The Interaction of Histone H5 and Its Globular Domain with Core

THE JOURNAL OF BIOLOGICAL CHEMISTRY (d 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 3, Issue of January 25, pp. 1502-1508,1991 Printed in U. S.A.

The Interaction of Histone H5 and Its Globular Domain with Core Particles, Depleted Chromatosomes, Polynucleosomes, and a DNA Decamer”

(Received for publication, July 9, 1990)

Alain SegersS, Serge Muyldermans, and Lode Wyns From the Instituut uoor Molekulaire Biologie, Vrije Uniuersiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium

Certain features of linker histone behavior were analyzed using a precipitation and a nitrocellulose filter binding assay. Chromatosomes, depleted of the linker histones, present one unique binding site to the globular domain of histone H5 (GH5) which involves the two 10-base pair DNA ends of the chromatosome. Additional binding to lower affinity sites is intrinsi- cally different and results in aggregation as does all binding to core particles. These findings, as well as the binding study on a synthetic DNA decamer, lend sup- port to earlier hypotheses of more than one DNA binding site on the globular domain. Our studies provide a deeper insight into the long standing question of H5/ nucleosome stoichiometry. A salt dependence analysis of GH5 binding to H5-depleted chromatosomes indicates that GH5 displaces a number of ions similar to the total H1 linker histone, suggesting a delocalized binding of the carboxyl- and amino-terminal tails.

The linker histones H1/H5 are involved in the organization and maintenance of chromatin higher order structure (Thoma et al., 1979; Butler and Thomas, 1980; Igo-Kemenes et al., 1982) and play an active role in the control of DNA replication and cell proliferation (Sun et al., 1989).

These histone proteins have a three-domain structure con- sisting of an amino-terminal unfolded “nose” of about 20-40 residues, a central globular domain of approximately 80 residues, and a 100 amino acid long carboxyl-terminal “tail” which contains an inducible a-helical region (Bradbury et al., 1975; Clark et al., 1988). The tertiary structure of GH5 has been shown to contain three a-helices by NMR studies (Zar- bock et al., 1986; Clore et al., 1987), and its recent crystalli- zation should lead to a complementary x-ray structural study (Graziano et al., 1990).

The globular domain may be prepared by trypsin digestion of the histones Hl/H5. This resistant polypeptide called GHl/GH5 retains all the native a-helical structure (Aviles et al., 1978) and is thought to interact with the nucleosome particle at the site at which DNA enters and exits from the chromatosome unit (Allan et al., 1980).

From our knowledge of the core particle, the chromatosome structure, and the dimensions of the globular domain we might expect GHl/GH5 to interact with more than one DNA duplex simultaneously (Crane-Robinson & Ptitsyn, 1989). However,

* This work was supported in part by the Belgian N.F.W.O. and I.I.K.W. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of a grant from the I.W.O.N.L. To whom correspond- ence should be sent. Tel.: 2-359-02-09; Fax: 2-359-03-90.

a biochemical analysis of both the number and the properties of these DNA binding sites has not yet been reported.

A further persistent uncertainty concerns the actual stoichiometry of linker histones bound to nucleosomes. Since, depending on the cell-type, stoichiometries are found between 0.8 and 1.3 (Bates and Thomas, 1981), one can imagine that in some instances a fraction of the nucleosomes binds more than one H1/H5 molecule. Indeed, in vitro studies indicate the possibility of reconstituting chromatin or isolated nucleosomes with H1/H5 so that nucleosomes accommodate several linker histones (Allan et al., 1981; Hofmann et al., 1980; Watanabe, 1989). On the other hand, chromatin reconstituted with one H1 molecule/nucleosome, displaying native characteristics, has been prepared (Klingholz and Stratling, 1982; Graziano et al., 1988).

We are thus dealing with a system in which apparently each of the interacting molecules, the Hl/H5 linker histone and the nucleosome, possesses multiple binding sites. In order to elucidate some of these problems we have developed a filter binding assay that allows us to determine the binding constant of H5 and GH5 to cbromatosomes and core particles. These data, in conjunction with a precipitation study of H5 and GH5 with chromatin-derived nucleoprotein particles, provide us with a coherent data set describing some major features of H5 binding to nucleosomes. In our experiments we have extended the work of Ali and Singh (1987) who studied core particles only. In contrast we used a variety of nucleoprotein particles and complementary techniques.

From our experiments we conclude that GH5 is able to recognize specifically the region on the nucleosome formed by the three DNA duplexes near the pseudo-2-fold axis and that only one GH5 molecule binds, centrally located on the nucleosome particle. Although other sites on the core particle exist, they have completely different characteristics, and binding to these leads to aggregation.

