Reconstitution of nucleosome core particles containing glucosylated DNA

J. Mol.

Biol.

(1982)

158, 685-698

Reconstitution of Nucleosome Core Particles Containing Glucosylated DNA

Laboratory Xational

Institute

of Molecular Biology Diabetes, and Digestive Bethesda, Md 20205, U.S.A.

of Arthritis,

(Received

15 January

and Kidney

Diseases

1982)

In the DNA from bacteriophage T4. all cytosine residues are replaced by hydroxymethylcytosine residues. from which a glucose group extends into the large groove of the DNA helix. We show that T4 DNA can be reconstituted with chicken rrythrocyte core histones and that micrococcal nuclease can digest this complex to release an - 11 S nucleoprotein particle containing - 145 hasp-pairs of glucosylated DNA. This T4 core particle appears completely “normal” by the criteria of h&one content. molecular weight. sedimentation properties, circular dichroism, thermal denaturation and DNAase I digestion. The results demonstrate that core histones do not interact extensivel,v with the large groove of nucleosomr DNA.

1. Introduction We have been interested in the interactions between histones and DNA that stabilize the structure of nucleosomes. It has been shown previously (McGhee &r Felsenfeld, 1979) that the N7 position of guanine residues in the large groove of nucleosome DNA appears to be almost completely accessible to the small chemical probe, dimethyl sulfate ; the N3 position of adenine in the small groove appears to be unblocked as well, although the chemistry of the reaction makes this statement somewhat less certain. Goodwin et al. (1979) have also concluded, from Raman spectroscopy, that guanine X7 positions remain unbonded in the nucleosome core particle. In this paper, we confirm the above conclusions by a completely independent approach; namely, by forming nucleosomes on a DNA in which the large groove is sterically occluded. The DNA used is from bacteriophage T4, in which every cytosine residue is replaced by a glucosylated hydroxymethylcytosine with the glucose group protruding into the large groove (Lehman & Pratt, 1960; Revel $ Luria, 1970). Our principal finding is that nucle&ome core particles can indeed be formed with glucosylated DNA and seem normal by all criteria examined. The implication is that the core particle histones do not lie in the major groove of the DNA.

