Figs 4 and 6.
during S phase are shown in Figure 2, and the average values for these histograms are presented in Table 1. It is clear that the average replication rate increases by a factor of about three from very early to mid or late S phase. Although the experimental procedure used in experiment 1 has the advantage that measurements are simple and consequently rapid, it also has the disadvantage that the lengths measured may be the result of fusion of adjacent replication forks during the pulse or initiation of replication after the start of the pulse. The short pulse length of 20 minutes was chosen to minimize such possibilities, but the best argument that the method used in experiment 1 leads to reasonable valuea for the rate of fork movement is that the results of experiment 1 are in qualitative agreement with those of experiment 2, which was designed to avoid the problems of initiation or temination during the pulse. The rationale of experiment 2 can be understood by reference to Figure 3, which shows in diagram form one kind of autoradiographic pattern that might be produced by two adjacent bidirectional replication units in a cell exposed first to a pulse of
FIQ. 3. Diagram illustrating methods for measuring fork movement rate end oenter-to-center distanoe between replication units. Solid lines indiaate high grain density regions in a DNA fiber autoradiogram, while dotted lines indiaate low grain density regions. The top braokets indicate the labeled regions used for fork rate measurements, and the bottom bracket shows the type of oenter-to-center distance measured to give an estimate of replioation unit size. The oonfiguration diagrammed is typioal for mammalian DNA labeled with a low specific activity to high specific aotivity shift (for more examples see Huberman & Tsei, 1973).
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[3H]thymidine at low speoific activity and then to a pulse of [3H]thymidine at high specific activity. The pattern diagrammed in Figure 3 is the one expected for replication units that initiated replication before the low specific activity pulse and did not complete it until after the high specific activity pulse. Such patterns are found to occur at a sufficiently high frequency to be useful for measurements when the pulse time is short enough (less than 30 minutes each for low and high specific activities; Huberman & Tsai, 1973). The labeled regions, indicated by the upper brackets, can be used to measure replication rate without interference from problems of termination or initiation; clearly each such region must have been labeled for the full length of the pulse period. The procedure for our experiment 2 was to synchronize the cells by mitotic selection, as for experiment 1, and then to label them at various times during S phase, first with [3H]thymidine (8 C!i/mmol) for 16 minutes and then with r3H]thymidine (SO Ci/mmol) for 15 minutes. The cells were then lysed and their DNA was autoradiographed. As shown in Plate I(d) and (e), regions of low and high grain density, oorresponding to the early and late labeling periods, can be distinguished in the resulting autoradiographs. We made rate measurements on configurations of the kind diagrammed in Figure 3. Lengths of suitable regions were measured and then divided by 15 to give the rate in pm/min. The results of this experiment are in qualitative agreement with those of experiment 1, as shown by the histograms in Figure 4 and the average rate values in Table 1.
I-25 175 O o’250 5°‘75 I 1.5 Replication rote (pm/min)
FIG. 4. Replication rates in synchronized Chinese hamster ovary aells at various times during S phase. Cells were synohronized, pulse-labeled with a low spe&ic aotivity to high speoi& activity shift and automdiogrephed &8 described in the text and Plate I. The procedures desoribsd in Fig. 2 for ensuring randomness of the segments measured were followed here as well. Measured lengths were divided by 15 to obtain replioation rates in q/min.
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Again the increase in rate is nearly threefold from very early to mid or late S phase. We note, however, that the rate values obtained in experiment 2 are higher than those obtained in experiment 1. This may be due to the frtct that the lengths measured in experiment 1 include lengths that initiated replication after the start of the pulse, as noted previously. It may also be due to bias in selection of lengths for measurement in experiment 2 ; it is more difficult to distinguish regions of low and high g&n density when both regions are short. Whatever the reason for this minor quantitative disagreement between the two experiments, the data from both esperiments clearly demonstrate a significant increase in rate from very early to mid and late S phase. The size of replication units could also be determined accurately in the autoradiogmphs of experiment 2. Measurements of center-to-center distances were made as illustrated by the bottom brackets in Figure 3 (Hubermcm 6 Riggs, 1968). Results are presented in histogram form in Figure 5. In striking contrast to the changes in the replication rate observed in the sa.me experiment, the size of replication units does not change significantly during S phase. At all times the average center-to-center distance is about 30 r;m (Table 1) and the range is from less than 10 to greater than 50 pm, very similar to the earlier findings of Huberman & Riggs (1968) for unsynchronized Chinese hamster B14FAF28 cells.
