The sites of deposition of newly synthesized histone.

Volume 9 Number 18 1981

Nucleic Acids Research

Tlhe sites of deposition of newly synthesized histone

Vaughn Jackson, Steve Marshall and Roger Chaildey Department of Biochemistry, University of Iowa, Iowa City, IA 52242, USA Received 12 May 1981 ABSTRACT The chromosomal fragments produced by nuclease digestion of freshly replicated chromatin migrate more rapidly relative to bulk chromatin when analyzed in nucleoprotein gels. The cause of the anomolous migration has been studied and the evidence indicates that rather than reflecting a shorter nucleosomal repeat in vivo that it may be a consequence of nucleosome sliding during the digestion itself. The distinct electrophoretic characteristics of nucleosomal material containing newly replicated DNA have enabled us to examine their histone composition by two dimensional electrophoresis. We find that nucleosomes containing new DNA also contain newly synthesized histones H3 and H4. In contrast more than 50% of newly synthesized H2A and H2B, and essentially all of new Hi, are deposited at sites on the bulk chromatin distinct from that material containing newly replicated DNA. In addition we show that newly synthesized histones H3 and H4 are bound unusually weakly when they first become associated with the chromatin.

INTRODUCTION The bulk of the DNA within a eukaryotic cell has a regular nucleosomal repeating structure (see review 1). The nucleosome contains two copies each of H2A, H2B, H3, H4 associated with 145 base pairs of DNA. The remaining 30-50 base pairs of DNA in the nucleosome are complexed with histone Hi in some fashion (2,3,4). Several attempts have been made to study nucleosomal packaging during eukaryotic DNA replication. There is general agreement that nucleosome-like structures exist on both daughter strands very soon after the replication fork has passed and that these structures are approximately two-fold more sensitive to attack by staphylococcal nuclease (5,6,7,8). These structures have been visualized on both strands near the replication fork by electron microscopy (9). The spacing between nucleosomes of newly replicated DNA, however, appears to be less than that of mature DNA. Bonner has reported that the spacing is 20 base pairs shorter (10,11). Levy and Jakob report that the nucleosomal dimer size is 410 bp for mature DNA and 305 bp for newly replicated sea -

©) IRL Press Umited, 1 Falconberg Court, London W1V 5FG, U.K.

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Nucleic Acids Research urchin chromatin (12). The presence of a significantly decreased nucleosomal repeat in newly replicated chromatin poses several complex problems. Particularly, how does such chromatin rearrange to the normal nucleosomal repeat characteristic of non-replicating chromatin? An area of much investigation and some controversy has been the source of the histones which package the newly replicated DNA. A wide variety of experimental procedures have been applied to this question with different conclusions. Initial studies using formaldehyde crosslinking of newly synthesized histones onto chromatin containing density-labeled, newly replicated DNA, suggested that the new histones become bound to the old DNA (13,14,15). Further studies using ultraviolet light to crosslink new histones to chromatin containing Budr-labeled new DNA suggested that no more than 5-22% of the new histones segregated specifically with new DNA (16). Studies using density labeled DNA in metrizamide density gradients suggested that only H3 and H4 are selectively deposited on new DNA (17). In contrast, studies using selective enrichment for the replicative form of SV40 suggested that all of the non-Hi histones become associated with new DNA (18). Further support for this selective deposition came from studies using density-labeled amino acids. By crosslinking density labeled histones as oligomers and determining the density of the product, the conclusion was drawn by the authors that new histones deposit and stay together, thereby implying deposition occurs on new DNA (19). In this report we have investigated in further detail the spacing of the newly replicated DNA. We conclude that the unusual spacing is produced during the nuclease digestion itself by selective sliding of the nucleosomes on new DNA. By utilizing this altered spacing we are able to partially separate newly replicated from mature chromatin using polyacrylamide gels and to demonstrate selective deposition of newly synthesized H3 and H4 on new DNA, deposition of H2A and H2B on both old and new DNA and no enrichment of Hi on newly synthesized DNA.

