Maturation of newly replicated chromatin of simian virus 40 and its host cell

J. Mol. Biol. (1980) 136, 359-374

Maturation

of Newly Replicated Chromatin and its Host Cell

of Simian Virus 40

KARL-HEINZ KLEMPNAUER,ELLEN FANNING BERND OTTO AND ROLFKN~PERS Fachbereich Biologie, D-7750 Konstanz,

Konstanz West Germany

Universitiit

(Received 23 July 1979, a& in revised

form

22 October 1979)

The DNA in replicating simian virus 40 chromatin and cellular chromatin was labeled with short pulses of r3H]thymidine. The structure of pulse-labeled nucleoprotein complexes was studied by micrococcal nuclease digestion. It was found that in both newly replicated viral and cellular chromatin, a structural state appears which is characterized by an increased sensitivity to nuclease and a faster than usual rate of cleavage to DNA fragments of monomeric nucleosome size and smaller. Pulse-chase experiments show that each of these effects requires a characteristic time to disappear in both systems, suggesting the existence of different sub-processes of chromatin maturation. One of these processes, detectable by the reversion of the unusually fast production of subnucleosomal fragments, is delayed in SV40 chromatin replication.

1. Introduction DNA in eukaryotic cell nuclei exists as a nucleoprotein complex, chromatin, which consists of repeating units called nucleosomes (Kornberg, 1977; Felsenfeld, 1978). The replication of the chromatin structure is of considerable interest to understand the molecular mechanisms by which the genetic material of the cell is duplicated and to evaluate possible mechanisms of transmission of information that may reside in the structure of chromatin itself (Tsanev & Sendov, 1971; Weintraub et al., 1977). Numerous reports (reviewed by Sheinin et al., 1978; Cremisi, 1979) indicate that chromatin containing newly replicated DNA transiently acquires a structure which differs from the structure of bulk chromatin. Most of the data are in accord with the notion that the nucleosomes do not leave the DNA during replication (Hewish, 1976; Seale, 1976,1978; Leffak et al., 1977), implying that the repeat structure of chromatin presents no great hindrance to the replication machinery, but that structural alterations, whose molecular nature is only poorly understood, occur in newly replicated chromatin . The DNA of simian virus 40 (SV40) exists in infected monkey cells as a nucleoprotein complex (White & Eason, 1971; Green et al., 1971) whose replication depends totally on the cellular replication apparatus except for the viral gene A product (T-antigen) which is required for the initiation of replication (Tegtmeyer, 1972). The architecture of the viral nucleoprotein complexes appears to be very similar to that 359

0022-2836/80/040359-16 $02.00/O

0 1980Academic PressInc.(London)Ltd.

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360

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AL.

of cellular chromatin (Hall et al.. 1973: Griffit,h. 1975: Meinke et al., 1975; Bellard et al., 1976; Cremisi Pt ul., 1976: Varshavsky d al., 1976; Miiller et al.. 1978; Shelton et al., 1978). SV40 DNA rrplicatjion is a convenient, model s,ystem for t,he study of cellular DNA replication since viral nucleoprotcin complexes offer several advantages. Replicating SV40 nucleoprotein complexes can be considered to be single isolated replicons whose relatively small size allows experimental manipulation without, much danger of breakage. Moreover. replicating SV40 nucleoprotein can be easily separat)ed by zone velocity sedimentation from non-replicating nucleoprotein, To evaluate the usefulness of viral nucleoprotein as a model system for the analysis of chromatin replication, we investigated some properties of newly replicated viral and cellular chromatin using digestion by micrococcal nuclease to explore the structural state of chromatin. We found that chromatin containing nascent DNA is transiently more accessible t,o nuclease at,tack. Newly replicat)ed DNA was more quickly converted to acid-soluble products and accumulated more rapidly in monomeric and shorter DNA fragments than DNA from non-replicating chromatin. These alterations, observed in both viral and cellular chromatin, arc revcrt,ed at, characteristic times after replication in bot,h systems.