Repiication
umt !er:qt> (UP )
FIQ. 6. Lengths of replioetion units. Measurements of center-to-oenter diatanaes between replioetion units (an approximation to replioation unit size; Huberman & Riggs, 1968) were made by the ariteria of Fig. 3 on the same autorediograms used for Fig. 4.
4. Discussion We have shown that the rate of DNA replication fork movement increases about threefold during S phase, while the size of the replication unit remains constant (although heterogeneous). We note that our results may underestimate the magnitude of the change in fork movement rate during S phase, both because of imperfect synchrony and because the labeled lengths in very early S phase are at the limit of resolution of the technique. Shorter lengths, if present, would have been missed.
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Our demonstration of an increase in fork movement rate during S phase is in basic agreement with the results of Painter & Schaefer (1971), who used HeLa cells synchronized by mitotic selection or with excess thymidine, and in apparent disagreement with the earlier finding by Huberman & Riggs (1968) of no significant variation in rate of fork movement during S phase in Chinese hamster cells synchronized with FdUrd. However, Huberman & Riggs’ flnding can now be explained partly as the result of imperfect synchronization by FdUrd (this compound does not inhibit DNA synthesis completely in Chinese hamster cells; Amaldi et al., 1972) and partly as the result of their measuring rates at one to two hours after the supposed start of S phase at the earliest (the most aigni6cant changes in rate, we now know, occur in the iirst hour of S phase ; 8ee Figs 2 and 4). It is interesting to compare our conclusions about regulation of DNA synthesis during the S phase in mammalian cells with the findings of other investigators for different eukaryotes. Blumenthal et al. (1973) have measured the rate of fork movement during the very short 8 phase (only 1 to 2 min) of very early Dmphila embryos Their conclusions, based on the electron microscopic appearance of replicating DNA, suggest that the rate of fork movement during this very short S phase is constant. Callan (1972) has measured the rate of fork movement and replication unit size during different developmental stages of a number of amphibians. In contrast to our findings, he concludes that the rate of fork movement is approximately constant in the different development stages, but that the size of the replication units varies considerably. Of course his measurements compare a different time-scale from ours (different developmental stages verszlS different times during a single S phase), but since the number of measurements of this sort is still small, we feel that the most reasonable conclusion is that both the rate of fork movement and replication unit size can vary within cells of a single eukaryotic species. It is not yet clear whether all developmental variations involve only replication unit size and all S phase variations involve only fork movement rate. The implications of our results with respect to the earliest events in the S period of Chinese hamster ovary cells are quite clear. Rather than assembling all the element8 required for sustaining fork movement at a maximum rate during a period in which initiation of new rounds of DNA synthesis in all replication units is forbidden, the cell permits initiation of DNA synthesis to take place in some replication unit8 before fork movement can be sustained at maximum rate. This interpretation of our data raises the question: which cellular components are rate-limiting for DNA synthesis in early S phase? One likely candidate for this role is the pool of deoxyribonucleoside triphosphates. Walters et al. (1973) and Skoog et al. (1973) have shown that the dNTP pools are very low during the 01 phase. These pools gradually increase in size as the S period progresses. These observations are consistent with our observation of an increase in rate of fork movement during S phase. However, a cause and effect relationship between dNTP pool size and DNA fork movement rate will have to be demonstrated directly. Other mechanisms for regulating fork movement rate, such as variations in the activity of proteins required directly for DNA 8yIIthe8iS, could well be operative. The results of Klevecz (1969) and Klevecz & Kapp (1973) on variations in the overall rate of DNA synthesis in WI-38 (human diploid) and Chinese hamster ovary cells (heteroploid) suggest that regulation of DNA synthesis in the human diploid cells may be more precise and complex than in the heteroploid hamster cells. These