MATERIALS AND METHODS Labeling of Cells and Preparation of Nuclei. One liter of Hepatoma Tissue Culture (HTC) cells at mid log (400,000/ml) were harvested by centrifugation at 400 xg for 5 min. For pulses with 3H thymidine the cell pellet was immediately resuspended in 2 mCi of 3H methyl thymidine (Schwarz-Mann, 53 Ci/mmole) at 0.5 mCi/ml in Swimms S-77 without serum and incubated at 370C for the appropriate time. The pulse was terminated by lysing the cells directly in 3 volumes of cold Nuclei Digestion Buffer (NDB) which consists of 0.25 M 4564

Nucleic Acids Research sucrose, 0.06 M KCl, 0.015 M NaCl, 0.005 M MgCl2, 0.001 M CaCl2, 0.01 M MES (2[N-morpholino]ethane sulfonic acid), 0.006 M sodium butyrate, pH 6.5 with Tris Base, plus 0.5% Triton x-100, 1 mM PMSF (20). This mixture was vortexed vigorously for 5 minutes and centrifuged at 2000 xg for 5 minutes (4°C). The nuclear pellet was washed 3 more times in NDB and resuspended in same at 2 mg/ml DNA for digestion with Staphylococcal nuclease. For pulses with 3H lysine the cell pellet was preincubated by resuspension in Dulbecco's Modified Eagles Medium (DMEM, lysine free and serum free) at 37°C for 10 min, centrifuged 400 xg for 5 min and then resuspended in 1 mCi 3H lysine (Schwarz-Mann, 60 Ci/mmole) at 0.5 mCi/ml in DMEM. The pulse was terminated and the nuclei isolated as described above. Staphylococcal Nuclease Digestion. Nucleoprotein particles for electrophoresis on acrylamide gels were prepared by digesting of the nuclei with 10 units/ml staphylococcal nuclease at 37°C for times up to 10 minutes. The reaction was terminated by the addition of 1/10 volume 0.2 M EDTA, pH 6.5 and dialyzed at 40C for 12 hrs against 2000 volumes of 10 mM MES, pH 6.5. The sample was then adjusted to 5% glycerol, 0.005% Bromophenol Blue (BPB) with a 5x concentrate and electrophoresed on a 4.5% acrylamide, 0.1% bisacrylamide, 10 mM MES, 2 mM EDTA, pH 6.5 gel at 150 volts for 5 hrs at 4°C (21). The gel was stained with 20 pg/liter ethidium bromide, photographed, and impregnated with PPO by the procedure of Laskey and Mills (22). The gel was dried and exposed to presensitized Kodak X-Omat R film and stored at -700C. Two dimensional analysis of the fragments involved a first separation of the nucleosomal fragments in the nucleoprotein system using a tube gel. The tube gel was then applied to the surface of the SDS gel system which is primarily a modification of the Laemmli system (25). The electrophoresis buffer was 0.1% SDS, 0.025 M Tris, 0.20 M glycine (pH 8.3) and the separating gel was 18% acrylamide, 0.09% methylene bisacrylamide, 0.1% SDS, 0.75 M Tris (pH 8.8). The stacking gel was 2.5% acrylamide, 0.125% bisacrylamide, 0.1% SDS, 0.125 M Tris (pH 6.8). Electrophoresis was at 150 V for 18 hr at 40C. After electrophoresis, the gels were stained in 0.1% Coomassie brilliant blue R, 40% methanol, 10% acetic acid for 12 hr, destained in 40% methanol, 10% acetic acid, and scanned in a RFT-II scanning densitometer. Gels were fluorographed by the procedure of Laskey and Mills (22). Sizing of DNA Fragments. The nuclei were digested with 10 units/ml Staphylococcal nuclease at 37°C for up to 45 minutes. Samples are taken at appropriate times and added to 2 ml of 0.5% SDS, 20 mM EDTA and the protein digested with Proteinase K (50 pg/ml) at 37%C for 3 hrs. DNA was extracted 4565