2. Materials and Methods (a.)Cells and virus CV-1-P cells were grown at 37°C in a 5y0 COz atmosphere in 145 mm diameter dishes in Dulbecco’s modified Eagle’s medium (DME, Gibco) with lOoi0 calf serum The preparation and storage of SV40, &rain 777 (Gerber, 1962), has been (Baumgartner et al., 1979). (b)

Labeling

and

preparation

qf viral

nucleoprotein

plastic (Flow).

described

complexes

CV-1 cells grown to confluency were infected with 1 to 5 plaque-forming units of SV40 per cell. In most experiments the infected cells were labeled with 0.1 &i [14C]thymidine/ml in DME between 24 and 36 h after infection. Infected cells were pulselabeled at 36 h after infection with 0.1 mCi [3H]thymidino/ml in DIME. During pulselabeling the culture dishes were floated on a 37°C water bath. Labeling was stopped by either of the following methods. The cells were washed 3 times with ice-cold Hanks’ balanced salt solution or, in pulse-chase experiments, were washed once with prewarmed DME containing 20 @-unlabeled thymidine and incubated for an additional period in that medium in an incubator at 37°C. At the end of the chase period, the cells were washed 3 times with ice-cold Hanks’ balanced salt solution. To prepare viral nucleoprotein complexes, the cells were washed once with hypotonic buffer (10 mw-Tris.HCl, pH 7.8, 1 mM EDTA), scraped off the plate and collected in 2 ml/plate of hypotonic buffer. After swelling for about 5 min, the cells were broken by 8 strokes with a tight-fitting Dounce homogenizer. The nuclei were pelleted (5 min at 3000 g), resuspended in 0.1 ml/plate of hypotonic buffer and kept at 0°C for 1 to 2 h. The nuclei were then pelleted as above. About 30 to 50% of the viral DNA present in the cells was recovered in the nuclear eluate, though only 15 to 20% of replicating DNA was extracted as reported previously (Baumgartner et al., 1979; Seidman & Salzman, 1979). This fraction of eluted DNA seems to be representative, however, since the distribution of denatured DNA species in alkaline sucrose gradients was identical to that of the DNA in a Hirt supernatant. The viral nucleoprotein complexes were routinely analyzed by zone velocity sedimentation in linear neutral sucrose density gradients (5% to 30% sucrose in 10 mM-Tris.HCl, pH 7.8, 1 mm-EDTA) for 1 h at 45,000 revs/min and 4°C in the Sorvall AH 650 rotor. When an analysis of the alkaline denaturation products was required, the sample was centrifuged through a linear 5% to 20% sucrose density gradient in O-3 M-NaGH, 0.7 M-N&~, 2.5 m&f-

CHROMATIN EDTA and rotor or for

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361

0.015% Sarcosyl for 1.5 h at 49,000 revs/min and 4°C in the 12 h at 40,000 revs/min and 4°C in the SW 41 rotor.

(c) Nucleolytic

cleavage of SV40 nucleoproteia

Sorvall

AH

650

complexes

Micrococcal nuclease (EC 3.1.4.7.) (Boehringer, Mannheim) was added either direct,ly to the nuclear eluate containing the SV40 nucleoprotein complexes or, in some experiments, after dilution of the eluate with “incubation buffer” (10 mivf-Tris-HCl, pH 7.8, 1 mMEDTA, 0.2 mg bovine serum albumin/ml, 10 mnf-2-mercaptoethanol and 10% sucrose).

The mixture

was transferred

t,o a 37°C water

bath and the reaction

started

by addition

of CaCl, to final concentration of 2 mM. After the desired incubation times, the reaction was stopped by addition of 10 mm-EDTA (final concn). The amount of acid-precipitablr radioactivity was determined in portions taken from t,he mixture and precipitated by 100/b trichloroacetic acid. The precipitate was collected by cent)rifugation (5 min, 10,000 rcvs/min), dissolved in 0.05 ml NCS (Amersham/Senrlc) and counted in a tolucne-based scintillation mixture.

(d) Nuclease treatment of nuclei from uninfected Subconfluent CV-1 nuclei from uninfected with hypotonic buffer nuclei/ml. Incubation eluatcs.

cells were labeled as described cells were prepared essentially and resuspended in this buffer with micrococcal nuclease was

(c) Agarose gel electrophoresis

of

cells

above for SV40-infected cells. The as described above, washed once at a concentration of about 1 x lo7 performed as described for nuclear