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authors observed several peaks in the rate of overall DNA synthesis in WI-38 cells but only a single broad rise and fall in synthetic rate in hamster cells. It would be of great interest to determine whether significant fluctuations in the rate of fork movement could be observed during the WI-38 cell cycle. This research was supported by research grants from the National Science Foundation and National Institutes of Health. One of us (D. H.) was a recipient of a postdoctoral research fellowship from the Jane Coffin Childs Memorial Fund for Medical Research, and the other (J. A. H.) is a recipient of a Career Development Award from the National Institutes of Health. Alice Tsai provided invaluable technical assistance. REFERENCES Amaldi, F., Carnevali, F., Leoni, L., & Mariotti, D. (1972). Ezp. Cell Res. 74, 367-374. Blumenthal, A. B., Kriegstein, H. J. & Hogness, D. S. (1973). Cold &m&q Harbor Symp. Qumt. Bid. 38, 206223. Callan, H. G. (1972). Proc. Roy. Sot., eer. B, 181, 1941. Hand, R. & Tamm, I. (1974). J. Mol. BioZ. 82, 175-183. Huberman, J. A. & Riggs, A. D. (1906). Proc. Na.t. Acud. Sci., U.S.A. 55, 599-606. Huberman, J. A. t Riggs, A. D. (1968). J. Mol. B&l. 32, 327-341. Huberman, J. A. & Tsai, A. (1973). J. Mol. Bid. 75, 5-12. Klevecz, R. R. (1969). Science, 166, 15361538. Klevecz, R. R. t Kapp, L. N. (1973). J. CeZZBid. 58, 564-573. Lark, K. G., Consigli, R. t Toliver, A. (1971). J. Mol. Biol. 58, 873-875. McFarlane, P. W. t CaUtm, H. G. (1973). J. Cell Sci. 13, 821-839. Painter, R. B. & Schaefer, A. W. (1969). J. Mol. Bid. 45, 467-479. Painter, R. B. & Schaefer, A. W. (1971). J. Mol. Bid. 58, 289-295. Skoog, K. L., Nordenskjold, B. A. & Bjursell, K. G. (1973). Eur. J. B&hem. 33,428-432. Stubblefield, E. (1968). In Method in Cell Phy.GoZogy (Prescott, D. M., ea.), vol. 3, pp. 26-43, Academic Press, New York. Walters, R. A., Tobey, R. A. & Ratliff, R. L. (1973). Biochim. Biophys. Acta, 319,336-347.
Changes in the Rate of DNA Replication Fork Movement During S Phase in Mammalian Cells A. HTJBERMAN
DAVID HOUSMANANDJOEL
Department of Biokgy Mamachueetts Inetitute of Technology Cam.bridge, Maes. 02139, U.X.A. (Received 25 July 1974) DNA fiber autoradiography was used to measure the rate of replication fork movement and the size of replication units as a function of time during the S phase of synchronized Chinese hamster ovary cells. The rate of fork movement increased by about threefold from early S to later S phase, with the most dramatic change occurring in the f%st hour of S phase. On the other hand, the size of replication units did not vary signif?cantly during S phase.
1. Introduction Each DNA molecule in a eukaryotic chromosome is replicated by a large number of separate replication forks. These forks generally occur in pairs; each pair starts at a common origin and moves outward bidirectionally, forming a bubble-like loop of replicated DNA (Huberman & Riggs, 1968; Huberman & Tsai, 1973; Blumenthal et al., 1973). The stretch of DNA replicated by a single pair of replication forks has been called a “replication unit” (Huberman & Riggs, 1968). The size of replication units and the rate of replication fork movement have been measured in a number of different eukaryotic species (Huberman t Riggs, 1968; Painter & Schaefer, 1969; Callan, 1972; McFarlane & Callan, 1973; Hand & Tamm, 1974). Within unsynchronized populations of mammalian cells, both replication unit size and rate of fork movement are quite heterogeneous. For instance, in Chinese hamster cells, Huberman & Riggs (1968) found replication units ranging in size from less than 10 pm (of extended DNA; 1 pm is approximately equivalent to M, = 2 x 10s) to greater than 100 pm, with a modal value of about 30 pm, and they found rates of fork movement ranging from O-5 to 1.2 pm/mm. The heterogeneity of replication unit size and fork movement rate within unsynchronized populations suggests the possibility that these parameters might be more homogeneous in synchronized populations and might vary during the cell cycle. Indeed, both Huberman & Riggs (1968) and Painter & Schaefer (1971) have attempted to measure fork movement rate at different times during S phase. Huberman & Riggs (1968) used Chinese hamster cells partially synchronized with FdUrdt and could detect no significant difference in the rate of fork movement between cells t Abbreviations used: FdUrd, 5-fluorodeoxyuridine; 173
dNTP, deoxyribonucleoside triphospbste.