MATERIALS AND METHODS

Histone Purification-Chicken histone H1 and H5 are prepared by the acid extraction method of Murray et al. (1968) and purified by chromatography on a Bio-Gel P-60 column, eluting with 20 mM HC1, 50 mM NaCI.

Tris, 1 M NaCl, pH 8.0 (enzyme:substrate ratio, 1:lOOO). GH5 was The globular domains are prepared by trypsin digestion in 50 mM

purified further by HPLC’ on a 21.5 X 150-mm TSK SP-5PW Ultropac column (LKB). The HPLC buffer contained 10 mM sodium phosphate, pH 7.1, and GH5 was eluted using a linear gradient of NaC1. At a flow rate of 4 ml/min the salt concentration was raised from 400 to 800 mM in 30 min.

Chromatin Samples-Core particles were prepared as described by Pennings et al. (1989). Chromatosomes depleted from H1/H5 were

‘ The abbreviations used are: HPLC, high pressure liquid chromatography; SDS, sodium dodecyl sulfate; bp, base pair(s).

1502

Histone HS-DNA/Nucleosome Binding 1503

obtained through two different procedures. The first procedure consists of an ultracentrifugation in the presence of 650 mM NaCI, in conditions under which the linker histones are released from the core nucleosome. This procedure, however, can give rise to "sliding," a repositioning of the DNA around the octamer proteins. The second technique employed is a modification of that of Muyldermans et al. (1980) and consists of a treatment of the chromatosomes with resin AG 50W-X2 (Bio-Rad) in the presence of 30 mM NaC1. The latter preparation strategy does not induce sliding as shown from DNase I cutting profiles of the isolated chromatosomes. Unless stated other- wise, the chromatosomes used were obtained through the former method. The term chrornatosomes will be used further on to indicate Hl/HB-depleted chromatosomes.

The mixture of polynucleosomes of different lengths was depleted of Hl/H5 by ultracentrifugation on sucrose gradients containing 650 mM NaCl to produce oligonucleosomes, which are primarily composed of particles having the length of tetranucleosomes or shorter. It should be noted that this preparation also contains a considerable amount of mononucleosomes.

DNA Oligonucleotide-The self-complementary decamer duplexes (B'CGGGATCCCGB') (Eurogentech, Liege, Belgium) were formed by heating the oligonucleotide at 90 "C for 5 min followed by cooling to room temperature in 1 h.

Concentration Determinations-Concentrations of protein and nu- cleoproteins were determined spectrophotometrically assuming an extinction coefficient a t 280 nm of 1367 M" cm-I for tyrosine and an extinction coefficient a t 260 nm of 7000 M" cm" for DNA.

Radioactive Labeling-H5 and GH5 were radioactively labeled with ["Clformaldehyde by reductive methylation (Glazer et al., 1975). The specific activities of GH5 and H5 were approximately 5 X lo5 cpm/ nmol.

Labeling of the decamer, the chromatosomes, and the core particles was carried out with [./-*'P]ATP (Amersham Corp.) using T4 poly- nucleotide kinase (Amersham) as described in Maniatis et al. (1982). pBR328 was digested with HpaII, and the resulting fragments were end labeled with [o-'*P]dCTP (Amersham) using Klenow DNA po- lymerase I (Amersham).

Precipitation Study-To avoid severe losses of protein, all samples were diluted in the detergent-containing buffer A 10 mM sodium phosphate, pH 7.1,60 mM NaCI, 0.1% Triton X-100 (Sigma). Sodium phosphate was included since it has been reported that low concentrations of phosphate ions induce folding of H1/H5 histones (De Petrocellis et al., 1986).

In all experiments the concentration of nucleoprotein particles was kept constant and sufficiently high so that the radioactivity in samples having low ratios of H5 or GHB/particle could still be easily detected.

A typical centrifugation took 10 min at 12,000 rpm in an Eppendorf centrifuge, after which an aliquot of the supernatant was counted.

Filter Binding-HAWP nitrocellulose filters (Millipore), 0.45-pm pore size, were pre-wetted with 10 mM sodium phosphate, pH 7.1, and the appropriate NaCl concentration. All samples were diluted, a t least 40 times, in this buffer in the holes of the filtration manifold in order to lower the Triton X-100 concentration sufficiently.

The diluted samples were given 2-3 min to equilibrate before filtration was carried out in 5-10 s. Filters were rinsed with 8 ml of buffer. Dried filters were counted in Aqua Luma Plus (Lumac).