DNA fragments

Nuclease digestion products to be analyzed by gel electrophoresis were deproteinized by incubation with 0.5 mg nuclease-free Pronase/ml (Schwarz/Mann) at 37°C for 30 min. After addition of sodium dodecyl sulfate (0.5% final concn), the sample was extracted several times with Tris-buffered phenol, twice with chloroform and then precipitated with ethanol. The precipitate was dissolved in Tris/EDTA buffer and incubated for 30 min at 37°C in the presence of 50 pg RNAase A/ml which had been preincubated at 80°C for 10 min. The DNA was extracted again with phenol and precipitated with ethanol. For electrophoresis the DNA was dissolved in a buffer containing 10 mM-Tris.HCl, pH 7.8, 1 mM-EDTA, 10% sucrose and 0.1% sodium dodecyl sulfate and loaded directly on to l-7:7/, agarose slab gels. Electrophoresis was carried out at 10 mA for 10 to 12 h at 4°C. The electrophoresis buffer contained 40 mnn-Tris*HCl, pH 7.8, 5 mMa-sodium acetate and 1 mM-EDTA. After the run the gels were stained with ethidium bromide and photographed. The distribution of radioactive DNA fragments throughout the gel was determined in 2-mm slices, which were incubated in O-1 ml 0.1 N-HCl at 100°C to dissolve the agarose.

The radioactivity Aquasol-

(New

was measured England

Nuclear)

by liquid to each

scintillation

counting

after addition

of 1 ml

slice.

3. Results (a) Forms

of SV40

nuckoprotein

complexes

Two forms of SV40 nucleoprotein complexes were recovered by a hypotonic extraction procedure from nuclei of virus-infected cells: replicating complexes that contained replicative intermediates of SV40 DNA and non-replicating complexes that contained closed circular supercoiled SV40 DNA (form I) (White & Eason, 1971; Green et al., 1971). These two forms were labeled selectively by growing virusinfected monkey cells from 24 to 36 hours after infection in the presence of [‘“Cjthymidine and then pulse-labeling for three minutes with [3H]thymidine. Nuclear eluates prepared from these cells were analyzed by zone velocity sedimentation in neutral or alkaline sucrose density gradients (Fig. 1). The sedimentation coefficients

K.-H.

BLEMPNAUER

h’T

Al,.

(b)

To Fraction

number

FIG. 1. Zone velocity sedimentation of SV40 nucleoprotein complexes in neutral and alkaline sucrose density gradients. (a) SV40-infected CV-1 cells were prelabeled with [iW]thymidine and then pulse-labeled for 3 min with [3H]thymidine as described in Materials and Methods. A portion of the nuclear eluate prepared from these cells was sedimented through a neutral sucrose density gradient as described. The gradients were fractionated from the bottom and each fraction was assayed for acid-precipitable radioactivity. The arrow indicates the sedimentation of T7 DNA in a parallel gradient (34 S). (0) sH cts/min x 103; (0) r4C cts/min x 102. (b) Nuclear eluates prepared from SV40-infected CV-1 cells, pulse-labeled for 3 min (0) or for 30 s (A) with [sH]thymidine, were sedimented through alkaline sucrose density gradients as described. The arrow indicates the position of SV40 form II DNA in a parallel gradient. [iW]thymidine continuously labeled viral DNA sedimented under these conditions to the bottom of the centrifuge tube (not shown), as expected for supercoiled circular DNA.

were approximately 90 to 95 S for the replicating and about 75 S for the non-replicating complexes in neutral sucrose gradients. Most of the pulse-labeled material sedimented in alkaline sucrose gradients faster than 10 S; only a very small fraction of the newly replicated DNA appeared to be in the form of 4 to 5 S Okazaki fragments (Fig. l(b)). (b) Degmdation

kinetics

of viral and cellular

chromutin

As a first approach to obtain information on the structure of pulse-labeled chromatin, nuclear eluates prepared from doubly labeled SV40.infected monkey cells were treated with micrococcal nuolease. After various times of incubation, the amount of radioactivity remaining acid-precipitable was determined. Similarly,

CHROMATIN

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363

nuclei from uninfected cells, labeled continuously for 12 hours with [14C]thymidine and pulse-labeled for three minutes with r3H]thymidine, were also digested with micrococcal nuclease. As shown in Figure 2, the [3H]thymidine-pulse-labeled DNA was more sensitive to nuclease digestion than the [14C]thymidine-labeled DNA, both in viral and cellular chromatin. The form of the digestion curves, however, differed significantly between viral and cellular chromatin under many different experiment,al conditions. The degradation of viral DNA in nucleoprotein complexes was eventually almost complete. In contrast,