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pulse-labeled at 1,3*5 or 7 hours after release of the FdUrd block. On the other hand, when Painter t Schaefer (1971) synchronized HeLa cells by mitotic selection or excess thymidine double block, they did detect a two- to threefold increase in fork movement rate during S phase. Our curiosity about this question was again aroused during the course of our recent DNA fiber autoradiographic experiments on the direction of DNA replication in mammalian cells (Huberman & Tsai, 1973). For these experiments we adopted a new and simpler method of lysing the cells and spreading the DNA for autoradiography (Lark et al., 1971). In this method the cells are lysed on the surface of a glass microscope slide, and their released DNA is spread over the surface of the slide, then autoradiographed. In scanning the slides after autoradiography we noticed that frequently the labeled regions in one small area of the slide were more uniform in length than on the slide as a whole. Occasionally two such small areas of uniform labeled lengths, each containing a different length, were found very close to each other, as in Plate I(a). We guessed that each such uniform region might be due to labeled DNA molecules originating from a single cell. The presence of these relatively uniform labeled regions emphasized the possibility that within single cells all of the replication forks might move at a much more uniform speed than in the unsynchronized cell population as a whole. If so, then we reasoned that the heterogeneity of fork movement rate seen for the total unsynchronized population might be due to differences in fork movement rate at different times during the S phase. In order to test this hypothesis, as well as to distinguish between the previous conclusions of Huberman & Riggs (1968) and those of Painter & Schaefer (1971), we used DNA autoradiography to measure the rate of replication fork movement at different times during S phase in carefully synchronized cells. We felt that it was important in this study to use a method of cell synchronization that does not interfere with or alter the pattern of DNA synthesis. Selection of cells in mitosis is such & method. In this paper we report our studies on fork movement rate and replication unit size in Chinese hamster ovary cells synchronized by mitotic selection. In agreement with the previous conclusion of Painter & Schaefer (1971), we detected a significant increase (at least threefold) in fork movement rate during S phase. In contrast to the dramatic change in fork movement rate, however, we found no sign&ant change in the size of replication units. We discuss the significance of these findings in relation to the overall control of DNA synthesis.
2. Materials and Methods Chinese hamster ovary cells (obtained from D. Baltimore)
were grown on Petri plates
at 37°C in a 6% CO2 atmosphere in Joklik-modified minimal essential medium (Grand Island Biological Co.) supplemented with non-essential amino acids (Grand Island Biological Co.) and 7% fetal calf serum. Cells were synchronized by a modification of the Colcemid reversal procedure of Stubblefield (1968). The growth medium was removed after gentle swirling of the plates to dislodge dead or loosely attached cells, then replaced with medium (2 parts fresh medium and 1 part conditioned medium) containing Colcemid (Cibs) at a final concentration of 60 rig/ml. Incubation was continued at 37°C for 3 h. The Colcemid-containing medium was then removed and replaced by ELsolution of 0.06% trypsin in Tris-buffered isotonic saline (TD buffer; Huberman & Riggs, 1966) at 20°C. The plates were gently agitated for 3 to 4 minutes at 20°C and the supernatant wms carefully
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decanted. The cells removed in this way had a mitotic index of > 99% in both experiments described in this paper. The oells were then concentrated by centrifugation andmmmpended in 2 parts fresh medium plus 1 part conditioned medium at a concentration of 2 X lOs/ml. The cell suspension was distributed in l%ml portions into 35 mm x 10 mm plastic Petri plates (Falcon). The oells attached firmly to these plates within 1 h. Labeling of the cells was sooomplished by the introduction of [aH]thymidine et the specific aotivity indicated in the Figure legends to a final concentmtion of 2.6 &ml. Increasee in speoi& activity of [3H]thymidine were carried out by removing the medium containing [3H]thymidine at low specific activity, washing the cells once with TD buffer at 37°C and then adding back medium conteining QH]thymidine at high specifl~~ a&iv&y. Labeling was terminated by removing the [sH]thymidine-oont&ing medium from the plate and weshing 3 times with ioe-oold TD buffer. Cells were removed from the plate into TD buffer, either by scraping with a rubber policeman or by trypsinization. The DNA from 500 to 1000 cells was spread on glass microscope slides and autorediographed ae described previously (Huberman t Tsai, 1973). Analysis of automdiogrtuns was carried out as described in the text and Figure legends.