Nucleic Acids Research twice with an equal volume of chloroform:isoamyl alcohol (24:1) and precipitated in 3 volumes of ethanol at -200C. The DNA was pelleted by centrifugation at 12,000 xg for 10 min, dissolved in 10 mM Tris and treated with RNAase A (50 pg/ml) at 37°C for 60 min. The sample was adjusted to 5% glycerol, 0.005% BPB, 0.05% SDS and electrophoresed on the following three gel electrophoresis systems: 20 mM Tris, 5 mM Na Acetate, 0.5 mM EDTA, pH 7.4 (TAE) in 1.7% agarose (23), 90 mM Tris, 90 mM Borate, 2.5 mM EDTA, pH 8 (TBE) in 1.7% agarose (24) and 0.75 M Tris, pH 8, 6% acrylamide, 0.075% bisacrylamide (25). The gels were stained with ethidium, photographed, impregnated with PPO and fluorographed by the procedure of Laskey and Mills (22). RESULTS Oligonucleosomes Containing Newly Synthesized DNA change Spacer Lengths as a Function of Digestion Time. HTC cells were labeled with 3H-thymidine for either a short pulse (2 min) or for a full cell generation (18 hr). Nuclei were isolated from these cells. In agreement with the observations of others the newly synthesized DNA (identified by the 2 min pulse of 3H thymidine incorporated) is more rapidly digested by Staphylococcal nuclease than the bulk DNA labeled during the long term exposure to radioactivity (data not shown). In order to attempt to learn more about the additional sensitivity of newly synthesized DNA to nuclease we analyzed the DNA sizes of the oligomer products at various stages in the digestions. These results are shown in Figure 1A and 1B. The size of the fragments obtained from preexisting DNA is indicated by the ethidium stain and that of oligomers of newly replicated 3H DNA is obtained from the fluorogram of the same gel. As shown in figure 1A, in general the size of the 3H-DNA fragments produced by nucleosome digestion is smaller than those from bulk DNA. This is particularly evident when the comparison is made at the same digestion time. However, since newly replicated DNA is digested approximately twice as fast as bulk DNA (see figure 1), a more proper comparison would be between the repeat length of bulk DNA at 30 min and the repeat length observed in a 15 min digest of newly replicated DNA. As shown in figure 1B, after 15 min, the new DNA repeat length is - 150 bp and is significantly different from the 173 bp repeat observed at 30 min for the bulk DNA. Therefore the different rates of digestion are not the cause of the difference in repeat length. However, if we analyze the data at very early digestion times as shown in figure 1B, we observe that the repeat length of newly replicated DNA is not significantly different from that of bulk DNA. This result suggests that the apparent repeat length of newly replicated chro4566

Nucleic Acids Research matin changes during the digestion. One possible interpretation for these observations is that the nucleosomes in newly replicated DNA are able to slide together during the digestion until a 150 bp repeat is produced at which point further sliding is prevented. This effect is not dissimilar to that observed when chromatin stripped of Hi is digested with staphylococcal nuclease. Under these conditions a minor component of the digest, a spacerless dimer is produced which remains relatively resistant to further nuclease cleavage (26). Such resistance to further cleavage may be the origin of the observation reported by Levy and Jakob (12) for the dimer cleavage product in newly replicated material from sea urchin nuclei. These observations are also entirely consistent with those reported by Bonner and his colleagues (10,11) who studied newly synthesized chromatin in Friend cells. However as they did not analyze a detailed time course of digestion, they were not able to observe the origin of this unusual phenomenon. Oligonucleosomes Containing Newly Synthesized DNA have Unusual Electrophoretic Properties. After a modest amount of digestion of the total chromatin, newly synthesized DNA is organized in shorter nucleosome repeat lengths. We wondered if we could resolve nucleoproteins containing newly synthesized DNA by exploiting the decreased repeat length observed after digestion. If this proved to be possible, clearly we could then ask what newly synthesized proteins become associated with newly synthesized DNA. Initially we analyzed the nucleoprotein products obtained after short digestion times. The digestion products were separated on the nucleoprotein electrophoretic system described by Nelson et al. (21). The results of such an analysis are shown in figure 2. Ethidium bromide staining shows a typical nucleoprotein repeat pattern. The dinucleosome region is split into two bands, the slower of which contains two nucleosomes and 2 Hi molecules, the faster band consists of a dinucleosome containing one Hi molecule (27 and also below). Core material lacking Hi is present, as is a monomucleosome consisting of core material with an associated Hi molecule. Nucleoproteins, labeled in vivo by a long term exposure to 3H-thymidine and identified by fluorography (fig. 2, lane b) migrate in an identical manner to that seen for the ethidium stained material. However, if the nucleoproteins were labeled by pulsing the cells for 2 minutes with 3H-thymidine, followed by digestion of such labeled nuclei with staphylococcal nuclease, this gives rise, as we had anticipated, to a repeating pattern of nucleoprotein oligomers which are quite distinct from that seen for long term thymidine labeled nucleoproteins. As seen in the fluorogram in figure 2, lane a, the higher oligomers of the 2 minute labeled 4567


