Early initiation of bovine satellite I DNA replication

Copyright @ 1982 by Academic Press. Inc. All rights of reproduction in any form reserved 00 14-4827182/070047-08$02.00/0

Experimental

EARLY

INITIATION

Cell Biology 140 (1982) 47-54

OF BOVINE

SATELLITE

LLOYD H. MATSUMOTO Brown University,

Division

I DNA REPLICATION

and SUSAN A. GERBI

of Biology and Medicine, Providence, RI 02912, USA

SUMMARY Synchronized bovine cells were used to studythe replication of satellite I DNA during the first 3 h of S phase. Following isopyknic ultracentrifugation, we compared the specific activities of pulselabelled satellite I and main band DNA. In addition, the specific activity of the Eco RI fragment of satellite I DNA was determined auantitativelv and visualized aualitatively. All methods showed an increase in the specific activity-of satellite I concurrent with that of main band DNA near the beginning of S phase, first detectable at I-14 h into S phase. Thus, although most satellite DNAs replicated late in S phase [l], we report here that bovine satellite I initiates its replication early in S phase. The implications of this result for the control of DNA synthesis are discussed.

In order to fully appreciate the control of DNA replication, a detailed understanding of initiation of DNA synthesis is required. In eukaryotic cells, initiation occurs at multiple points of origin along the length of each chromosome [2-51. Since there are numerous origins of replication in a eukaryotic genome, separate control may be exercised over each of them. Thus, it would be possible to begin synthesis of some DNA sequences earlier than for others. It is, in fact, known that not all DNA sequences are replicated simultaneously during S phase. For example, simple sequence satellite DNA which is found in heterochromatic portions of virtually all eukaryotic genomes, generally replicates later in S than the bulk DNA of the euchromatin [ 11.Since the majority of satellite DNA is replicated late in S, it has been a tacit assumption by most that it must also initiate replication late in S. This is an important point, as it suggests that a specific control exists to delay initiation of satellite DNA replication. We have reinvestigated this issue, using 4-821808

methods of restriction endonuclease digestion and gel electrophoresis, which are more sensitive than methods previously used. In this paper we report our finding that a satellite DNA initiates its replication early in S phase at the same time as bulk DNA. This was an unexpected result, and we discuss its implications. A possibility suggested by our data is that all functionally active origins of the entire genome initiate replication fairly synchronously, but that continuation of replication is blocked for heterochromatic sequences until later in S. Thus, temporal control would be exerted over progression of DNA replication rather than over its initiation. As a model system, we have studied bovine satellite I DNA. This sequence comprises 5% of the genome, has a buoyant density in CsCl of 1.715 g/cm3 [6], and is located in the centromeric heterochromatin of all autosomes [7]. It was the first satellite DNA to be cut into specific fragments by restriction enzymes, and an excellent restriction map now exists [g-14]. RestricExp Cd Rcs 140 (1982)

48

Matsumoto

and Gerbi

dem array of short radioactive stretches of DNA [ 15, 161. In this communication we show that the bovine satellite I origins initiate replication early in S phase.

m v) 0 .E m m al D m J *

80

60

MATERIALS

Cell culture and labelling

40

20

1

3

5 Time

7 in

9

1 1

hours

Fig. 1. The percentage of labelled mitotic figures as a function of the time after labelling an asynchronous log phase culture as described in Materials and Methods. Zero time was when the T3H]TdR was added. The time recorded in the y-axis therefore represents the 25 min pulse plus variable length chase; it excludes the 1 h colcemid treatment after the chase. 200-300 cells were scored for each time point. The estimates for the lengths of G2 and S are indicated. Mitosis was observed to be 1 h. The length of Gl can be estimated by subtracting the sum-of G2, S and M from the doubling or generation time, determined to be about 15.6 h.