I

I

IO

20

Time

(min)

Fro. 2. Nuclease digestion kinetics of vim1 and cellula;r chromatin. (a) SV40-infected CV-1 cells were labeled with [‘W]thymidine from 24 to 36 h after infection and then pulse-labeled for 3 min with [3H]thymidine. The viral nucleoprotein complexes were prepared from these cells and sedimented through a linear 5% to 30% neutral sucrose density gradient. Fractions containing replicating and non-replicating SV40 nucleoprotein complexes were combined, diluted approximately Z-fold with incubation buffer, to which 30 units of micrococcal nuclease had been added, to yield a final volume of 4 ml. This mixture was then distributed in 0.1 -ml portions and digested as described in Materials and Methods. The time point at zero was determined in samples before addition of C&l, to start the digestion and the data were plotted as a percent++ of the zero time value. Each point represents the average of 4 digestions. (b) Subconfluent CV-1 cells were labeled with [‘Wlthymidine and [sH]thymidine as described in Materials and Methods. About 1 x lo6 nuclei from these cells were suspended in 4 ml of incubation buffer containing 30 units of micrococcal nucleate and digested as in (a). 3H label (0); 14C label (0). In control experiments nucleate was omitted from the reaction mixture: 3H label ( n ); r*C label (0).

364

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ET

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the degradation of cellular chromatin proceeded at a much reduced rate when about 50% of the continuously labeled DNA and about 75% of the pulse-labeled DNA had become acid-soluble, in agreement with published data (Seale, 1975,1976; Hildebrand & Walters, 1976; Levy &
fractionation of nuclease digestion cellular chromatin

products

of viral and

Further evidence for structural differences between pulse-labeled and continuously labeled chromatin was obtained from examination of the micrococcal nuclease digestion products at various stages of the digestion. When nuclear eluates prepared from doubly labeled SV40-infected monkey cells were digested, the continuously labeled DNA was cleaved to yield a typical band pattern of mono-, di-, tri-nucleosomal etc. DNA fragments and finally accumulated in mononucleosomal DNA pieces. The behavior of pulse-labeled SV40 chromatin, however, differed in several ways. First, [3H]thymidine-pulse-labeled DNA of monomeric length was generated faster than [14C]thymidine-labeled DNA of the same size. Second, upon prolonged digestion, [3H]thymidine-labeled DNA fragments appeared as small “subnucleosomal” DNA fragments when most of the [14C]thymidine-labeled DNA pieces were still of monomeric or oligomeric size (Fig. 3). While this trimming of nucleosome fragments does occur to a small extent in continuously labeled material, it is obvious that trimming proceeds at a much faster rate in chromatin containing pulse-labeled DNA (Fig. 3(c)). Examination of the digestion products from cellular chromatin indicates that, as for viral chromatin, DNA of monomeric length is generated more quickly from three minute pulse-labeled than from uniformly labeled chromatin (Fig. 4). However, in contrast to the results obtained with SV40 chromatin (Fig. 3), no extra peak of faster migrating pulse-labeled DNA was detected at any stage of the digestion. At least two interpretations of these results are conceivable: either the structural feature in pulselabeled SV40 chromatin which gives rise to the fast production of small “subnucleosomal” DNA fragments never appears during DNA replication in uninfected monkey cells or, alternatively, such a structure exists but is much more transient during cellular than during viral DNA replication. To distinguish between these possibilities, uninfected cells, prelabeled for 12 hours with [14C]thymidine, were pulse-labeled for only 40 seconds with [3H]thymidine. Nuclease digestion of the nuclei from these cells followed by analysis of the digestion products on 1.7 o/0 agarose gels demonstrated that a significant part of the pulse-labeled DNA migrated as an extra peak of small DNA fragments, analogous to that observed with pulse-labeled SV40 chromatin (Fig. 4(c)). Since some of the pulse-labeled DNA after a 40 second pulse would be expected to appear in Okazaki fragments, it is conceivable that Okazaki fragments

CRROMATIN

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365

1604 I

a)

5 Distance

migrated

(cm)