3. Results Although the population of Chinese ha;mster ovary cells we selected for stsrting our synchronous cultures was >99% in mitosis, their degree of synchrony diminished with progress through the cell cycle. Thus, entry of cells into S phase occurred over a span of more than four hours (Fig. l), and departure from S phase took place over a spsn of more than ten hours (Rig. 1). Nevertheless, there was sufficient synchrony in the population to allow detection of significant changes in the rate of fork movement.
0 FIG. 1. Passage of synchronized Chinese hamster ovary aells through S phase. Cells were 3yn. ohronized as deaoribed in Materials and Methods. At 2-h intervals after release from mitosis the
oells were pulse-l&&d for 10 min with [3H]thymidine (20 Ci/nunol; 17 &i/ml). The cells were then wsshed twioe with cold isotonic ssline, oolleoted by trypsinimtion, &owed to swell in hypotonic medium for 10 min, pelleted, fixed with methenol/aoetio soid (3: l), spread onto g&tin. coated glass slides, and allowed to dry. The slides were costed with Kodak AR-10 autoradiographic stripping film and exposed for 7 days. After development the slides were stsined with Uiemss stein, end at least 312 oells were sosnned to determine the peroentsge of cells lsbeled at each time point.
Our initial experiment (experiment 1) was designed to give a rapid estimate of the rate of replication fork movement by measuring the lengths of DNA labeled during a 20-minute pulse with [3H]thymidine. The basic procedure was to label synchronized cells for 20 minutes with [3H]thymidine at various times during S phase, subject the DNA to autoradiography, and then measure the lengths of labeled regions of DNA in
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the autoradiographs. Examples of such autoradiographs are shown in Plate I(b) and (c). A striking feature of the slides from which the examples in Plates I(b) and (c) were taken is that, on each slide, the distribution of labeled lengths appears muoh more homogeneous than would be the case for unsynchronized cells, and the average length of labeled region appears to increase during S phase. This trend is documented in the histograms to be described below. An estimate of the fork movement rate for each labeled region can be made by dividing the length of the labeled region by 20 (to give the rate in pm/n&). Histograms showing the relative frequency of different replication rates at several times
dl-I
LI
’ ‘5h
Repllcatlon
rate
(pm/mln)
FIQ. 2. Apparent replktion rates in synohronized Chinese hamster ovary aells at various times during S phase. Cells were synohronized, pulse-lebeled for 20 min at the indicated times, and autoradiographed aa desoribed in the legend to Plate I. Measurements were made ouly in the internal regions of taudem arrays. Au effort was made to ohoose areas of the slide free from aggregation, but onoe the ohoioe of area was made, all interual segments of tandem arrays in that area were measured. M easurements were made using an eyepiece micrometer at a final mqpifieation of 400 x . Measured lengths were divided by 20 to obtein replioation rates in -/min.