RESULTS

Characterization of the Samples-We analyzed the quality of labeled H5 and GH5 by SDS-polyacrylamide gel electrophoresis (Fig. lB), using different preparations of H5 and different fractions of GH5 without influencing the experiments. This is important with regard to the GH5 purification. The HPLC procedure employed enabled us to purify several GH5 subfractions, differing in length by a few residues as determined by amino-terminal sequencing. All of the fractions tested, labeled or not, behaved identically in our assays.

The nucleoprotein particles were checked for their DNA length by gel electrophoresis (Fig. lA). Since during the removal of Hl/H5, which occurs ,in the presence of elevated salt concentrations, the DNA can slide on the octamer core, we also checked the positioning of these macromolecules. The overall DNase I digestion pattern for the chromatosome is

A B a b c a b c

FIG. 1. Characterization of the samples. Panel A, 4% polyacrylamide gel electrophoresis in the presence of 0.1% SDS. a, oligonucleosomes; b, chromatosomes; c, core particles. Panel B, 17.5% SDS-polyacrylamide gel electrophoresis. Proteins were stained with Coomassie Blue. a, H5; b, GH5; c, total histones of chicken erythrocyte chromatin. The respective core histones are indicated.

C? SI. NS

- so

20 - .e* - 20

FIG. 2. Autoradiography of DNase I digestions. 8% polyacrylamide gel electrophoresis under denaturing conditions in 7 M urea. Single time points from DNase I digestion of CP, core particle; SL, slided chromatosome; NS, nonslided chromatosome. At the sides, the distance to the DNA ends of the preferred cleavage sites is indicated in base pairs.

similar to that of the core particle with 10 nucleotides added to each of the fragments. The main features of the digestion were: (a) prefered cleavage sites at 20,40,50,90,100, and 120 nucleotides from the end; ( b ) limited cleavage at 30 and 110 bases from the ends; and (c) a central valley with low frequency of DNase I cutting in the region 60-80 nucleotides from the ends of the DNA (Simpson, 1978). It is clear that our nonslid chromatosome preparation fulfilled all these requirements (Fig. 2). The slid chromatosomes, on the other hand, retain only one characteristic, namely a low frequency of cutting at the site 70 bp from the end, since a shift by 10 bp of the DNA with respect to the octamer, brings site 70 at 60 or 80 bp from the ends, and these sites are cut by DNase I also at low frequency. This feature is also easily discerned from the DNase I digestion pattern.

Adsorption of GH5 to Surfaces-In the course of our inves- tigation we noticed that purified GH5 was considerably lost when it was diluted in plastic tubes. For example, vortexing 50 pl of a 5 X M 14C-labeled GH5 solution in the absence of detergents resulted in a loss of 60%, meaning that approximately 10 pg of GH5 could be adsorbed on the surface of an Eppendorf tube. Other surfaces, such as acrylamide gels and Sephadex columns, also showed high binding capacities for GH5. Wright et al. (1987) showed that the only H1 peptide fragment generated by protease or chemical cleavage, which failed to be transferred t o the nitrocellulose filter, was the globular domain. Thus, GHl/GH5 displays an unusual adsorption behavior.

Therefore, in our work we used nonionic detergents in most of our manipulations and established that as much as 0.1% Triton X-100 was required in order to prevent losses. This is 10 times higher than the concentrations normally used for similar proteins such as the sperm-specific protein from Spi- sula solidissima (Libertini et al., 1988). However, we obtained

1504 Histone H5-DNA/Nucleosome Binding

evidence that 0.1% Triton X-100 does not alter the histone structure substantially, since a trypsin digestion of H1 and H5 in the presence of 0.1-2% Triton X-100 resulted in the same digestion profile as that obtained in the absence of detergent (data not shown). We further noticed that once bound to any kind of nucleic acid or nucleoprotein particles used in this study, GH5 did not then adsorb to plastic surfaces any more. This indicates that the presumably hydrophobic patch on the GH5 molecule responsible for adsorption to surfaces is somehow covered or altered by the bound ligand.

Precipitation Study with Core Particles, Chromatosomes, and Oligonucleosomes-Ionic interactions between histones and DNA are crucial for binding (Clark and Kimura, 1990). In these experiments the NaCl concentration was chosen low enough as to promote strong ionic interaction between H5/ GH5 and the different nucleoprotein particles. We have shown that the interaction of GH5 with linear DNA is dras- tically changed above 200 mM NaCl (see below). In contrast, omission of the salt gave irreproducible data so that an intermediate value of 60 mM NaCl was used. This ionic strength allows formation of stable chromatin filaments re- sembling those found in nuclei (Thoma et al., 1979; Widom, 1986). In addition to the NaCl we also included a sufficient concentration of phosphate in all buffers to ensure the folding of GH5 at all ionic strengths (De Petrocellis et al., 1986).