tion analysis indicates great homogeneity of sequences in satellite I; for example, Eco RI cuts virtually all of satellite I into a 1400 base pair (bp) fragment [S]. The sequence of the 1400 bp Eco RI fragment of satellite I has been determined from cloned fragments [34, 351 as well as for uncloned Eco RI fragments from bulk DNA [36]. These results show only 3% variation in sequence among the 105000 copieslgenome of the satellite I Eco RI repeat unit, both within one individual as well as between organisms. This homogeneity can be of experimental advantage if multiple, identical origins are located in this satellite. It is reasonable to assume that there will be multiple origins in bovine satellite I, by analogy with mouse satellite which was observed by DNA fiber autoradiography to have a tanRes 140 (1982)

of cells

MDBK cells [17] were obtained from Dr Peter Palese (Mt Sinai Hosnital, N.Y.) and were grown in Eagle’s MEM media supplemented with lO%fetal calf serum (FCS) but without penicillin and streptomycin. Isoleutine-deficient media, containing 10% dialysed FCS was oreDared accordinrr to Lev & Tobev r181. Cells were’ seeded at a conc&tration of 2x 16 celislml in 150 mm Nunc tissue culture dishes. For synchronization, cells at 80% confluence were washed twice with Earle’s balanced salt solution and grown for 39 h in isoleucine-deficient media followed by growth for 13 h in normal media containing 1 mM hydroxyurea. Since this synchronization regime reversibly arrests cells at the GllS border Tl9211, reulacement of the hydroxyurea-containing media with normal media constitutes the beginning of S phase for the majority of the cells. [3H]TdR (sp. act., 28.4 Ci/mM; New England Nuclear) was added to a final concentration of 1 uCi/ ml to synchronized cells for 30 min; [3PP]orthophosphoric acid (carrier-free, New England Nuclear) was added to a final concentration of 15 &/ml to synchronized cells for 5 min.

Measurement

Exp Cd

AND METHODS

of cell cycle parameters

The cell cycle parameters for this cell line were autoradiographically determined as previously described bv others r22. 231. Brieflv, asvnchronous populations of dells in Id phase were pulsed for 25 min with 1 &i/ml [3H7TdR followed by varying periods of chase in 2x lo-’ mM unlabeled thymidine. Each chase period was terminated by culturing cells in 1x 10e6M colcemid for 1 h. Mitotically arreited cells were collected, fixed and mounted onto glass slides. The slides were dipped in NTB-2 emulsion (Kodak) which had been diluted with an equal volume of distilled water, and were air-dried. After exposure for 3 days the slides were developed, and the frequency of labelled metaphase plates at each time point was surveyed. To determine the doubling time, cells were initially seeded as described in the preceding section, and at 12 h intervals were collected by trypsinization and counted in a hemocytometer.

Isolation

of labelled satellite I DNA

The isolation of [3H]TdR-labelled satellite I DNA from a synchronous population of cells began with a differential lysis of cells on the tissue culture plate. Media was first removed and the cells washed with three changes of Earle’s balanced salt solution. This and all subsequent steps were performed at 4°C. About 25 ml of a lysis buffer (0.25 M sucrose, 20 mM tri-

Bovine satellite DNA replication 100 60I.0) 0 = 60,P al 73 $40

_) s

*ON .4

6 Time

a

in

1”

12

14

hours

Fi.g. 2. The minimum time in 1 mM hydroxyurea to-arrest cells at the Gl/S border. Following growth for 39 h in isoleucine-free media, at ‘zero-time’ on the y-axis, control cells (A) on coverslips were released into media, and experimental cells (0) on coverslips were released into 1 mM hydroxyurea in n&ma1 media. One hour before each time-point experimental cells were washed in normal media to remove hydroxyurea, and then both control and experimental cells were incubated in media containing rH]TdR for 1 h. The coverslips were immersed in two changes of methanol-acetic acid (3: 1) and two changes of methanol, air-dried and prepared for autoradiography as described in Materials and Methods.