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Nucleic Acids Research material all migrate more rapidly than their counterparts from the long term labeling. The dimer region is again split, but taken as a pair, the dimers move faster than the long term labeled dimer. Essentially no monosome (nucleosome + H1) is present in the short term labeled nucleoprotein. Finally, and in sharp contrast to these observations, the core material from both short and long labeling has a constant migration rate independent of the length of labeling of the DNA which it contains. It was necessary to determine whether the change in electrophoretic mobility of newly replicated DNA seen in nucleohistone gels disappears in the same time period during which freshly replicated DNA loses hypersensitivity to nuclease digestion (15-30 min). We pulsed HTC cells with 3H thymidine for increasing times (ranging from 2 min to 30 min) and from nucleohistone gels determined the rate of return to normal repeat lengths. These results are shown in figure 3 and consist of densitometric scans of the fluorograms. Clearly the 3H-thymidine labeled DNA does mature such that within 30 minutes a majority of the material has returned to a normal spacing. Such a maturation rate correlates rather well with the loss of nuclease sensitivity for replicative DNA which has been characterized by a number of laboratories (5,6,7,8). The Nucleoprotein Organization of Newly Synthesized Nuclear Protein. Since the nucleosomal organization of newly synthesized DNA is sufficiently unique and distinct from that of bulk chromatin it was logical to ask how

Figure 1. Analysis of the staphylococcal nuclease digestion products from both newly replicated and mature chromatin. A. Agarose gel electrophoresis of the DNA fragments isolated from nuclease digests of 3H-thymidine labeled nuclei. HTC cells were labeled for either (a) 1 min or (b) 24 hours with 3H-thymidine. Nuclei were isolated and partially digested with staphylococcal nuclease. Electrophoresis was on a Tris-acetate-EDTA agarose gel (1.7%). The DNA standard is a HpaII digest of PBR322 end labeled with 3H-dCTP and 3H-dGTP by DNA polymerase I (28,29). The ethidium stain shows the DNA fragments from the mature chromatin and the fluorogram shows the DNA fragments from the 3H-thy labeled DNA. B. Graphical analysis of the repeat length for newly replicated (0-0) and mature chromatin (-e-*-). The size of the DNA fragments in figure 1A was determined for each digestion time. The repeat length was determined by calculating the molecular weight difference between each DNA fragment (up to a 5-mer). The average repeat length and standard deviation was then determined. This process was repeated also using two other gel systems, Tris-BorateEDTA-1.5% agarose and 0.75 M Tris-6% acrylamide. The average repeat length from these gels were averaged for all three systems and the standard deviation determined. 4569