When we added increasing amounts of H5 to core particles and the mixture was centrifuged, the solubility of H5 decreased gradually, reaching a minimum between 1 and 3 H5/ core particle (Fig. 3A). For H5/core particle ratios above 4 the percentage H5 in solution rose again and eventually approached 100%. The titration with H5 of chromatosomes also resulted in a decrease in solubility of H5 but started at a higher ratio of H5 to particle compared with the titration of core particles (Fig. 3B) . Again the maximum of H5 precipitation occurred between 1 and 3 H5/chromatosome. Using oligonucleosomes as a ligand, H5 stayed maximally in solution up to 0.4 H5/nucleosome (Fig. 3C); thereafter its solubility decreased and reached a minimum in the range of 3-5 H5/ nucleosome.

The above experiments were repeated with GH5 for com- parison. The GH5 core particle complexes showed the same behavior as those of H5; even at low ratios (e.g. 0.2 GH5/core particle) the precipitation of GH5 was pronounced (Fig. 30) . The minimum fell between 2 and 6 GH5/core particle. On the other hand, addition of GH5 to chromatosomes resulted only in precipitation of GH5 beyond the point of a 1:l stoichiometry (Fig. 3E). At lower ratios GH5 stayed in solution for 100%. This result is in sharp contrast with the behavior of H5. Minimal solubility is reached at approximately 4-5 GH5/ chromatosome. A comparable observation was made when GH5 was added to chromatosomes that had been prepared in such a way as to prevent repositioning of the octamer core with respect to the DNA. Thus, ordinarily prepared chromatosomes and their nonslid counterparts behaved in a similar manner. GH5-oligonucleosome complexes remained soluble up to a ratio of 1 GH5/nucleosome (Fig. 3F). At higher ratios GH5 precipitated, reaching a broad minimum between 4 and 10 GH5/nucleosome.

These precipitation phenomena were also studied from the point of view of the nucleoprotein. In order to do SO we used labeled nucleosomes and core particles and unmodified H5 and GH5. This provided a complementary study of the precipitation behavior of the complexes. Fig. 4A represents the precipitation curve of labeled core particles and shows that both H5 and GH5 precipitated core particles even a t low ratios of protein to particle. H5 was more effective than GH5

and precipitated core particles completely at 2 H5/particle whereas 6-7 GH5 molecules/core particle were required in order to precipitate the latter completely. Chromatosomes were rendered completely insoluble at a ratio of 1.5 H5/ chromatosome and higher (Fig. 4B). The titration with GH5 followed another curve; the chromatosomes stayed soluble up to 0.7 GH5/particle after which they gradually precipiated, and the chromatosomes became completely insoluble at 5-7 GH5/particle.

The globular domain and the carboxyl-terminal tail of linker histones interact differently with the nucleosome (Thomas & Wilson, 1986). A similar observation was made for the interaction of GH5 with core particles (Ali and Singh, 1987) suggesting that the globular domain binds specifically whereas the carboxyl-terminal tail binds in a delocalized way. Consequently their binding properties are affected differently by salt. This led us to conduct a series of experiments in which we followed the salt concentration dependence of the precipitates. In Fig. 5A it is clear that the GH5-chromatosome aggregates resolubilized above 150 mM NaCl whereas those of H5 persisted as precipitate up to 400 mM NaCl, indicating that H5 and GH5 form different types of complexes. In analyzing the salt sensitivity of H5-DNA and GH5-DNA precipitates we observed the same dependence as for the chromatosome complexes (Fig. 5B).

Precipitation Study with a IO-bp DNA Fragment-We have used a double-stranded IO-bp oligonucleotide (5’ CGGGATCCCG) as a tool to probe the possible DNA binding sites on GH5. 32P-Labeled decamer was maximally precipitated when we mixed GH5 and decamer in a ratio of 1 GH5/ DNA strand (Fig. 6A). This may be a result of charge neutralization since GH5 bears somewhat more than 10 positive charges as judged from its amino acid composition. This assumption is supported by our observation that GH5 precipitated maximally when added to supercoiled or linear pUC18- DNA at a ratio of 11-12 bases/GH5 molecule (data not shown).