2. Materials

and Methods

Bacteriophage T4D were collected by centrifugation of.5 I of Iysat (kindly provided 1)~ I )I N. Nossal) and resuspended in 60 ml of 50 mM-Tris. HC’I. I m&t-EI)TA (pH 8.0) by gentle rocking for 20 h at 0 to 4°C”. Magnesium chloride was added to a final concentrat,iott of 5 tnlt. pancreatic deoxyribonuclease. ribonuclease and hen egg-white lysozyme were each added to 50pg/ml and the mixture incubated for I h at 37°C. Af%er a low-speed clarifying centrifugation. bacteriophage were purified on a cesiurn chloride strap gradient followed 1)~ banding to equilibrium in cesium chloride, dialyzed into 50 mM-Tris. HCI. I ~M-EI)TA (pH 8.0), heated for 10 min at 50°C in the presence of OP,, sodium dodecyl sulfate, digested with 1OOpg of proteinase K/ml for 1 h at 37°C and then with a second addition of 100 pg of proteinase K/ml for I4 h at 37°C. After 3 rxtractions with an equal volume of phettol/chloroform/isoamyl alcohol (X5 : 24 : I by vol.). 3 taxtractions with 2 vol. of chloroform/isoamyl alcohol (24 : I , v/v). sodium acet,at,e MXH added to 0.3 M and the I)R;A spooled out after adding 3 vol. of cold ethanol. The 1)NA we Rdissolved and t,hen shtbarcad by passing several times through a 21 ga,uge syringe net~dle. and the entire series of proteinase K digestions and organic ext,raction steps was repeattd. I)SA preparations had double-stranded molecular weights of 16 x lo6 to 24 x IO6 with no dct,ectablc singk-strand nicks. as determined by neutral and alkaline centrifugation. respectively (Studier. 1965). Enzymic analysis (see below), as well as equilibrium densitjy centrifugation in neutral cesiunr sulfate (Erikson & Szybalski. 1964) verified that the DNA was fully glucosylated. Several experitnents were also repeated with 1’4 DNA obtained commercially (Miles). with essentially identical results. Sveral tnethods of histone preparation were used : (I ) acid extraction of stripped t1uc.k erythrocyte chromatin (kindly provided by I)r K. I). (‘amc,ritti-Otero): (2) acid extraction of isolated chicken erythrocyte core particles: and (3) salt dissociation of chicken erythroc>,tch chromatin according to the tnethod of Simon & Felsenfeld (1979) (kindly provided by I)rs .I. Nickel and B. Emerson). All preparations gave essential!~ identical rest&s. Micrococcal nuclease (Worthington) was furt,her purthed as described by Fuchs cf crl. (1967) and assayed as described by Cuatrecasas ut cl/. (1967). Pancreatic deox~ribonucleast~ (Worthington) was further purified by passage through a lima-bean t,rTpsin inhibitor ctrlutnlt (Otsuka & Price. 1974) followed by a, phosphocellulosr c&tmrt (Salnlkow C/ (I/.. 1970). Sediment,ation velocity. sedimentation equilibriunt and equilibrium density gradients were performed in a Beckman tnodel E analytical ultracentrifuge equipped with scauner and absorption optics. and int,erfaced either to a Hewlett-Packard 2lOOA or 1000 comput~er. Sedimentation coefficients were determined in a solvent of 0.1 M-NaU. 5 mnz-Tris. HC’I (pH 8.0). 0.1 mM-EDTA at t,emperatures of 17 to IHY’ and rotor speeds of 24.000 to 28.000 revs/mitt. Thertnal melting profiles were obtained in cit,hrr a Beckman Acta III or a Car. %I!) spectrophotometer interfaced t,o the above computers. Samples \vere extensively dialyzed against I mM-sodium cacodylate, (PI rn,w-EDTA (pH 7.0). buhblrd with helium. overlaid with dodecane and heated at a rate of 05 deg.(‘/miu. Urcular dicahroism spectra were obtained on a Jasco .I-5OOA spectropolarimeter. The coupled hexokinase/glurose-6-phosphate-dehvdrogc,rtase assay ((,‘albioc~hetn) nas used bo determine glucose concentrations. DNA samples were’ susprndrd in I M-HCI and heated at 100°C for 2 h to allow complete hydrolysis of the glycosidic bond (I’igman & Goepp, 1948: Lehman & Pratt. 1960): after cooling arid ttc~utralization with a c~alculated amount of concentratid atnmonium hydroxide. samples were Iyophilized repeatedly and finally redissolved in 1 &r-‘I’ris. HCl (pH 8.0) at a DNA concent,ration of 0.5 to 1 mg/ml. Histone-DNA reconstitutions were generally performed using the salt/urea dialysis method described by Camcrin-Otero ii! nl. (1976). with mitt& tttodilications. DNA concentrations were 0.2 to 0% trig/ml and core histones were usually present at I g/g of DNA. Reco~tstif;ution was also perfortned by overnight inculcation of histones and I)NA in 0.2 M-NaCY at 37°C: however. the yield of core particles was low. Micrococcaal nuclease dinestions of reconstitutes were net-formed at 37-C’ for 15 min : appropriate enzyme concent,rations wet-r determined 1)~ pilot digestions and wet-f usually in

NI’CLEOSOME

(‘ORE

PARTICLE

KECOXSTITI’TION

ti87

the range of‘ @l to 0.4 unit/ml (see e.g. Fig. 1). Digestion buffer was either 5 mhr-Tris. HU (pH 8.0). 0% mm-CaCl,. @I mM-EDTA, or 75 mM-NaCl. 25 mM-Tris’HCl (pH 8.0). 1 mMCaCl,. 05 m&r-spermidine, 015 mM-spermine. The former buffer gave higher vields of core particles. Digestions were stopped by addition of excess EDTA and digestion products separated on isokinetic sucrose gradients containing 0.4 M-NaCI, 50 mM-Tris HCI (pH %O) ~ 1 rnM-EDT&A. Digestion at 0°C gave poor yields of core particles. T4 core particles were digested by DNAase I at 37°C for 15 min m a buffer of 50 mM-Tris. HCI (pH SO). 4 mruY&&l,. 1 mM-Call,, @I mM-EDTA. The DE-4 in T4 core particles was end-labeled with two consecutive additions of T4 polvnucleotide kinase (05 to 2 units/pg DNA) followed bv incubation at 37°C for 15 min, in a. buffer of .5 mM-Tris.HCl (pH 8.0), 5 rnM-dithiothreitol. 5 mm-MgCl,. and an ATP concentration of @3 to 1 pM (1000 to 3000 Ci/mmol). Core particles were m-isolated on sucros~~ gradients and had 10 to 30% of their DNA termini labeled.