Figure 2. Newly made nucleoprotein has altered electrophoretic properties. HTC cells were labeled for either (a) 2 min or (b) 24 hours with 3H-thymidine. Nuclei were isolated and partially digested with staphylococcal nuclease (approximately 5-10% acid soluble). The nucleoprotein fragments were analyzed on nucleoprotein gels and the digestion products visualized either by ethidium stain or by fluorography. newly synthesized nuclear proteins are organized with respect to new DNA. HTC cells were grown in the presence of 3H lysine for a range of times from 1 to 5 minutes. The cells were harvested, nuclei isolated and digested with staphylococcal nuclease. The nucleoprotein fragments were then analyzed electrophoretically and visualized both by ethidium bromide and by fluorography as shown in figure 4. Clearly the distribution of short pulse lysine-labeled protein is very different from that of short pulse thymidine-labeled DNA. However, both direct visualization and graphical analysis indicate that the overall distribution of bulk DNA (ethidium stain) and 3H-protein are the same. This is seen in identical mobility of higher oligomers, of the two dimer bands and of the core material. In addition lysine label is found in the mononucleosome region from which newly synthesized DNA is almost completely lacking as well as in a band moving more rapidly than core material. The origin of this band 4570


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Figure 3. Effect of 3H-thymidine pulse length on detection of anomolous migration. Cells were labeled with increasing pulse of 3H-thymidine for 2 min, 5 min, 10 min, 30 min, and 20 hrs. Nuclei were isolated and digested with staphylococcal nuclease. The digestion products were then electrophoresed on acrylamide nucleoprotein gels. The gels were fluorographed and the fluorogram scanned at 540 nm. The location of the various nucleoprotein particles is indicated as follows. TE-tetramer, T-trimer, D1/D2 - Dimer 1/Dimer 2, N-nucleosome, NC-nucleosome core.

is unknown. It is also seen in the fluorogram that the bands labeled by 3H-lysine are broader than those identified either by eithidium bromide or the fluorogram of 3H thymidine in DNA. This is a result of the complex distribution patterns of new proteins and is discussed below. During nuclease digestion, the newly synthesized DNA is rapidly cut to a size so that it enters the gel efficiently. However, under similar conditions (see fig 4) newly synthesized protein associated with nucleosomal material seems to be much larger and less likely to enter the gel. These results confirm earlier observations (13,14,15,16,11) that new DNA and the bulk (though not necessarily all) of new proteins are on different sets of nucleosomes. The Nucleoprotein Organization of Newly Synthesized Histones. Although a significant fraction of the nuclear protein incorporating 3H lysine in short pulse periods is histone, it is possible that a part of the protein, which 4571


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Figure 4. Electrophoretic properties of nucleoprotein containing newly synthesized material. HTC cells were labeled for 1 min, 2 min or 5 min with either 3H-thymidine or 3H-lysine. Nuclei were.isolated and digested with staphylococcal nudlease as previously described. The nucleoproteins were electrophoresed on an acrylamide gel and the gel fluorographed. The dotted line indicates the position of the various nucleoprotein particles. The abbreviations are the same as in figure 3. clearly is in a different nucleosome organization from new DNA, is not histone in nature. Accordingly we have asked how each newly synthesized histone is distributed with respect to nucleosomes containing either new or old DNA using our ability to distinguish between these materials as discussed above. The strategy used in these experiments is very simple. HTC cells were labeled with 3H lysine for short time periods. The cells were harvested and nuclei isolated. After a brief nuclease digestion the resulting nucleoprotein was separated electrophoretically in a tube gel. This gel was then soaked in SDS to dissociate DNA and attendant proteins which were then identified by a second dimension electrophoresis into a SDS slab gel. After staining and photography the gels were treated for fluorography following the procedures of Laskey and Mills (22). The results of such an experiment are shown in figure 5 which shows the data obtained after a 5 minute pulse of 3H lysine. The 4572