Since we observed the precipitation of both protein and decamer in a double-labeling experiment, we were able to determine the composition of the pellet after centrifugation. As described above, GH5 and the decamer formed maximally insoluble complexes at a ratio of 1 GH5/decamer strand. At the point of 1:l stoichiometry it can be extrapolated that more than 90% of the material is in the pellet. Another phenome- non, however, was apparent in the precipitation curve of GH5; a sharp decrease in GH5 solubility, between 0.1 and 0.2 GH5/ strand, where the composition of the pellet abruptly changed. For mixtures with less than 0.15 GH5/strand, the pellet had stoichiometries with a mean value close to 0.15. The precipitates formed in mixtures with more than 0.2 GHS/strand consisted only of aggregates with a stoichiometry of at least 0.9 GH5/strand.

The influence of salt on the amount of DNA precipitated shows that the percentage of DNA in solution gradually increased as the salt concentration was raised. Above 30 mM NaCl no oligonucleotide could be precipitated with GH5, and this explains the absence of NaCl in the above described precipitation study, in contrast to the one with the nucleoprotein particles.

Determination of the Association Constants-The fraction of free GH5 could be determined as a function of the concentration of added nucleoprotein particle by a nitrocellulose filter assay (Fig. 7), in which GH5, when not complexed to nucleic acid, is retained on the filter. Typically 60-70% of the protein input was bound to the filter. The rather low specific activity ( lo6 cpm/nmol) of our labeled proteins prevented us

Histone H5-DNA/Nucleosome Binding 1505

120 A 100 *

0 ' I 0.1 1 io

1 2 0 2 100 -

80 -

eo -

40 -

20 -

0 0.1 1 10

120 0

80 -

80 -

40 -

20 -

0 ' J 0.1 1 10

120

D/

0 0.1 1 10

l2O] 100 0

80 -

80 -

40

0 ' 0.1 1 10

120

F 100 -

80 -

eo -

40 - 20 -

0

0 0.1 1 10

Ratio (protein/padicle) FIG. 3. Precipitation of 14C-labeled H5 and GH5 upon titration of nucleoprotein particles. The

concentration of nucleoprotein particles was kept constant a t 4.2 X lo-* M. Increasing amounts of A-C, H5 and 0 - F , GH5 were added to nucleoprotein solutions in buffer A. Mixtures were equilibrated for 20 min a t room temperature before centrifugation. Three different preparations were used: A and D, core particles; B and E , chromatosomes; C and F, oligonucleosomes. E, GH5 was added to nonslided chromatosomes (0) and slided chromatosomes (0).

from reaching very low concentrations. Our limit was around somes, even at high concentrations (see Fig. 5A). 2 X 10"" M GH5 for 8-ml samples (about 1000 cpm). Although From the precipitation assay we concluded that there are we used very low concentrations of GH5, which reduce aggre- at least two classes of binding sites on chromatosomes for gation, we still worked with a molar excess of chromatosomes GH5: ( a ) the putative native site, with a high affinity for in order to avoid precipitation. Moreover our binding experi- GH5; and ( b ) the extra sites, which lead to aggregation. Sites ments were performed in the presence of 200 mM NaC1, a salt ( b ) have a much lower affinity for GH5 than ( a ) , since GH5- concentration at which GH5 hardly precipitates chromato- chromatosome complexes stayed in solution for 100% up to a


60 -

40 -

20 -

U‘ \

“4 \ \ \

A

8 20 - 0

n J 0 100 200 300 400 500 600 700

Ratio (protein/particle) Ratio (protein/particle)

100‘ O

O

B 1

60[ ’ \ b I

h 40 b s

I \ \ I \

\o \ a -

0 0.1 1 10

Ratio (protein/particle) FIG. 4. Precipitation of 32P-labeled nucleoprotein particles

upon H 5 and GH5 addition. 8.5 X lo-* M core particles ( A ) and chromatosomes ( B ) were mixed with increasing amounts of GH5 (0) and H5 (El), equilibrated for 20 min, and centrifuged.

1:1 ratio, filling up first this class of sites. We assume a 1:l binding stoichiometry in order to calculate

the concentration of bound chromatosome or core particle, with which we titrated, since we could only observe the free fraction of GH5 which is quantitatively retained on the filters. Our data were reexpressed after the classical Scatchard procedure. Let @ be the number of mol of nucleoprotein particle bound per mol of GH5. Then, a plot of @/[free particle] uersus p should give a linear relationship if there is only one class of sites. The resulting plots fitted the expected linear relationship.