PLATE 1 (a) Fiber autoradiograph of HeLa cell DNA showing 2 distinct classes of replicated regions. Unsynchronized HeLa cells growing on Petri plates were pulse-labeled with [eH]thymidine at 15 Ci/mmol for 16 min and then with [aH]thymidine at 51 Ci/mmol for an additional 15 min. The cells were lysed with sodium dodecyl sulfate and then their DNA was spread onto a glass microscope slide and autoradiographed. Details of the procedure are in the Materials and Methodi section and in Huberman & Tsai, 1973. The exposure time was 7.75 months. (b) and (c) DNA fiber autoradiographs from synchronized Chinese hamster ovary cells pulselabeled at various times during S phase. Cells were grown and synchronized as described in Materials and Methods. At 2 h (b) or 7.6 h (c) after mitosis the cells were pulse-labeled for 20 min with [eH]thymidine (60 Ci/mmol). The pulse was terminated and the cells were lysed and autoradiographed as described in Materials and Methods. Autoradiographic exposure time was for 2 to 3 months. (d) and (e) DNA fiber autoradiographs from synchronized Chinese hamster ovary cells pulselabeled with a low to high specific activity shift at different stages of S phase. Cells were grown and synchronized as described in Materials and Methods. At 2.3 h (d) or 10.3 h (e) after mitosis the cells were labeled for 16 min with [3H]thymidine at 8 Ci/mmol followed by 16 min at 50 Ci/mmol. The labeling was terminated and the cells were lysed and autoradiographed as described in Materials and Methods. Exposure time was 8 months.
[facing p. 176
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S PHASE
TABLE 1
Average fork movement rates ad replication unit sizes at various times after mitosis in sy?whronized Chinese hamder ovary Gel& Time after mitosis 04 Experiment
Experiment
Average fork movement r&e b.dmW
Average replication tit size (P-4
lt 2 3 4 6 6 7-b 9 10.5
0.21 O-28 O-36 0.42 0.42 0.63 0.64 0.69
2.3 4.3 6-3 10-3
0.27 0.67 0.70 O-68
2$
t C&ml&ions $ Calculations
32 31 28
baaed on d&a from Fig. 2. based on d&a from Figs 4 and 6.
during S phase are shown in Figure 2, and the average values for these histograms are presented in Table 1. It is clear that the average replication rate increases by a factor of about three from very early to mid or late S phase. Although the experimental procedure used in experiment 1 has the advantage that measurements are simple and consequently rapid, it also has the disadvantage that the lengths measured may be the result of fusion of adjacent replication forks during the pulse or initiation of replication after the start of the pulse. The short pulse length of 20 minutes was chosen to minimize such possibilities, but the best argument that the method used in experiment 1 leads to reasonable valuea for the rate of fork movement is that the results of experiment 1 are in qualitative agreement with those of experiment 2, which was designed to avoid the problems of initiation or temination during the pulse. The rationale of experiment 2 can be understood by reference to Figure 3, which shows in diagram form one kind of autoradiographic pattern that might be produced by two adjacent bidirectional replication units in a cell exposed first to a pulse of
FIQ. 3. Diagram illustrating methods for measuring fork movement rate end oenter-to-center distanoe between replication units. Solid lines indiaate high grain density regions in a DNA fiber autoradiogram, while dotted lines indiaate low grain density regions. The top braokets indicate the labeled regions used for fork rate measurements, and the bottom bracket shows the type of oenter-to-center distance measured to give an estimate of replioation unit size. The oonfiguration diagrammed is typioal for mammalian DNA labeled with a low specific activity to high specific aotivity shift (for more examples see Huberman & Tsei, 1973).
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[3H]thymidine at low speoific activity and then to a pulse of [3H]thymidine at high specific activity. The pattern diagrammed in Figure 3 is the one expected for replication units that initiated replication before the low specific activity pulse and did not complete it until after the high specific activity pulse. Such patterns are found to occur at a sufficiently high frequency to be useful for measurements when the pulse time is short enough (less than 30 minutes each for low and high specific activities; Huberman & Tsai, 1973). The labeled regions, indicated by the upper brackets, can be used to measure replication rate without interference from problems of termination or initiation; clearly each such region must have been labeled for the full length of the pulse period. The procedure for our experiment 2 was to synchronize the cells by mitotic selection, as for experiment 1, and then to label them at various times during S phase, first with [3H]thymidine (8 C!i/mmol) for 16 minutes and then with r3H]thymidine (SO Ci/mmol) for 15 minutes. The cells were then lysed and their DNA was autoradiographed. As shown in Plate I(d) and (e), regions of low and high grain density, oorresponding to the early and late labeling periods, can be distinguished in the resulting autoradiographs. We made rate measurements on configurations of the kind diagrammed in Figure 3. Lengths of suitable regions were measured and then divided by 15 to give the rate in pm/min. The results of this experiment are in qualitative agreement with those of experiment 1, as shown by the histograms in Figure 4 and the average rate values in Table 1.