FIG. 3. Agarose gel electrophoresis of nuclease digestion products from viral chromatin. Double labeled SV40 chromatin was prepared from infected CV-1 cells and digested without further dilution at a concentration of 125 units of micrococcal nuclease/ml for 1 min (a), 5 min (b) and 15 min (c) at 37°C. The DNA was extracted from the digestion mixture and fractionated on 1.7% agarose gels as described in Materials and Methods. The ethidium bromide-stained Ha&II restriction fragments of SV40 DNA, used to calibrate the gel are shown at the top of the Figure. The numbers indicate their lengths (in base-pairs) calculated from the published relative lengths of these fragments (Yang et al., 1976) and the length of the SV40 genome (Fiers et al., 1978). The continuously labeled DNA was about 100% (a), 100% (b) and 85% (c) acid-precipitable after digestion, whereas about 100% (a), 95% (b) and 7 1 o/0 (c) of the pulse-labeled DNA remained acid-precipitable. (0) 3H cts/min x 102; (0) r4C cts/min x 11)~.

K.-H.

366

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366

329

ET

A.&.

319

a) l-5

I-0

0.5

b)

3

2

I

5

x .5 E > 2 ” 0

0.4

o-2

Distance

migrated

km)

FIG. 4. Agarose gel electrophoresis of nuclease digestion products from cellular chromatin. (a) and (b) Subconfluent CV-1 cells were labeled for 12 h with [14C]thymidine and then pulselabeled with [JHlthymidine for 3 min. About 0.3 ml of a suspension containing 1 x 10’ nuclei/ml was digested with 30 units of micrococcal nuclease. Portions were removed from the mixture after 1 min (a) and 6 min (b) of incubation at 37°C. The DNA was extracted from each sample and analyzed by electrophoresis on 1.7 yo agarose gels. The continuously labeled DNA was 86% (a)

CHROMATIN

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could be responsible for the fast degradation of pulse-labeled DNA to subnucleosomal fragments. However, this explanation is unlikely since pulse-labeled SV40 chromatin, which contains little or no DNA comparable in size to Okazaki fragments (Fig. l(b)), is also quickly digested to such small DNA fragments (Pig. 3). (d) Reversal of the differences between pulse-labeled and continuously labeled viral chromtin In the experiments described above, we have operationally defined three characteristics of pulse-labeled chromatin (the increased nuclease sensitivity, the faster rate of cleavage of DNA to monomeric fragments and the accumulation of significant amounts of small subnucleosomal DNA fragments) which reflect structural differences between pulse-labeled and continuously labeled viral and cellular chromatin. In an attempt to correlate these operationally defined differences with t,he replicative state of the chromatin, pulse-chase experiments were carried out. Cultures of virusinfected cells labeled from 24 to 36 hours after infection with [i4C]thymidine were pulse-labeled for three minutes with [3H]thynlidine followed by an additional incubation of 5 to 25 minutes in medium containing excess unlabeled thymidine (“chase”). Analysis of the nuclear eluates prepared from these cultures by zone velocity sedimentation in alkaline sucrose gradients showed that pulse-labeled replicative intermediate forms of SV40 DNA gradually matured to form I DNA during incubation in the presence of unlabeled thymidine. The amount of [3H]thymidine-labeled form I DNA depended linearly on the length of the chase up to 25 minutes when all pulse-labeled DNA sedimented as form I DNA (Table 1). The nuclease sensitivity of nuclear eluates prepared from cultures of virus-infected cells labeled with [14C]thymidine and [3H]thymidine and chased for either 5, 10 or 25 minutes as described above is summarized in Table 1. This experiment demonstrates that the nuclease sensitivity of pulse-labeled chromatin was restored to the same level as that of continuously labeled chromatin in roughly the same time period needed to complete replication. Similar results were obtained when the nuclease digestion products of such SV40 chromatin preparations were analyzed on 1.7% agarose gels (Fig. 5). A chase period of about 25 minutes was required to obtain the same cleavage pattern of DNA from pulse-labeled and from continuously labeled chromatin. In contrast, a chase period of only five minutes was sufficient to suppress an enhanced accumulation of small subnucleosomal DNA fragments.