ethanolamine, 25 mM NaCl, 5 mM MgClp, 1% Nonidet P-40, pH 8.0) was pipetted onto each plate. A few minutes following the lysis of the cell membrane, the buffer with ruptured cell membranes and liberated cytoplasm was removed, but nuclei remained attached to the dish [24]. Nuclei were collected from the plates by washing with 0.14 M NaCl, 0.01 M Tris-HCI, 0.01 M EDTA, pH 8.0, pelleted by centrifugation and resuspended in a small volume of the same buffer. To optimize the yield of high molecular weight DNA, the DNA from nuclei was isolated by a complex sucrose/CsCl gradient [25], with a modified sucrose nuclear lysis buffer of 3% sucrose, 3% Sarkosyl, 3% sodium deoxycholate, 2% SDS in 10 mM CAPS buffer (Sigma Biochemicals), pH 10.4; centrifugation was carried out in a Beckman SW27 rotor for 6 h at 25 000 rpm at 18°C. Aliquots of the extracted DNA were adjusted to a density of 1.715 g/ml CsCl and centrifuged at 31000 rpm for 60 h in a Beckman 40.3 rotor. Enhanced senaration of satellite I DNA from main band DNA, probably due to a ‘relaxation’ effect [26], was obtained if gradients were allowed to remain unfractionated at room temperature for 24 h after the first equilibrium centrifugation, and were re-run at 31000 rpm for an additional 12 h and then fractionated. Fractions containing satellite I DNA were pooled and the purity with respect to main band contamination was assayed by analytical ultracentrifugation at 39460 rpm for 18 h in a Beckman Model E

49

ultracentrifuge, using Micrococcus lysodeikticus DNA as a marker (ecsc,= 1.731 g/cm”), and also by gel electrophoresis in 1% agarose (70 y for 6 h in 0.04 M Tris, pH 8.0, 0.02 M sodium acetate, Z’mM disodium EDTA, 0.018 M NaCl) after digestion with the restriction enzyme, Eco RI. The specific activity of the Eco RI fragment of satellite I was determined by cutting out the 1400 bp fragment and counting an aliquot of this melted agarose (Sea-Plaque; MarineCohoids Div.) fragment (KKPC, 10 min) in Aquasal II, in a Beckman LS200 liquid scintillation counter. To unambiguously delineate the region containing satellite I DNA in a preparative isolation of satellite I DNA from main band DNA, a marker DNA was used. Satellite I DNA was isolated from calf thymus DNA by a malachite green affinity chromatography column (Boehringer-Mannheim), as previously described by Bunemann & Miiller [27]. ‘The isolated DNA was shown by CsCl analytical ultracentrifugation and restriction digestion to be .highly purified (>95%) satellite I DNA. Aliquots bf this DNA were labelled with a y-[3ZP]ATP by T4 polynucleotide kinase (New England Biolabs) according to the procedures of Maxam & Gilbert [28]. A specific activity ot 1x 106 cpm/pg DNA was routinely obtained.

RESULTS The initiation of bovine satellite I DNA replication was studied in synchronized cells. Before synchronizing cells, it was first necessary to determine the length of each phase of the cell cycle. This was done by following the percentage of labelled metaphase plates at various times after labelling [22]. The cell doubling time was observed by counting in a hemocytometer to be 15.6 h. Fig. 1 shows the percentage of labelled mitoses at various times after labelling. Under these labelling conditions, G2 and S were shown to be 2.2 and 8 h respectively. M was determined to be 1 h by observing the length of time that cells took to divide once individual cells had rounded up and lifted off the plate to undergo mitosis. Gl was determined to be 4.4 h by adding the times of M, G2, and S and subtracting the sum from the doubling time. The synchronization protocol involved the exposure of cells first to isoleucine-free media to arrest cells in Gl, followed by exposure to hydroxyurea to arrest cells EXP Cd Res 140 f 1982)

Matsumoto and Gerbi

0 o

Fig. 3. Isolation of calf thymus satellite I DNA by malachite green afftnity column chromatography (0.9~ 15 cm). 1.0 mg purified calf thymus DNA was loaded onto the bottom of the column and eluted from bottom to top by pumping with a 40 ml O-O.8M sodium perchlorate gradient in 10 mM sodium phosphate buffer, pH 6.0, followed by an equal volume of 2.0 M sodium perchlorate. The flow rate was 0.5 ml/mm and 1 ml fractions were collected. The pooled area indicated by the arrows was assayed for satellite I DNA purity (see fig. 4).