I-25 175 O o’250 5°‘75 I 1.5 Replication rote (pm/min)
FIG. 4. Replication rates in synchronized Chinese hamster ovary aells at various times during S phase. Cells were synohronized, pulse-labeled with a low spe&ic aotivity to high speoi& activity shift and automdiogrephed &8 described in the text and Plate I. The procedures desoribsd in Fig. 2 for ensuring randomness of the segments measured were followed here as well. Measured lengths were divided by 15 to obtain replioation rates in q/min.
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Again the increase in rate is nearly threefold from very early to mid or late S phase. We note, however, that the rate values obtained in experiment 2 are higher than those obtained in experiment 1. This may be due to the frtct that the lengths measured in experiment 1 include lengths that initiated replication after the start of the pulse, as noted previously. It may also be due to bias in selection of lengths for measurement in experiment 2 ; it is more difficult to distinguish regions of low and high g&n density when both regions are short. Whatever the reason for this minor quantitative disagreement between the two experiments, the data from both esperiments clearly demonstrate a significant increase in rate from very early to mid and late S phase. The size of replication units could also be determined accurately in the autoradiogmphs of experiment 2. Measurements of center-to-center distances were made as illustrated by the bottom brackets in Figure 3 (Hubermcm 6 Riggs, 1968). Results are presented in histogram form in Figure 5. In striking contrast to the changes in the replication rate observed in the sa.me experiment, the size of replication units does not change significantly during S phase. At all times the average center-to-center distance is about 30 r;m (Table 1) and the range is from less than 10 to greater than 50 pm, very similar to the earlier findings of Huberman & Riggs (1968) for unsynchronized Chinese hamster B14FAF28 cells.
Repiication
umt !er:qt> (UP )
FIQ. 6. Lengths of replioetion units. Measurements of center-to-oenter diatanaes between replioetion units (an approximation to replioation unit size; Huberman & Riggs, 1968) were made by the ariteria of Fig. 3 on the same autorediograms used for Fig. 4.
4. Discussion We have shown that the rate of DNA replication fork movement increases about threefold during S phase, while the size of the replication unit remains constant (although heterogeneous). We note that our results may underestimate the magnitude of the change in fork movement rate during S phase, both because of imperfect synchrony and because the labeled lengths in very early S phase are at the limit of resolution of the technique. Shorter lengths, if present, would have been missed.
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Our demonstration of an increase in fork movement rate during S phase is in basic agreement with the results of Painter & Schaefer (1971), who used HeLa cells synchronized by mitotic selection or with excess thymidine, and in apparent disagreement with the earlier finding by Huberman & Riggs (1968) of no significant variation in rate of fork movement during S phase in Chinese hamster cells synchronized with FdUrd. However, Huberman & Riggs’ flnding can now be explained partly as the result of imperfect synchronization by FdUrd (this compound does not inhibit DNA synthesis completely in Chinese hamster cells; Amaldi et al., 1972) and partly as the result of their measuring rates at one to two hours after the supposed start of S phase at the earliest (the most aigni6cant changes in rate, we now know, occur in the iirst hour of S phase ; 8ee Figs 2 and 4). It is interesting to compare our conclusions about regulation of DNA synthesis during the S phase in mammalian cells with the findings of other investigators for different eukaryotes. Blumenthal et al. (1973) have measured the rate of fork movement during the very short 8 phase (only 1 to 2 min) of very early Dmphila embryos Their conclusions, based on the electron microscopic appearance of replicating DNA, suggest that the rate of fork movement during this very short S phase is constant. Callan (1972) has measured the rate of fork movement and replication unit size during different developmental stages of a