We observed that the association constant for GH5-chromatosome binding at 200 mM NaCl is about 4 times lower than at 150 mM NaCl. Titrations with core particle revealed that the so-called low affinity sites indeed had at least a 10 times reduced affinity as compared with the putative native site. We further noticed that the K,, for chromatosomes hardly increased upon going from 150 to 60 mM NaCl.

Additional determinations in the range of 150-200 mM were feasible. By fitting a straight line through these points in a log-log plot of K, uersus [Na+], we could estimate the number of counterions released from the DNA upon complex formation. The slope of the line is described as follows (DeHaseth et al., 1978).

0 100 200 300 400 500 600 700

Ratio (protein/particle) FIG. 5. Salt dependence of precipitates formed by H 5 and

GH5 with DNA and chromatosome. A , a 10-fold molar excess of H5 (0) and GH5 (0) was mixed with 32P-labeled chromatosomes a t different ionic strengths. The chromatosome concentration was 1.7 X M. B, mixtures of H5 (0) and GH5 (0) with 32P-labeled pBR328-HpaII fragments were made a t different ionic strengths and centrifuged. H5 and GH5 were present at 1.3 X M, and the DNA concentration was 7 X M nucleotides.

-dlog KJ8log [NaCI] = m X 9

Where \k is the fraction of counterion bound per phosphate charge, being 0.88 for double-stranded DNA; m is then the number of ions released from the DNA, assuming that no ions are released from the protein surface upon binding. Our fit gave a value for m = 5.2, which is close to the number determined by Watanabe (1986), who found m to be 6.3 for the binding of calf thymus H1 to DNA in the same range of salt concentrations.

DISCUSSION

Chromatosome-GH5 complexes up to a 1:l ratio do not give rise to precipitates whereas core particle/GH5 mixtures result in a 70% loss of GH5 into precipitate a t a 1:l ratio (Fig. 3, D and E ) . We therefore conclude that chromatosomes provide GH5 with a binding site which, upon binding, does not lead to precipitation of the complex. Although precipitation of nucleoprotein particles and aggregation of chromatin can be caused by several, sometimes poorly understood, phenomena such as partial neutralization of the charged groups of DNA (Manning, 1978) or bridging between chromatin fibers by interactions between DNA and H1 (Jin and Cole, 1986), we

Histone H5-DNA/Nucleosome Binding 1507

Ratio (GH51 10 nucleotides)

0- 0.01

, , JK, 0.1 1

Ratio (GH5/ 10 nucleotides) FIG. 6. Solubility of DNA decamer-GH5 complexes. A , a "P-

labeled decamer solution having a concentration of 2 X M strands was titrated with increasing amounts of 14C-labeled GH5 in 10 mM sodium phosphate, pH 7.1. After centrifugation, aliquots of the supernatant were analyzed by liquid scintillation counting for their 14C (0) and '"P (0) content. All manipulations were carried out at 4 "C. H , transformation of the data presented in Fig. 6A.

0 0.2 0.4 0.6 0.8

D

FIG. 7. Nitrocellulose filter binding assay. Scatchard plot of titrations of 14C-labeled GH5 with chromatosomes a t 200 mM NaCl (O), a t 150 mM NaCl (O), and with core particles a t 200 mM NaCl (0). The GH5 concentration was 9.7 X 10"' M or lower. (3 is the fraction of GH5 bound by a particle.

believe that in our case the cross-linking capacities of both H5 and GH5 are responsible for the precipitation. Although differing in net charge by a factor of 5 both GH5 and H5 precipitate maximally at roughly the same ratios (Fig. 3), indicating that charge neutralization does not play a deter- mining role. We thus conclude that the precipitation of H5 and GH5 occurs through oligomerization of the nucleoprotein particles, suggesting that both proteins possess more than one DNA binding site.

The results of the decamer binding (Fig. 6A) reflect the ability of GH5 to interact with the oligonucleotide in two different ways. At sufficiently high concentrations of GH5 all

possible binding sites on the DNA are filled, and the complexes, having a stoichiometry around 1 GH5/DNA strand, precipitate completely. On the other hand at ratios below 0.2 GH5/DNA strand the precipitate has a stoichiometry of about 0.15 GH5/10 nucleotides ( i e . roughly 3 DNA duplexes/GHS) indicating that GH5 has more than one DNA binding site.