and 73% (b) acid-precipitable after the digestion, whereas about 80% (a) and 68% (b) of the pulselabeled DNA remained acid-precipiteble. (c) About 5 x lo5 nuclei from cells labeled for 12 h with [‘*C]thymidine and about 6 x 10s nuclei from cells pulse-labeled for 40 s with O-1 mCi of [3H]thymidine/ml were mixed and digested at a concentration of 1 x 1Or nuclei/ml in the presence of 75 units of micrococcal nuclease for 3 min at 37°C. The DNA was extracted from this mixture and investigated by electrophoresis on a 1.7% agarose gel. 3H label (a); l*C label (0). Calibration of the gels was performed using Hoe111 restriction fragments of SV40 DNA. Their positions and lengths (in base-pairs) are given on top of the graph (cf. Fig. 3). DNA fragments from micrococcal nuclease-treated CV-1 nuclei continuously labeled with [3H]thymidine were run in parallel slots of the gel. Fluorograms of DNA fragments prepared after digestion of nuclei by micrococcal nuclease to 51% (a) and 85% (b) acid-precipitable radioactivity are shown at the top of the Figure.

368

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KLEMPNAUEH TABLE

N&ease

Time of chase? (mm)

0

5 10

25

sensitivity

Percentage of SV40 DNA in form I$

3 22 29 87

ET

AL.

1

of pulse-labeled

and chased XV40

chromatin

Percentage of acid-precipitable radioactivity after treatment with micrococcal nuclease§ 3H label r4C label 10 min 20 min 10 min 20 min digestion digestion digestion digestion

60 67 75 75

45 57 64 58

74 76 79 76

64 64 66 58

t SV40-infected cells, prelabeled with [‘Wlthymidine from 24 to 36 h after infection, were pulse-labeled with [3H]thymidine for 3 min (Materials and Methods) and either used immediately for the preparation of nuclear eluates or chased in medium containing an excess of unlabeled thymidine. $ Nuclear eluates were centrifuged in linear alkaline sucrose gradients for 2 h at 4°C and 49,000 revs/min in the Sorvall AH 650 rotor. The amount of acid-precipitable radioactivity in viral form I DNA was calculated from the area under the corresponding peaks. About 96% of the continuously 14C-labeled DNA sedimented as form I DNA. $0.1 ml of each nuclear eluate was diluted with 0.1 ml incubation buffer containing 120 units of micrococcal nuclease. The mixture was distributed in 0.02 ml portions and digested as described. The numbers given in the Table are averages of 3 independent digestions.

Recent evidence has shown that in the buffer system used here for the preparation of nuclear eluates, virion-like particles decompose to give rise to 75 S nucleoprotein complexes (Garber et al., 1978; Nedospasov et al., 1978; Fernandez-Munoz et al., 1979; Baumgartner et al., 1979). Thus the continuously labeled 75 S complexes described in this study contain significant amounts of these decomposition products. Two lines of evidence suggest that the nucleoprotein structure of these complexes is similar to that of 75 S complexes which have not yet undergone packaging to complete virions. First, the nuclease digestion kinetics and cleavage pattern of continuously labeled SV40 nucleoprotein complexes and the immediate chase products, which have just completed replication and have not yet been converted to virionlike particles (Garber et aE., 1978; Baumgartner et al., 1979), are identical. Second, the kinetics of codigestion of [l*C]thymidine-labeled 75 S nucleoprotein complexes prepared as described in Materials and Methods and [3H]thymidine-labeled 75 S nucleoprotein complexes, prepared under conditions in which virion-like particles do not break down (Baumgartner et al., 1979), are identical (data not shown). Therefore, the possibility that packaging of viral nucleoprotein to complete virions is responsible for any of the described differences in susceptibility to nuclease between pulse-labeled and continuously labeled SV40 chromatin is unlikely. (e) Reversal

of differences

between pulse-labeled chronratin

and continuously

labeled cellular

Pulse-chase experiments were also performed with uninfected monkey cells, using the same labeling and chase program as described above (Table 2; Fig. 6). Both the increased nuclease sensitivity of pulse-labeled chromatin and the faster

CHROMATIN

369

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2.5

Dastonce

migroted

(cm)

FIG. 5. Agarose gel electrophoresis of the nuclease digestion products from pulse-chased SV40 chrometin. SV40-infected CV-1 cells that had been prelabeled with [‘%]thymidine were pulse-labeled with [3H]thymidine for 3 min and then used either immediately ((a) and (b)) or after a chase period of 5 min ((c) and (d)) or 25 min ((e) and (f)) for preparation of nuclear eluates. 0.5 ml of each eluate was diluted with 0.5 ml of hypotonic buffer and digested with a total of 600 units of micrococcal nuclease for 30 s ((a), (c) and (e)) or 3 min ((b), (d) and (f)). The DNA was extracted from each digestion sample and analyzed on 1.7% agerose gels. The gels were calibrated using HaeIII restriction fragments of SV40 DNA whose lengths (in base-pairs) are indicated at the top of the Figure (see Fig. 1). (0) 1% ots/min x 1Oe; (a) 3H cts/min x 103.