0.200

0.100

10

20

30

40 Fraction

50

60

at the GllS border [19]. After 44-46 h in isoleucine-free media, cells exhibited morphological changes (e.g., vacuolated appearance) which later led to cell death. To insure that these morphological changes would not occur, a 39-h exposure time to isoleucine-deficient media was selected. Fig. 2 presents the results of studies to de-

a

aat

I

10

00

Number

termine the minimum length of time in hydroxyurea necessary to arrest cells at the Gl/S border. Under the conditions of isoleucine deprivation and exposure to hydroxyurea for 13 h, 99 % of cells enter S phase within 1 h of removal of hydroxyurea. In the absence of hydroxyurea as a synchronizing agent, only 60% of control

pBR322

2483

1400 1108 772

Lkt.In Lsat.llit* I band

Y. DNA

Fig. 4. Assay for purity of calf thymus satellite I DNA isolated from malachite green column. (a) Gel electrophoresis after Eco RI digestion of this DNA. The plasmid pBR322 cut with Hint II+Ava I was used as a marker. The Eco RI fragment of satellite I DNA Exp Cd Res 140 (1982)

lysodaiktlcus

oonponentr

(omlem3)

is 1400 bp in length. (b) tion in CsCl of an aliquot lysodeikticus DNA &cl= a marker. The satellite I cmS.

Analytical ultracentrifugaof this DNA. Micrococcus 1.731 Jcm3) is included as peak has a pcac,=1.715 g/

Bovine satellite DNA replication

Fig. 5. CsCl equilibrium centrifugation of MDBK cell DNA pulse-labelled with [3H]TdR after 1.5 h into S phase. A 3ZP-satellite I marker was used to delineate the region of [3H]TdR-replicating satellite DNA in the gradient. The satellite I-containing region (A) and the main band region (B) were pooled as indicated by the arrows. The specific activities of the DNAs in these regions were determined and are presented in table 1.

cells are able to enter S phase synchronously. In addition, an equivalent frequency of labelled nuclei is achieved earlier in hydroxyurea-treated vs control cells as evidenced by the leftward shift of the hydroxyurea curve in fig. 2. This effect of hydroxyurea has previously been observed in CHO cells by Hamlin & Pardee [21]. To determine the interval of time during which satellite DNA replication is initiated, it was necessary to isolate satellite I DNA. To unambiguously identify fractions from a neutral CsCl gradient containing satellite

51

I, labelled satellite I DNA was used as a marker. This marker was obtained by malachite green affinity chromatography (fig. 3). Aliquots of the pooled satellite I fractions were assayed for purity by two methods. As shown in fig. 4a, when this DNA was digested with Eco RI a single band (1400 bp) characteristic of satellite I DNA was generated. In fig. 4b, the isolated satellite showed a density in CsCl of 1.715 g/cm3 which is typical of calf satellite I DNA. In addition, fig. 4b clearly shows that the malachite green purified satellite I DNA is free from main band contamination; the latter would have a ecscl=1.697 g/cm3. An aliquot of this purified satellite DNA was end-labelled with y-[82P]ATP by T, polynucleotide kinase, and used as a density marker. We studied replication of satellite I DNA by using the synchronization regime of 39 h in isoleucine-free media and 13 h in hydroxyurea, after which cells taken at halfhour intervals since the beginning of S were pulse-labelled with 1 &i/ml r3H]TdR for 30 min or 15 pCi/ml [32P]orthophosphate for 5 min. DNA which had been purified from lysed nuclei on a complex sucrose/CsCl gradient was adjusted to a CsCl density of 1.715 g/cm3 and centrifuged with an aliquot of the purified 32P-satellite I marker DNA.

Table 1. Specific activities of satellite I and main band DNA during the first 3 hours of S phase ‘Specific activity ([3H]TdR cpm/pg) Time into S phase (hours)

Pooled satellite I region from CsCl gradient

Pooled main band region

Satellite I as Eco RI fragment

Bulk DNA prior to loading gel

0.5 1.0

417 2 705

910 3 029

1 704 3 523

4 301 11 535

::i

43 278 041

43 819 707

44466 352

16 561 15 013

;:;

74408 383

5008 8 835

78 952 076

13 17 750 174 Exp CdRes

140 (1982)

52

Matsumoto and Gerbi

345676

12

2453 2150

070 772

Fig.