We thus conclude that H5 and GH5 possess more than one DNA binding site as postulated previously by Clark and Thomas (1986, 1988) and Clark et al. (1988), whose electron micrographs show H1-DNA complexes that appear to be composed of H1 molecules bridging two aligned DNA molecules. Our conclusion agrees also with the hypothesis put forward by Crane-Robinson and Ptitsyn (1989) that GH5 would bear one major and two subsidiary DNA binding sites. A GH5 molecule having more than one DNA binding site would thus be able to oligomerize separate DNA molecules and DNA-containing macromolecules, provided the ligand does not cover all the binding sites. On the other hand, binding of GH5 to its natural site (the entry and exit position of DNA on the nucleosome) is expected to block all its DNA binding sites since a high degree of complementarity should exist between both regions.

Binding of GH5 in a 1:l ratio without forming aggregates is only observed with particles of structural complexity higher than core particles, implying that the site completely covering the DNA recognition region of GH5 must lie outside the core particle DNA and reinforcing the idea that the two 10-bp extensions from core particle constitute the putative native site (Allan et al., 1980). The fact that slid and native chromatosomes behaved much alike indicates that in the former all requirements for correct GH5 binding are still fulfilled. So it seems that GH5 needs the correct DNA framework but that the location with respect to the core histones is of minor importance, indicating that a shift by 10 bp of the DNA around the octamer does not greatly influence the local ar- rangement of the different DNA strands.

When all chromatosomes are loaded with one GH5 molecule, the only sites left for GH5 binding are those present on the core particle DNA, and these sites, as the filter binding experiments indicate, have at least a 10 times lower affinity that the putative native site. Binding to these sites inevitably leads to aggregation since only a fraction of the nucleosome binding site of GH5 is now covered by DNA and may, therefore, bind an additional DNA duplex. Our results are thus compatible with the view of Nelson et al. (1979) that one molecule of Hl/histone octamer confers the 160-bp nuclease digestion barrier and that a second, low affinity, H1 binding site exists on nucleosomes. Linker histones bind efficiently to linkerless core particles, having one binding site for the carboxyl-terminal tail and a second for the globular domain (Ali and Singh, 1987). Furthermore, our results are not in conflict with this observation although they demonstrate clearly that the main binding site for the globular domain is not present on a core particle.

Thus, H5 is more effective than GH5 in the precipitation experiments because of the presence of the very basic carboxyl-terminal tail, which contributes strongly to the precipitation (Ali and Singh, 1987). H5 stays in solution much longer when titrating with oligonucleosomes than with chromatosomes (Fig. 3, B and C). The presence of linker DNA apparently makes the terminal domains bind in such a way that it reduces its cross-linking action. Chromatosomes (and core particles too) lack this linker region and therefore force the carboxyl-terminal tail to bind to the core particle DNA, giving rise to cross-linking and precipitation of the complexes. At high salt concentrations (e.g. 350 mM NaCl) GH5 is unable


to precipitate chromatosomes or linear DNA, whereas H5 is still very effective (Fig. 5). Thus, the tail domains, and espe- cially the carboxyl-teminal region, have the ability to cross- link DNA and nucleoprotein particles in accordance with earlier observations (Ali and Singh, 1987).

Concerning the C-tail binding, our studies show that when GH5 binds to chromatosomes it releases about five ions from the DNA surface whereas Watanabe (1986) found that H1 binding to DNA is accompanied by a displacement of about six Na' ions. This indicates that the amino- and carboxyl- terminal tails of H1 and H5 indeed bind in a delocalized manner and not through specific electrostatic linkages (Thomas and Wilson, 1986).

In short, using an in vitro study of the binding of GH5 and H5 to oligonucleotides, to core particles, to depleted chromatosomes, and to oligonucleosomes we provide evidence for the existence of more than one DNA binding site on GH5. We showed that a nucleosome can bind several H5 molecules although only one H5 or GH5 binds in the unique strong binding mode with its globular domain to the two 10-bp ends of the chromatosome, regardless of a reorientation of the core histone octamer. The carboxyl-terminal domain binds in a delocalized way to the linker DNA or to core particle DNA when free linker is absent. The precipitation assay is a useful and quick test for screening of the binding behavior of engi- neered GH5/GH1 mutants and could be a valuable alternative for the assay of Allan et al. (1980), which is based on micro- coccal nuclease digestion.

Acknowledgments-We thank Maria Vanderveken for excellent technical assistance and Remy Loris for the DNase I digestions. Thanks also go to Professor D. Strosherg (Institut Pasteur, Paris) for the amino-terminal sequence determination of the GH5 subfractions. We thank Dr. C. Reynolds for his kind help with the revision of the manuscript.