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FLUOROGRAM Figure 5. Two dimensional gel electrophoresis of the newly synthesized histones associated with nucleoprotein particl'es. HTC cells were pulsed for 5 min with 3H lysine, nuclei isolated and digested to produce the nucleoprotein particles. Electrophoresis was the same as previously described in fig.ures 2-4 except that an ii cm tube gel was used. After electrophoresis this gel was laid above an SOS stacking gel and electrophoresed in the second dimension (see Materials and Methods). The stained protein indicates the relative positions of the various mature nucleoprotein particles and the histones associated with them. The fluorogram indicates the location of the newly synthesized histones. The positions characteristic of newly replicated DNA are ind.icated by 'R'. The abbreviations used are as described in the legend to Figure 3. The band indicated with an arrow, although it migrates like Hi, does not appear to be a member of this class as judged by its insolubility in perchloric acid. 4573

Nucleic Acids Research coomassie stained gel shows that the major protein component is histone and that the organization of histone reflects the organization of the bulk nucleosomes. It is possible to discern oligomers up to the pentamer level. A split dimer is present, as is a monomer containing HI and a core particle lacking Hi. Analysis of the fluorogram of the same gel reveals a very different picture. The distribution of the newly synthesized histones varies, depending on the histone to be analyzed. Thus Hi clearly follows the pattern shown for bulk material in Coomassie stain. This is also demonstrated graphically in figure 6. However, the main regions of H3 and H4 intensity do not follow the same distribution pattern as Hi. After a brief nuclease digestion, H3/H4 are organized in a repeating pattern with a distribution identical to those seen for newly synthesized DNA with a shorter repeat due to the apparent decreased spacer length. This is particularly evident in the dimer region where the H3/H4 migrate more rapidly than D1 and D2, so that the distribution of the new histones is the same as the faster dimer doublet characteristic of new DNA. When the distribution of H2A/H2B is analyzed (fig 5) it appears to have a broad distribution, a major portion migrating with Hi and old DNA as well as a fraction running more rapidly with new DNA. The data shown in fig. 5 can be analyzed graphically as shown in fig. 6. From scans of the fluorography and the Coomassie stained gel one can calculate the relative enrichment of any given histone at any point in the separation. The graph of fig. 6 shows the amount of a given histone in the "new DNA" region relative to that in the "old DNA" region. Obviously for a histone which shows no selective deposition on new DNA this ratio should be the same at all points (defined as unity at the monomer level). A value higher than unity indicates selective deposition on new DNA. Thus for H3 and H4 we see a high degree of selective deposition, whereas H2A and H2B do not show the same degree of selectivity. Hi is not deposited selectively on new DNA at all. We had previously stressed that new DNA is largely absent from the monomer nucleosome position and that accordingly any new histone found in this region must necessarily be associated primarily with old DNA. Examination of this region reveals that a good fraction of Hi, H2A and H2B are found at this point in the gel, whereas only a small amount of H3 and H4 migrate to this position. Newly deposited histones H3 and H4 are not strongly bound to chromatin. It is clear from the data of figure 5 that the yield of newly synthesized H3 and H4 from the nuclease-digested nuclei is small. This reflects an observation made several years ago in which the appearance of H3 and H4 in the nucle4574


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Figure 7. The selective release of newly synthesized H3 and H4 from nuclei. A. HTC cells were pulsed for 1 min with 3H lysine. Half of the cells were fixed with 1% formaldehyde according to procedures previously described (30). The remaining cells were immediately frozen. Both cell preparations were washed as described in Materials and Methods to isolate nuclei. In order to analyze the histones in the fixed material, the nuclei were heated at 100°C for 30 min in 4 M guanidine hydrochloride -0.5 M p mercaptoethanol according to procedures previously described (30,33). After dialysis against 50 mM P mercaptoethanol the sample was ready for electrophoresis on SDS gels. (a) HTC cells were disrupted and nuclei isolated. The nuclei were washed with washing medium and histones obtained from the pellet by acid extraction and suspension in 1% SDS. (b) The supernatant from the washed nuclei in (a) above was acid extracted, ethanol precipitated and adjusted to 1% SDS. (c) Nuclei isolated from cells fixed for 2 hrs with 1% formaldehyde. (d) Nuclei isolated from cells fixed for 18 hrs with 1% formaldehyde. B. HTC cells were pulsed 3 minutes with 3H lysine. Cells were inmediately quick frozen, thawed and nuclei prepared as previously described. These unfixed nuclei (-1 mg/ml DNA) were divided into equal aliquots and collected at 2000 xg for 10 min. The pellets were suspended in 0.5 ml 50 mM sodium phosphate at pH values of 5-10 as required. After 5 min at 40C with occasional vortexing the solutions were centrifuged and the supernatant adjusted to 0.4 N H2S04 and dialyzed overnight against 20 mM H2S04-5 mM 4576

.