3. Results (a) Micrococcal

nuctease can excise a “nucleosome containing glucosylated DNA

core-particle”

Chicken erythrocyte histones were mixed with T4 DNA at a weight ratio of 1 : 1. and then subjected to the reconstitution procedure described by Camerini-Otero et al. (1976). The reconstitute was then digested with increasing levels of micrococcal nuclease, the DNA freed of proteins and electrophoresed on a nondenaturing ST/b polyacrylamide gel (Fig. 1). Over a considerable range of enzyme concentrations, a prominent DNA fragment can be observed, which is of approximately the same size as the DNA isolated from chicken erythrocyte core particles. The DNA fragment obtained from micrococcal nuclease digestion of T4 DNA histone complexes migrates as if it. were slightly larger (apparent size of 155 bpt) than the 145 bp DNA fragment isolated from chicken erythrocyte core particles. This larger apparent fragment size is not a result of the reconstitution procedure. since the core particles isolated from a parallel reconstitution with Escherichia coli DNA yield a DNA fragment size of 147 bp (see Fig. 7, below). The slower rlectrophoretic migration undoubtedly reflects the added mass of glucose residues rather than an increase in the number of base-pairs contained in a core particle: this is made more clear in the denaturing gels shown below. A “dime? band as well as submonomer bands can also be detected. No bands are seen in digests of proteinfree T4 DNA (data not shown). Figure 2 shows a typical sucrose gradient profile of the material obtained after micrococcal nuclease digestion of either h&one-T4 DNA or histone-E. coli DNA reconstitutes (using enzyme levels shown in pilot experiments to yield maximal c&ore particle production). The major nucleoprotein peak sedimenting close to 1 I S in each case represents 30 to 350,; of the total input DNA, showing that core particle reconstitution is equally efficient with glucosylated and non-glucosylated DNA. (b) Composition of the 7’4 core partide Three kinds of measurements isolated from the T4-DX&hist’one t .Al)I)rpviafion usrd : hp. base-pairs

show that the - 11 8 nucleoprotein particles complexes (hereafter referred to as T4 core

abcdefghg

-?94bp - 118 bp s-

72bp

particles) contain DNA a-it,11 essentially the same base composition and glucoses cont,ent as t)he input Dr\‘A. First. acid hydrolysis of glycosidic bonds followed by enzymatic detection of any released glucose yields an estimabe of 20+_8($, (w/u.) of’ glucose in the DNA of T4 core particles. compared to 11 + 2”;, for t,he input DNA. Second, DNA purified from the T4 core particles has a melting temperature (f,) 3 deg.V lower than the t, of the high molecular weight starting DKA (data, not shown : see also Fig. 5. below). A t, depression of 2.5 to 3 deg.C’ would be expected. solely due to the lower molecular weight (estimated from Fig. 3 of Brothers P/ (I/. 1965). and thus DXA contained in the T4 core particles has the same hast composition (within -2”;) as overall ‘I’4 DNA. Third. DSA isolated from the T-4 core particles has the same median density in neut’ral c&urn sulfate gradients as does native T4 DKA, thus indicating the same average rxtent of glucosyla.tion (Erikson 8i Szghalski, 1964) : (this last estimate is only semi-yuantitative due to the size-dependent peak broadening). Figure 3 shows that the peak sedimenting at - 11 S in the sucrose gradients ot

Bottom

Fraction

number (0)

2r,

0

1

5

IO

15 Froctlon

20

25

30

number

(b)

FIG: 2. (‘ore histones ww reconstituted onto: (a) ‘I?4 DNA. or (1)) IC. co/i DNA; the complexes UWP digrstrd with micrococcal nuoleaxe and nucleoprotrin products WWP separated on isokinetic sucrose fq1dient.s.