rate of appearance of monomeric DNA fragments from pulse-labeled reversed simultaneously within a 15 minute chase period.

chromatin

are

4. Discussion DNA in soluble XV40 nucleoprotein complexes and in nuclei from uninfected host cells, both pulse-labeled and continuously labeled with different radioisotopes in viva, wss digested with micrococcal nuclease to characterize the structural state of newly replicated and non-replicating chromatin.

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TABLE 2 N&ease

sensitivity

of pdse-labeled

and chased cellular

chromatin

Percentage

Time of chase? (min)

0 5 15

of acid-precipitable radioactivity after nuclease digestion$ 3H label 14C label 10 min 20 min 10 min 20 min digestion digestion digestion digestion __-61 63 49 74 62 68 67 64 57 70 58 69

t Subconfluent CV-1 cells, prelabeled for 12 h with [‘Wlthymidine, were pulse-labeled for 3 min with [3H]thymidine. Nuclei were prepared either immediately after the pulse or after incubation in medium with excess thymidine for 5 or 15 min. $ 1 x 1Oe nuclei were suspended in 2 ml of incubation buffer containing 30 units micrococcal nuclear+ O*l-ml portions of this mixture were distributed and incubated under digestion conditions for 10 or 20 min. The percentages of acid-precipitable radioactivity after digestions are averages of 4 independent incubations.

The most obvious difference between newly replicated and non-replicating viral and cellular chromatin is the enhanced rate of digestion of pulse-labeled DNA to acid-soluble material compared to that of continuously labeled DNA (Fig. 2(a) and (b)). Pulse-chase experiments indicate that restoration to mature chromatin gradually takes place in viral chromatin within approximately 25 minutes, and in cellular chromatin within about 15 minutes after the incorporation of pulse-label (Tables 1 and 2). Additional evidence for a structural difference between newly replicated and nonreplicating chromatin was obtained through electrophoretic fractionation of the nucleasedigestion products (Figs 3 and 4). In viral replication, two structural differences between replicating and mature chromatin were distinguished operationally by their different lifetimes asdetermined in pulse-chaseexperiments (Fig. 5). (1) DNA in newly replicated chromatin is cleaved faster to yield fragments of monomeric size, and (2) significant amounts of subnucleosomal DNA pieces appear under conditions when most of the non-replicating DNA is still of monomeric or even oligomeric size. Similar steps in chromatin replication were even more clearly demonstrated in cellular DNA replication since an enhanced production of subnucleosomal DNB piecesfrom chromatin was only detected after a 40 second pulse-label, but not after a three minute label. On the other hand, the faster rate of production of monomeric DNA pieceswas also demonstrated using cellular chromatin labeled for three minutes. A chase period comparable to that for viral chromatin was required to restore the cellular chromatin to its mature structure. Based on these data, both viral and cellular chromatin appear to undergo two different structural transitions which occur at chracteristic times after replication in both systems. It is not possiblefrom the experiments presented to determine whether the fast production of monomeric DNA fragments and the increased sensitivity to nuclease, which both return to the mature state during a chase period of about 25 minutes (viral chromatin) or 15 minutes (cellular chromatin), both reflect the samebasic structural change in newly replicated chromatin.

CHROMATIN

REPLICATION

Distance

migrated

371

km)

FIG. 6. Agarose gel electrophoresis of nuclease digestion products from pulse-chased cellular chromatin. Subconfluent CV-1 cells, prelabeled with [14C]thymidine, were pulse-labeled with [3H]thymidine and then incubated in medium containing excess unlabeled thymidine for 5 or 15 min. Nuclei prepared from these cells were digested at a concentration of 1 x 10’ nuclei/ml and 150 units of micrococcal nuclease/ml for 1 min at 37°C. The DNA was extrected from the samples and subjected to electrophoresis on 1.7% agerose gels as described. (a) 3 min pulse, (b) 3 mm pulse and 5 mm chase, (c) 3 mm pulse and 15 min chase. Calibration of the gel was performed with Hoe111 restriction fragments of SV40 DNA whose lengths (in base-pairs) are indicated at the top of the Figure. (0) r4C ots/min x 1O3; (a) 3H cts/min x 103.