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Nucleic Acids Research p mercaptoethanol. The samples were adjusted to 0.1% SDS-5% glycerol-50 mM Tris for electrophoresis on 18% gel. The pH of the exposure of the unfixed nuclei is indicated directly on the figure. Lane T - total nuclei before the washer in 50 mM sodium phosphate.

us after a short pulse, seemed to be delayed (32). It was concluded that the transit time for these histones from cytoplasmic site of synthesis to the nucleus was longer than that seen for the other histones. However, an alternative explanation is that freshly deposited H3 and H4 are exceedingly easily dislodged from nuclei during preparation procedures even though H3 and H4 in bulk chromatin are the histones most strongly bound to DNA. Two lines of evidence now indicate that this latter explanation is correct. We have recently reported on a technique for crosslinking chromosomal components in situ within whole cells (30). Subsequent isolation of fixed chromatin and reversal of the crosslinks reveals, even after a short pulse of 3H-lysine, that normal quantitative yields of newly synthesized H3 and H4 are found within the nuclei as is shown in figure 7 (compare preparations from unfixed (A) and fixed nuclei (C,D)). Clearly then during a normal isolation a part of the new H3 and H4 and to a lesser extent H2B is being lost from unfixed nuclei and can be identified in the extract as such (see fluorogram B). This effect is not as extensive for new H2A. That newly synthesized histones H3 and H4 are more weakly bound than the mature forms of these histones was shown by the following experiment. HTC cells were exposed to 3H-lysine for a 3 minute period and then harvested immediately. Nuclei were isolated rapidly and carfully as described in Materials and Methods. Subsequently they were washed at a series of different pH values. Any extracted material was then analyzed on an SDS gel and examined by Coomassie staining (bulk histone) and by fluorography (newly synthesized histones). The results are shown in fig. 7B. Clearly the chromatin contains a normal complement of mature histones as seen from the stained gel of the unextracted chromatin. That this has lost some newly synthesized histone is demonstrated in the fluorogram of the same gel (7B, lane T). Extraction of bulk histone from chromatin as a function of pH follows a normal pattern (stained gel) in that none of the non-Hl histones are extracted over the range of pH 5-10. HI is increasingly dissociated above pH 8. However, newly synthesized H3 and H4 begin to be selectively released from pH 7 upwards as seen from the fluorogram, such that at pH 9-10 most of these newly synthesized histones have been dissociated.

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Nucleic Acids Research DISCUSSION After moderate digestion the oligonucleosomes containing newly synthesized DNA are organized about a shorter repeat length. This is a consequence of an apparently normal sized core and a spacer region which is approximately 25 ± 6 bp shorter than that seen in non-replicated chromatin. We have attempted to determine whether the difference in repeat length is a reflection of the chromosomal organization of new DNA in vivo or if it was produced in some way during the digestion process. Accordingly we have analyzed repeat lengths at various stages in digestion including very early time points where the extent of nucleolysis is very small. The analysis of the repeat length was conducted in two different agarose gel electrophoretic systems as well as in polyacrylamide. Analysis in agarose-TAE showed the best periodic repeat patterns (see fig 1) and gave a strong indication that the repeat size was the same between bulk and new DNA when digestion was minimal and that the different repeat size was generated during digestion. Somewhat similar effects were noted when the analysis was performed on polyacrylamide, though the measured repeat sizes were significantly smaller than that seen in agarose TAE even though the same DNA size standard was employed in both systems. At this stage however we feel that a firm commitment to the notion of digestion-induced decrease in repeat length would be premature, as the argument is critically dependent upon measurements of spacing at the earliest times of digestion, when the amount of material entering the gels is small and one is concerned about the precision of measurement. The solution to the problem would most likely lie in fixation of the histones to the DNA in intact cells followed by nuclear isolation and subsequent digestion. The techniques for this kind of analysis have been reported recently (30,31). We have not pursued this point further as it was not of pressing concern in as much as the main thrust of this work was to exploit the clearly reduced repeat length of oligonucleosomes containing newly synthesized DNA whether or not it reflects in vivo organization or is an artifact of digestion. As a consequence of the characteristic properties of chromatin which contains new DNA we find that the new nucleosomal oligomers all migrate faster than the comparable material from non-replicated DNA when the materials are analyzed on a nucleoprotein gel (see figure 2). The cause of the increased mobility of nucleoprotein oligomers containing new DNA may lie in the decreased size of the DNA they contain, though this is not at all certain since the loss of 26 bp per nucleosome will also lead to an overall reduced negative