Figure 2 contains all four core histones in apparently equimolar amounts. Peak areas of densitometer scans were measured and indicate that the content of each histone species in the T4 particle was, on average, 1%+0.13 times that of the same histone in the native chicken erythrocyte core particle. The molecular weight of the T4 core particles is -2.1 x 105, as estimated from sedimentation equilibrium. This value is sufficiently accurate to demonstrat,e that the particle contains a histone octamer. rather than a tetramer or dodecamer. (c)

Further

characterization

of the

T3 core particle

(i) Sedimentation The median sedimentation coefficient (s~~,~) of T4 core particles is 12.0+@08 S (mean + standard error, from four independent isolations). This is 5.00/O higher than the s20,W of chicken erythrocyte core particles measured under the same

a

solvent conditions. expected solely from DNA, with no other essentially the same

b

C

An increase of -Y), in the s value of core particles would be the increase in molecular weight due to glucose residues on the alterations in shape or hydration. Thus. the T-I core particle has frict’ional coefficient as a normal core particle.

Figure 1 shows that T1 core particles have the same depressed circular diohroic spectrum as native core particles at wavelengths greater than 260 nm. The mean residue ellipticity at 283 nm is 1500 (deg. cm’/dmol phosphate), slightly higher than the value of 1300 reported for calf thymus and chicken erythrocyte core particles (Simpson. 1978 : Watanabe & Iso. 1981) but’ certainly within the range

NUCLEOSOME

CORE

PARTICLE

RECOSSTTTI~TION

69 I

- 10,000 250

260

270

280 Wavelength

290

300

310

320

(nm)

VI<:. 4. (kwkr dichroir spectrum of T4 core particles (-n--A-) addition of @loi, sodium dodecgl sulFate. Mean deg. cm2/dmol phosphatr.

before residue

(- 0 ellipticitg

0 - ) and after IS] is in units of

from 1000 to 2000, reported by De Murcia et al. (1980) for individual core particle preparations. The spectrum of the T4 core particle also exhibits the expected shoulder at w 275 nm and the small negative peak at 295 nm (ellipticities of 400 and -900 deg. cm’/dmol, respectively). Thus. as judged by circular dichroism, glucosylated DNA can adopt essentially the same conformation in the core particle as unglucosylated DNA. Addition of O*lo/, sodium dodecyl sulfate causes the spe&um to revert to that of B form DNA. (iii) Thermal

devsaturation

Figure 5 shows that nucleosome core particles either reconstituted from T4 DNA (i.e. the sucrose gradient peak shown in Fig. 2) or isolated from chicken erythrocytes show biphasic thermal melting profiles, with 35 + 2% of the overall absorbance change occurring in the first phase. The midpoint temperatures of both phasesare 3 deg. lower for the T4 DNA-containing particle whereas a difference of approximately 4 deg. would be expected solely from the difference in DNA base composition. As shown previously, the first phase of the denaturation is reversible (Weischet’ et al., 1978) and can be analyzed thermodynamically (McGhee & Felsenfeld, 1980). The most important result obtained from the thermal denaturation of Figure 5 is that the t, of this first phase is 19°C + 1 deg.C higher than the t, of the naked DNA : this t, shift is the same within experiment’al error for the T4 and chicken erythrocyte core particles. That is, core particle histones stabilize glucosylated DNA to the same extent’ that they stabilize unglucosylated DNA. (iv) Digestion

with

DNr4ase

I

T4 core particles were digested with increasing concentrations of pancreatic deoxyribonuclease (DNAase I) and the DNA was isolated, denatured and labeled at

30

40

50 60 70 Temperature (“C)

80

90

the 5’ ends with [3zP]ATP and T4 polynucleotide kinase. An autoradiogram of a denaturing gel is shown in Figure 6(A). DNAasc I digestion of 7’4 core particles yields a regular array of subnucleosome bands, as first reported by No11 (1974) for nuclear digests. The T4 bands are spaced only roughly at ten-base intervals: a quantitative analysis of electrophoretic migrations using unglucosylated standards, reveals an apparent) spacing of ll.S_+O.l bases between band centers. I%,, higher than the 104base interval found in natural core particles (Lutter, 1979). Thirteen discrete subnucleosome bands can be counted and the intact T4 core particle DNA migrat)es as if it had a single-strand length of 166 bases. Most or all of this apparent 14$ increase in length can be reasonably ascribed to the lo?;, increase in mass per unit length arising from glucose residues. Gel resolution of T4 subnucleosome DNA fragments is poor. as would be expected if each fragment size contains a statistical distribution of glucose residues. It is known that even unglucosylated hydroxymethylcytosine residues can cause anomalous electrophoretic migration of oligonucleotides (van Ormondt & Hattman, 1976).