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AL.

An increased sensitivity of newly replicated DNA to nuclease, as well as the fast rate of cleavage of that DNA to fragments of monomeric size, have been observed in different, cell types (Seale, 1975.1976; Weintraub, 1976; Hildebrand & Walters, 1976; Hewish, 1977; Levy & Jakob, 1978; Shelton et al., 1978; Schlaeger & Klempnauer, 1978). Newly replicated DNA in shorter than monomeric fragments has been found in several experimental systems though the actual lengths of these fragments vary somewhat in different systems (Levy & Jakob, 1978; Seale, 1978; Schlaeger & Klempnauer, 1978; Schlaeger & Knippers, 1979). The structural basis of these effects is still not entirely clear. The general increase in nuclease sensitivity of newly replicated DNA (Fig. 2) could conceivably arise from a transient histone deficiency on the newly replicated chromatin, as proposed previously (Seale, 1975). If this interpretation is correct,, it’ can be concluded t,hat a time of 15 to 25 minutes is required to fill out the existing protein-depleted sections on the newly replicated DNA. The rapid production of monomeric DNA fragments from pulse-labeled chromatin may be related in some way to a more exposed linker region between the nucleosomes. It is conceivable that these linkers are covered with less or with modified proteins, including histone Hl, rendering them more susceptible to nuclease. Alternatively, it has been suggested that newly replicated chromatin as a whole acquires a more exposed localization in the nucleus (Hewish, 1977), an unlikely explanation since the nuclease-sensitive st.ructural state of newly replicated chromatin is also exhibited in viral nucleoprotein extracted from the nucleus. Furthermore, enlargement of spacers as the cause for the enhanced rate of monomer production can be ruled out since this would result in a larger nucleosomal repeat length in newly replicated chromatin, which was not observed. The most peculiar difference between newly replicated viral and cellular chromatin lies in the cleavage of three minute pulse-labeled viral but not cellular DNA to fragments of subnucleosomal size. This difference could be account,ed for by an altered conformation of isolated viral chromatin. However, this is unlikely since cellular chromatin exhibits this effect too, although after a shorter labeling period. Alternatively, it may be proposed that some structural features of newly replicated chromatin (i.e. those responsible for the fast appearance of subnucleosomal DNA fragments) are more transient in cellular than in viral chromatin. Since the replication of SV40 DNA, once initiated, depends totally on the cellular replication apparatus, such a difference in mechanism would seem unlikely. The obvious difference between viral and cellular chromatin replication with respect to the fast appearance of subnucleosomal DNA fragments could be related to the relatively small size of the SV40 replicon compared to the average size of cellular replication units (50 to 330 x lo3 base-pairs; Hand, 1978). If we assume that the rate of fork movement in viral chromatin is similar to that observed for mammalian cells (1 to 15 x lo3 base-pairs per min; Sheinin et al., 1978), replication of the viral DNA should be completed within a period shorter than the labeling time employed. The slow formation of SV40 form I DNA depends on time-limiting steps near the end of the viral replication cycle (Mayer & Levine, 1972; Tapper & DePamphilis, 1978; Seidman & Salzman, 1979). As a consequence, viral DNA labeled within a three minute pulse will, on the average, be located nearer to the replication fork than cellular DNA labeled for the same period. If this is related to the fast appearance of viral pulse-labeled sub-

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nucleosomal fragments, it implies that the reversion of some structural traits of newly replicated chromatin might be regulated as a function of distance from the replication fork. In summary, in both SV40 and cellular chromatin replication, a similar structural state appears transiently in newly replicated chromatin. This state is characterized by an increased sensitivity of the newly replicated DNA to nuclease and a faster rate of cleavage of this DNA to fragments of monomeric nucleosome size or smaller. Pulsechase experiments have shown that each of these effects requires a characteristic time to disappear in both systems, suggesting the existence of different subprocesses of chromatin maturation, one of which is delayed in SV40 DNA replication. We t,hank W. Deppert for a stock medium and cells, and E.-J. Schlaeger by t,he Deutschc Forschungsgemeinschaft

of SV40, strain 775, R. Mettke for helpful discussions. This (SFB 138/B4).

for work

preparation was supported

of

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