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Nucleic Acids Research charge density on the molecules which would have an opposite effect on mobility. It seems quite likely that the cause of the increased mobility may be a consequence of the decrease in spacer length in as much as it is entirely possible that less Hl may be bound to these nucleoproteins thereby increasing the overall negative charge substantially. However, since the fraction of such newly synthesized oligomers is small (much less than 1%) it has proved impossible to test this hypothesis. Nonetheless the important point is that nucleoprotein oligomers containing new DNA can be resolved from bulk oligomers. This is particularly clear for the faster nucleosomal dimer. This provided us with the means to probe the site of deposition of specific newly synthesized histones. There is a problem in experiments of this type which arises from the exciting observation that newly synthesized histones H3 and H4 appear to bind chromatin very weakly until there has been a maturation process. In fact the binding is so weak that these two new histones seem to be continuously dissociating throughout the isolation procedures. Clearly then any conclusions drawn from H3 and H4 refer to that fraction of the material which still remains bound to the chromatin during the nuclease digestion step. Using this approach we find that histones H3 and H4 are deposited primarily on newly synthesized DNA, that histones H2A and H2B are mostly deposited on old DNA though a portion does become associated with new DNA. Finally, histone Hi is almost exclusively deposited on pre-existing chromatin. Since newly synthesized DNA is ultimately organized into nucleosomes with an apparently normal sized 145 bp core it seems unavoidable that histones H2A and H2B must be available to the replicative region. Since a sizable fraction of this is not new H2A and H2B it follows that old H2A and H2B must be rearranged to provide the histone necessary for nucleosome formation. Obviously from these experiments we cannot exclude the possibility that some new H3 and H4 molecules were deposited and weakly bound to regions of non-replicating chromatin. In view of exactly such results with H2A and H2B this would not be surprising. However, we do not think that this is the main site of deposition of H3 and H4 because of recent work in which we studied deposition of H3 and H4 after a prior, efficient fixation of these molecules within chromatin using formaldehyde. In these experiments we found that most of the new H3 and H4 molecules behaved in a parallel fashion, depositing selectively on newly synthesized DNA (30,31). Additional experiments have been performed to check whether newly synthesized H2A-H2B are deposited on new DNA at a much later time than new H3 and H4 and this has been shown not to be the case (31).

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Nucleic Acids Research These results are consistent with the observations we have made using formaldehyde fixation to isolate replicative chromatin (30), using iododeoxyuridine to identify density labeled replicative chromatin (31) and from the isolation of the replicative form of SV40 minichromosomes (31). In all instances we find selective deposition of H3 and H4 on newly replicated DNA and a majority of H2A and H2B are deposited on pre-existing DNA as is all of histone Hi. A more detailed review of the various aspects of deposition, presenting a synthesis of the different points of view has been presented in a previous report (31). ACKNOWLEDGEMENTS We wish to thank our colleagues in the laboratory for their advice and criticism. This work was supported by grants from the USPHS CA-10871 and GM27228.

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