SI’(‘LEOSOME

(1

b

(‘ORE

c

PARTICLE

693

RECONSTITITTION

b

c

d

e

f Band

180 160

160 147 122

-I-

110

1_

90 76 67 .76 67

-3

26

-2

.26

9

FIG:. 6. Denaturing gels of DNAase l-digested T4 core particles. (A) After DNAase I digestion of T4 core particles. DNA was purified. denatured. 5’.termini dephosphorylated. and 32P-labeled with T4 polynucleotide kinase and electrophoresed on a loo/,0 polyacrylamide gel containing 7 M-urea. Nuclease concentrations were: lane a, no enzyme control: lane b, 0.02 unit/ml: and lane c. 12.5 units/ml. 1Tnglucosylated size standards (pBR322 restricted with Mql) are indicated (in bases). (R) T4 core particles were “P-labeled at 5’ ends with T4 polvnucleotide kinase. digested with DNAase I and the DNA purified. denatured and.electrophoresed on a'lO?& polq’acrylamidegel containing 7 M-urea. Suclease concentrations were: lane a. no enzyme control : lane b, @16: lane c. 0.32: lane d. 0.63; lane e, 1%: lane f. 2.5 units/ml. On the left single-strand unglucosylated DNA standards are indicated : on the right, hand numbers are indicated as multiples of - 11.5 bases (see text).

no.

6114

,I.

I).

MvC:HEE

AND

(:.

FfJLSENFEI,I)

As first described by Simpson & Whitlock (1976). cutting at individual sites within the core particle can be followed b-y end-labeling the DNA prior to digestion. T4 core particles were first labeled at t)he 5’ ends of the DIL’X. re-isolated on sucrose gradients. digested with DNAase I and then the DNA was isolat)ed. denatured anti electrophoresed. As shown in Figure 6(B). the T4 core particle shows the same wriw of bands and the same modulation of band int’ensities seen in cnontrols of chicken eryt)hrocyte core particles and first described by Simpson Hr W?hitlock (1976); i.e. lower intensity at bands number 3, 6, 8 and 11. The different fragments migrate with an apparent size-interval of 11.4 20.2 bases. measured relative to unglucosylated markers.

(v) Ihtribution

of glucoseresidues within the T4

(:(Jrc

part

iclr

Although the overall glucose content of the T4 DNA that was assembled into core particles is close to that of the initial bacteriophage DNA, there is the possibility that, at certain positions within the core particle. the presence of a glucose residue is forbidden. To determine bhe dist,ribution of glucose residues along the DNA, the T4 DNA fragment was isolated from the - 11 S part’icle, labeled at the 5’ ends with 32P, and subjecte d to the puaninr-specific sequencing resction described by Maxam & Gilbert (1977). The distribution of guanine residues starting from the 5’ end of the DNA should correspond exactly to t,he distribution of’ hydroxymethylcytosine and hence of glucose residues sta,rting from the 3’ end of’ the complementary strand. Positions where glucose is forbidden should correspond to blank areas on the gel autoradiogram. The result, of such an experiment is shown in Figure 7. The analysis is limited by t,wo fart,ors : (1) the poor resolution of the> glucosylated DNA fragmenm (even on the best 0.4 mm t,hick 15o/o gels, fragments of T4 DNA longer than 20 nucleotides are not resolved into individual bands. in contrast to non-glucosylated DNA, which loses resolution only for >70 nucleotides); (2) the presence of discrete subnucleosome bands arising from the initial micrococcal nuclease digest. These are also present and of approximately equal intensity in the E. coli DNA control and are much more prominent in reconstituted core particles than in core particles isolated from nuclear digests. Within these technical limitations, there seems to he no large region (longer than 3 to 5 nucleotides) where a glucose residue cannot reside.