REFERENCES

Ali, Z. & Singh, N. (1987) J. Biol. Chem. 262, 12989-12993 Allan, J., Hartman, P. G., Crane-Robinson, C. & Aviles, F. X. (1980)

Allan, J., Cowling, G. J., Harborne, N., Cattini, P., Craigie, R. &

Aviles, F. J., Chapman, G. E., Kneale, G. G., Crane-Robinson, C. &

Bates, D. L. & Thomas, J . 0. (1981) Nucleic Acids Res. 9, 5883-5894 Bradbury, E. M., Chapman, G. E., Danhy, S. E., Hartman, P. G. &

Nature 288,675-679

Gould, H. (1981) J. Cell Biol. 90, 279-288

Bradbury, E. M. (1978) Eur. J. Biochem. 88, 363-371

Riches, P. L. (1975) Eur. J. Biochem. 57, 521-528

Butler, P. J. G. & Thomas, J. 0. (1980) J. Mol. Biol. 140, 505-529 Clark, D. J. & Kimura, T. (1990) J. Mol. Biol. 211, 883-896 Clark, D. J. & Thomas, J. 0. (1986) J. Mol. Biol. 187, 569-580 Clark, D. J . & Thomas, J. 0. (1988) Eur. J . Biochem. 178, 225-233 Clark, D. J., Hill, C. S., Martin, S. R. & Thomas, J . 0. (1988) EMBO

Clore, G. M., Gronenhorn, A. M., Nilges, M., Sukumaran, K. &

Crane-Robinson, C. & Ptitsyn, 0. B. (1989) Protein Eng. 2, 577-582 DeHaseth, P. L., Lohman, T. M., Burgess, R. R. & Record, M. T., Jr.

(1978) Biochemistry 17, 1612-1622 De Petrocellis, L., Quadgliarotti, G., Tomei, L. & Geraci, G. (1986)

Eur. J. Biochem. 156,143-148 Glazer, A. N., Delange, R. J . & Sigman, D. S. (1975) in Chemical

Modifications of Proteins (Work, T. S. & Work, E., eds) pp. 71-73, Elsevier Science Publishers, Amsterdam

Graziano, V., Gerchman, S. E. & Ramakrishnan, V. (1988) J. Mol. Biol. 203,997-1007

Graziano, V., Gerchman, S., Wonacott, A., Sweet, R., Wells, J., White, S. & Ramakrishnan, V. (1990) J. Mol. Biol. 212, 253-257

Hofmann, K.-W., Arfmann, H.-A,, Bode, J. & Arellano, A. (1980) Znt. J . Biol. Macromol. 2, 25-32

Igo-Kemenes, T., Horz, W. & Zachau, H. (1982) Annu. Reu. Biochem.

Jin, Y. & Cole, R. D. (1986) J. Biol. Chem. 261, 15805-15812 Klingholz, R. & Stratling, W. H. (1982) J. Biol. Chem. 257, 13101-

13107 Lihertini, L., Ausio, J., van Holde, K. & Small, E. (1988) Biopolymers

27, 1459-1477 Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning:

A Laboratory Manual, pp. 113-122, Cold Spring Harbor Lahorato- ries, Cold Spring Harbor, NY

J. 7, 69-75

Zarbock, J. (1987) EMBO J. 6, 1833-1842

51,89-121

Manning, G. (1978) Q. Reu. Biophys. 11, 179-246 Murray, K., Vidali, G. & Neelin, J. M. (1968) Biochem. J. 107, 207-

Muyldermans, S., Lasters, I. & Wyns, L. (1980) Nucleic Acids Res. 8,

Nelson, P. P., Alhright, S. C., Wiseman, J. M. & Garrard, W. T.

Pennings, S., Muyldermans, S. & Wyns, L. (1989) Mol. Biol. Rep. 13,

Simpson, R. T. (1978) Biochemistry 17, 5524-5531 Sun. J.-M.. Wiaderkiewicz. R. & Ruiz-Carrillo. A. (1989) Science

215

731-739

(1979) J. Biol. Chem. 254, 11751-11760

191-196

245,68-71 Thoma. F.. Koller. T. & Klue. A. (1979) J. Cell Biol. 83. 403-427 Thomas, J. 0. & Wilson, C. M . (1986) &4BO J. 5, 3531-3537 Watanabe, F. (1986) Nucleic Acids Res. 14, 3573-3585 Watanahe, F. (1989) FEBS Lett. 249, 147-150 Widom, J. (1986) J. Mol. Biol. 190, 411-424 Wright, J. M., Wiersma, P. A. & Dixon, G. H. (1987) Eur. J. Biochem.

Zarhock, J., Clore, M. G. & Gronenborn, A. M. (1986) Proc. Natl. 168,281-285

Acad. Sci. U. S. A . 83, 7628-7632

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