6. Autoradioaranhic detection of initiation of satellite I DNA re$ication. DNA from pulse-labelled cells (see Materials and Methods) was digested with Eco RI; equivalent amounts of DNA were ioaded onto a 1.5% agarose gel and electrophoresed. The ethidium bromide-stained profile of the marker, plasmid pBR322 cut with Hint II and Ava I and mixed with pBR322

cut with Hinf I, is shown in lane I; the ethidium bromide stained profile of calf DNA in lane 2. 3-8, Autoradiographically detected pulse-labelled calf DNA profiles from 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 h into S respectively; the exposure was 5 days with an intensifying screen.

Fractions containing the 32P-marker DNA, and hence also [3H]TdR-replicating satellite I DNA were collected; an example of such a gradient is shown in fig. 5. The specific activities of the pooled regions of satellite I and main band DNAs indicated in fig. 5 were determined and are summarized in table 1. These data suggest that satellite I DNA replication may be initiated early in S phase. However, as it was possible that this result was due to main band contamination, a more precise assay of the specific activity of satellite I DNA was accomplished by determining the specific activity of the Eco RI fragment of this DNA after gel electrophoresis. These data are summarized in table 1, and concur that satellite I DNA replication begins early in S phase, like bulk DNA. This quantitative result can

be visualized qualitatively as shown in fig. 6. In this case, DNA from [32P]orthophosphoric acid pulse-labelled cells was digested with Eco RI and electrophoresed in a 1.5 % agarose gel. The resulting autoradiograms (fig. 6) show that the replication of the 1400 bp Eco RI fragment of satellite I was first detectable by label incorporation at 1.5 h into S phase. In order to confirm that the labelled 1400 bp Eco RI band was indeed derived from satellite I, this band was recovered, digested with Eco RII, and rerun on another gel. As shown in fig. 7, all the labelled bands of this secondary digest coincided with the expected sizes known from DNA sequence studies of satellite I. These observations support the results presented in table 1 that the initiation of satellite I replication can be detected as early as 14 h

Exp CdRes

140 f/982)

Bovine satellite DNA replication

310 243

141 133

53

matin is generally found to be late-replicating [I], and satellite I DNA is heterochromatic [7J. For example, mouse satellite DNA is replicated during the second half of the S phase [23, 29, 301. However, low levels of mouse satellite DNA synthesis have also been reported for early S phase [29]. Also, satellite DNA from Dipodomus or&i is late-replicating [31], yet Bostock et al. also detected low level D. or&i satellite DNA synthesis early in S, but discounted this finding [31]. Further, in three different kangaroo rat species, satellite DNAs are replicated throughout S phase, and include three cases where satellites have maximal rates of synthesis early in S

[321. Although the majority of a given satellite may replicate primarily late in S, it is 61 possible that it may initiate replication early in S phase, and hold these initiated seFig. 7. Autoradiographic detection of satellite I DNA quences for elongation and completion of replication. DNA from pulse-labelled cells (see Materials and Methods) was digested with Eco RI; the replication until near the end of the S phase. 1400 bp Eco RI fragment was recovered from a gel, The regulation of this temporally arrested recut with the enzyme Eco RII, and electrophoresed eye form and its later elongation may be in a 6% acrylamide gel. I is the ethidium bromidestained profile of the same marker as in fig. 6; 2 con- similar to the accumulation of mitochontains the ethidium bromide-stained Eco RI1 fragments drial D-loop DNA and its expansion at a derived from the parental 1400 bp Eco RI fragment. 3, autoradiographically detected labelled DNA of the later time [33]. Alternatively, satellite replimaterial run in 2. The sizes given for the Eco RI1 fragments agree with those exactly determined by cons may not behave synchronously; for sequence analysis of satellite I [34-361. example, some satellite replicons may initiate early in S phase and proceed to complete replication, whereas other replicons into the S phase of synchronized bovine may initiate and complete replication late in S phase. We are continuing our studies to cells. determine which of these two alternative possibilities is correct. DISCUSSION The results presented here from specific activities of DNA from CsCl gradients and in gels after Eco RI restriction indicate that in a highly synchronized population of cells the initiation of bovine satellite I DNA replication can be detected in early S phase. This is perhaps surprising, as heterochro-

This research was supported by ACS grants NP-227 and CD-76. We thank Carol King for typing this paper and Norma Messier for assistance with the cell culture.