4. Discussion We have demonstrated that core histones can be reconstituted onto glucosylated T4 DNA and a nucleoprotein particle can be excised by digestion with micrococcal nuclease. The overall particle yield is essentially the same with T4 DNA as it, is with E. coli DNA used as the non-glucosylated control. All physical and chemical properties examined are consistent with the conclusion that these T4 core particles are normal nucleosome core particles. with an apparently uniform probability that a glucosylated hydroxymethylcytosine residue occurs at all positions in the DNA. Because of the technical limitations described with respect to Figure 7. we cannot, rule out conclusively the possibilitp of a region perhaps three to five nucleotides

SI’CLEOSOME

CORE

PARTICLE

ti!).‘,

RECONSTITI’TLOS

J -

124 104

-

1z E .90 76 67

-

34 26

21 18

-

15

9

Fit:. i. Distribution of guanine residues in core particles. DNA. 3ZP-labeled at the 5’ termini. was purified from reconstituted T4 core particles, reconstituted E. coli core particles 01‘ native chicken rrythrocyte core particles, reacted with 50 miw-dimethglsulfate for 0. 10 or 30 min at 20°C (as indicated). the DNA backbone cleaved at methylgusnine residues and the single-stranded products separated on a lO’?,, polyacrylamide gel containing 7 ~-urea. Size standards are indicated. in bases (pBR322 cleavrd with Hcw11 I or MspI).

long, somewhere near the middle of the T4 core particle, in which the presence of a glucose residue is forbidden. The presence of a number of such regions becomes unlikely, purely on statistical grounds. For example (assuming random base composition), there is a probability of less than OGO4 (i.e. 0.67i4) of selecting a 140 base-pair piece of T4 DNA in which one position out of every ten does not contain a glucose in either strand. The actual degree of steric hindrance provided by these glucose residues can be appreciated from the drawings in Figure 8. In Figure 8(a) a five base-pair segment of DNA helix, with the sequence C-C-G-T-C, is viewed from the large groove. The glucose residues are arranged to minimize van der Waals’ contacts, but otherwise are in arbitrary positions. An impression of the maximum possible steric hindrance

WH

J.

I).

Mr(;HEE:

AN\‘)

(:.

~EI,SE:SFEI,I)

that could he provided by a glucose residue is shown in Figure X(b). Here. a glucosylated hydroxymethylcytosine . guanosine base-pa,ir is viewed along the helix-axis and the glucose residue is rotated around both (‘-0 bonds of thti glycosidic linkage as well as around the C-C’ bond joining the exocayclic methyl group to the cytosine ring. The actual disposition of the glucose residues in t,he DSA large groove is not known with any certainty and many of the conformations shown in Figure 8(b) art’ probably not allowed, due to interference with the remainder of the DNA helix. 11’~

(0 I

(b)

from i hv largf~ FII:. A. (a) A .i bp segment of tloul,lr-st,randed I)NA (sequenw 6’ (‘-(‘Y :-‘r-f’ :I’). viwwl groove. (~lwose rrsidues arc shown as solid sticks and halls: the (lrox~riI)osrphosphate backbone is shown as a thick line. I)NA co-ordinat,rs were taken from Arnott & Hukins (1972). (~lucosr co-ordinate were adapted from Berman & Kim (198X). (b) (:lucosylatrd h~drr)x~mrth~l(,vtosinr.Ruanine base-pair as viewed along the DXA helix-axis. The thin lines are a supwposition of 27 glu~~~sc configurations. resulting from 120” rotational stages around each of thr 3 rotatable bouds connecting the glucose rwidut~ to the qtosine ring.

can, however. put limits on the extent to which core particle formation is likely to constrain the glucose conformations. For example, if core parMe formation decreased the number of allowed glucose conformations by half. the stabilization free energy would be reduced by RT In 2 = 0.4 kcal/mol of glucosylated base-pair or -0.14 kcal/average base-pair. This lowered free energy should in t,urn lead to a -7 deg. shift in t,. which could have been detected in Figure 5 but was not. Since the melting profiles actually indicate that the overall stabilization free energy of the T4 core particle is essentially identical to that, of the native core particle isolated from chicken erythrocytes, there is no evidence t,hat the glucose residues are being pushed aside during core particle formation. (lonversely, if sections of histones have to be pushed aside in order to assemble onto glucosylated DNA. the

NI;(ILEOSO~~liIE

CORE:

PARTI(‘LE

695

KECOSRTITI’TTON

melting profiles indicate that these histone sections provide little to the net stabilization free energy?. Zn summary, we have been able to reconstitute nucleosome core particles in which the large groove of the DNA is occupied by glucose residues. This argues against histones or parts of histones occupying regions of the large groove of nucleosome DNA. Our previous study using a chemical probe also indicated small groove accessibility (McGhee & Felsenfeld, 1979). The combined results suggest that contacts between histones and DNA are localized to the phosphodiester backbone. Even the histone-phosphate interactions seem to be few (McGhee Kr Felsenfeld, 1980). Our experiments support a model for the nucleosome core part)icle in which the DNA is ext’remely accessible to the solvent and available for int,eraotion with other molecules. The current low-resolution models derived from diffraction studies (see. e.g. Klug rt al., 1980) certainly seem compatible wit,h t’his view. It has been argued elsewhere (McGhee ef ul.. 1980) that’ t’his exposed DNA might play an important’ role in the higher-order structure of chromatin. The aut,hors thank I>rs Camerini-Otero. materials as described in the text. They pr
Emerson. Nickel and Nossal for providing various also thank Dr S. Zimmerman for his help in the for expert, preparation of the manuscript.

RFFERENCES A Arnot,t. S. W. & Hukins, I). W. L: (1972). Riochem. Riophys. Res. Covnmun. 47. 1504&1509. Berman. H. M. & Kim, S. H. (1968). dctcl Crystnlbgr. sect. R. 24. 897-904. Camerini-Otero. R. D., Sollner-Webb. B. & Felsenfeld. G. (1976). Cell. 8. 333-347. Crothers. T). M., Kallenbach. N’. R. & Zimm. B. H. (1965). J. Mol. Biol. 11, 802-820. Cuatrecasas. J’., Fuchs. S. & Anfinsen, C. 8. (1967). J. Rio/. Chem. 242, 1541-1547. De Murcia. G.. Mazen. A.. Erard. M.. Pouyet,. M. 8: Champagne. M. (1980). Sucl. ilcida Rrs. 8. 767.-779. Erikson. R. L. & Szybalski. W. (1964). P’irology, 22, 111-124. Fuchs. S., Cuatrecasas. I’. $ Anfinsen. C. B. (1967). J. Biol. Chem. 242, 4768-4770. Goodwin. D. C.. Vergne. tJ., Brahms. J.. Defer. ru’. & Kruh. J. (3979). Biochemistry, 18.2057-

2064. Klug.

A.. Rhodes.

T).. Smith,

J..

Finch,

J. T. $ Thomas.

tJ. 0. (1980).

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287.

509-516. Lehman. I. K. 8r Pratt. E. A. (1960). .J. Rio/. Chrnr. 235, 3254-3259. Lut’ter. I,. C. (1979). Xucl. Acids R~s. 6. 41-56. Maxam, A. X. & Gilbert, W. (1977). Proc. :lixl. =Icnd. Sri.. V.S.,1. 74. 560-564. Mc(:hee, ,J. 1). & Felsenfeld. (:. (1979). f’roc. ht. Acnd. Sci.. 1rS.A. 76. 2133-2137. Mc(:hee. .J. 1). &I Felsenfeld. G. (1980). Sucl. Acids Re.s. 8. 2751-2769. McGhee. ,J. I).. R’au. D. C.. Charney. E. & Felsenfeld. U. (1980). &II. 22, 87-96. Nell. pu3. (1974). Sucl. Acids Rrs. 1, 1573-1578. Otsuka, A. S. 8: Price. P. A. (1974). dl&. Biochem. 62. 180-187. Pigman. W. W. B Goepp, IX. 31. Jr (1948). The Chemistry of the Carhoh~ydmtes. pp. 202-206. Academic Press. New York. Revel. H. R. & T,uria, S. E. (1970). ,Jnnzc. Rav. (:r~et. 4, 177-192. t This simple calculation applies most directly to the reversible melting of the termini of the core particle DSA (McGhee & Frlsenfeld. 1980). It can be applied to the melting of t,he interior DNA in so far as its f, is determined by the breakdown of the hixtone-DNA interactions and not by the breakdown of histone--histonc int,eractions.

698

.J. 1). MP(:HEE

AXI)

(:. FEl,S~SFEJ,l,

Salnikow. J.. Moore. S. Di St)ein. IV. (1970). ./. Rio/. Phrr,/. 245. T,KH;S-5690. Simon. R. H. d Felsenfeld. (:. (1!179). SW/. .-Irids 12~~s.6. W-696. Simpson. R. 7’. (1978). Hiochrmistry. 17. 5,X4--5531. Simpson. R. T. & Whitlock. .I. P.
Edited

by A. Kluy