number of amphibians. In contrast to our findings, he concludes that the rate of fork movement is approximately constant in the different development stages, but that the size of the replication units varies considerably. Of course his measurements compare a different time-scale from ours (different developmental stages verszlS different times during a single S phase), but since the number of measurements of this sort is still small, we feel that the most reasonable conclusion is that both the rate of fork movement and replication unit size can vary within cells of a single eukaryotic species. It is not yet clear whether all developmental variations involve only replication unit size and all S phase variations involve only fork movement rate. The implications of our results with respect to the earliest events in the S period of Chinese hamster ovary cells are quite clear. Rather than assembling all the element8 required for sustaining fork movement at a maximum rate during a period in which initiation of new rounds of DNA synthesis in all replication units is forbidden, the cell permits initiation of DNA synthesis to take place in some replication unit8 before fork movement can be sustained at maximum rate. This interpretation of our data raises the question: which cellular components are rate-limiting for DNA synthesis in early S phase? One likely candidate for this role is the pool of deoxyribonucleoside triphosphates. Walters et al. (1973) and Skoog et al. (1973) have shown that the dNTP pools are very low during the 01 phase. These pools gradually increase in size as the S period progresses. These observations are consistent with our observation of an increase in rate of fork movement during S phase. However, a cause and effect relationship between dNTP pool size and DNA fork movement rate will have to be demonstrated directly. Other mechanisms for regulating fork movement rate, such as variations in the activity of proteins required directly for DNA 8yIIthe8iS, could well be operative. The results of Klevecz (1969) and Klevecz & Kapp (1973) on variations in the overall rate of DNA synthesis in WI-38 (human diploid) and Chinese hamster ovary cells (heteroploid) suggest that regulation of DNA synthesis in the human diploid cells may be more precise and complex than in the heteroploid hamster cells. These
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authors observed several peaks in the rate of overall DNA synthesis in WI-38 cells but only a single broad rise and fall in synthetic rate in hamster cells. It would be of great interest to determine whether significant fluctuations in the rate of fork movement could be observed during the WI-38 cell cycle. This research was supported by research grants from the National Science Foundation and National Institutes of Health. One of us (D. H.) was a recipient of a postdoctoral research fellowship from the Jane Coffin Childs Memorial Fund for Medical Research, and the other (J. A. H.) is a recipient of a Career Development Award from the National Institutes of Health. Alice Tsai provided invaluable technical assistance. REFERENCES Amaldi, F., Carnevali, F., Leoni, L., & Mariotti, D. (1972). Ezp. Cell Res. 74, 367-374. Blumenthal, A. B., Kriegstein, H. J. & Hogness, D. S. (1973). Cold &m&q Harbor Symp. Qumt. Bid. 38, 206223. Callan, H. G. (1972). Proc. Roy. Sot., eer. B, 181, 1941. Hand, R. & Tamm, I. (1974). J. Mol. BioZ. 82, 175-183. Huberman, J. A. & Riggs, A. D. (1906). Proc. Na.t. Acud. Sci., U.S.A. 55, 599-606. Huberman, J. A. t Riggs, A. D. (1968). J. Mol. B&l. 32, 327-341. Huberman, J. A. & Tsai, A. (1973). J. Mol. Bid. 75, 5-12. Klevecz, R. R. (1969). Science, 166, 15361538. Klevecz, R. R. t Kapp, L. N. (1973). J. CeZZBid. 58, 564-573. Lark, K. G., Consigli, R. t Toliver, A. (1971). J. Mol. Biol. 58, 873-875. McFarlane, P. W. t CaUtm, H. G. (1973). J. Cell Sci. 13, 821-839. Painter, R. B. & Schaefer, A. W. (1969). J. Mol. Bid. 45, 467-479. Painter, R. B. & Schaefer, A. W. (1971). J. Mol. Bid. 58, 289-295. Skoog, K. L., Nordenskjold, B. A. & Bjursell, K. G. (1973). Eur. J. B&hem. 33,428-432. Stubblefield, E. (1968). In Method in Cell Phy.GoZogy (Prescott, D. M., ea.), vol. 3, pp. 26-43, Academic Press, New York. Walters, R. A., Tobey, R. A. & Ratliff, R. L. (1973). Biochim. Biophys. Acta, 319,336-347.