REFERENCES 1. Lima-de-Faria, A & Jaworska, H, Nature 217 (1%8) 138. 2. Kriegstein, H J & Hogness, D S, Proc natl acad sci us 71(1974)135. Exp Cell Rcs 140 (1982)

54

Matsumoto and Gerbi

3. Burks, D J & Stambrook, P J, J cell biol77 (1978) 762. 4. Baldari, C T, Amaldi, F & Buongiomo-Nardelli, M, Cell 15 (1978) 1095. 5. Kurek, M P, Bill&, D & Stambrook, P J, J cell biol 81 (1979) 698. 6. Filipski, J, Thiery, J & Bernardi, G, J mol biol 80 (1973) 177. 7. Kurnit, D M, Shafit, B R & Maio, J J, J mol biol81 (1973) 273. 8. Botchan, M R, Nature 251 (1974) 288. 9. Mowbray, S L, Gerbi, S A & Landy, A, Nature 253 (1975) 367. 10. Philippsen, P, Streeck, R E & Zachau, H G, Eur j biochem 57 (1975) 55. 11. Roizes, G, Nucl acids res 3 (1976) 2677. 12. Gautier, F, Mayer, H & Goebel, W, Molec gen genet 149 (1976) 23. 13. Gautier, F, Biinemann, H & Grotjan, L, Eur j biochem 80 (1977) 175. 14. Kopecka, H, Macaya, G, Cortadas, J, Thiery, J P & Bernardi, G, Eur j biochem 84 (1978) 189. 15. Hori, T A & Lark, K G, J mol biol88 (1974) 221. 16. - Nature 259 (1976) 504. 17. Madin, S H & Darby, N B, Proc sot exp biol med 98 (1958) 574. 18. Ley, K D & Tobey, R A, J cell biol 47 (1970) 453. 19. Tobey, R A & Crissman, H A, Exp cell res 72 (1972) 460. 20. Parris, D S, Bates, R C & Stout, E R, Exp cell res 96 (1975) 422. 21. Hamlin, J L & Pardee, A L, Exp cell res 100(1976) 265.

Exp Cell Res 140 f/982)

22. Bostock, C J & Prescott, D M, J mol biol60 (1971) 151. 23. - Exp cell res 64 (1971) 267. 24. Buetti, E, J viral 14 (1974) 249. 25. Kaback, D B, Angerer, L M & Davidson, N, Nucl acids res 6 (1979) 2499. 26. Anet, R & Strayer, D R, Biochem biophys res commun 34 (1969) 328. 27. Bunemann, H & Mtiller, W, Nucl acids res 5 (1978) 1059. 28. Maxam, A M & Gilbert, W, Methods in enzymology (ed L Grossman & K Moldave) vol. 65, p. 259. Academic Press, New York (1981). 29. Tobia, A M, Schildkraut, C L & Maio, J J, J mol biolS4 (1970) 499. 30. Flamm, W G, Bernheim, N J & Brubaker, P E, Exp cell res 64 (1971) 97. 31. Bostock, C J, Prescott, D M & Hatch, F T, Exp cell res 74 (1972) 487. 32. Restock, C J, Christie, S, Lauder, I J, Hatch, F T & Mazrimas, J A, J mol biol 108(1976) 417. 33. Kasamatsu. H & Vinoerad. J. Ann rev biochem 43 (1974) 695.’ ’ 34. Taparowsky, E J & Gerbi, S A, Nucl acids res 10 (1982) 1271. 35. Gaillard, C, Doly, J, Cortadas, J & Bemardi, G, Nucl acids res 9 (1981) 6069. 36. Plucienniczak, A, Skowronski, J & Jaworski, J, J mol biol (1982). In press. Received October 8, 1981 Revised version received January 11, 1982 Accepted January 14, 1982

Printed

in Sweden