Proc. Natl. Acad. Sci. USAVol. 92, pp. 7060-7064, July 1995Biochemistry

High-affinity binding sites for histone Hi in plasmid DNAJULIA YANEVA*t, GARY P. SCHROTH*, KENSAL E. vAN HOLDE*, AND JORDANKA ZLATANOVA*t*Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331-7305; and Institutes of tMolecular Biology and tGenetics, BulgarianAcademy of Sciences, 1113 Sofia, Bulgaria

Contributed by Kensal E. van Holde, April 6, 1995

ABSTRACT The interaction of histone HI isolated fromchicken erythrocytes with restriction fragments from plas-mids pBR322 and pUC19 was studied by gel electrophoresis.Certain restriction fragments exhibited unusually high affin-ity for the histone, forming high molecular mass complexes atprotein to DNA ratios at which the other fragments did notshow evidence for binding. The highly preferred fragments areintrinsically curved, as judged by their electrophoretic mo-bility in polyacrylamide gels, by computer modeling, and byimaging with scanning force microscopy. However, controlexperiments with either curved portions of the same frag-ments or highly curved kinetoplast DNA fragments showedthat the presence of curvature alone was not sufficient forpreferential binding. By using various restriction fragmentscentered around the highly preferred sequence, it was foundthat the high-affinity binding required in addition the pres-ence of specific sequences on both sides of the region ofcurvature. Thus, both curvature and the presence of specificsites seem to be required to generate high affinity.

The members of the lysine-rich histone family are known tointeract with the linker DNA between nucleosomes, sealingtwo complete turns ofDNA around the histone octamer (1, 2).By binding to the DNA entering and exiting the nucleosome,the linker histones fix the entry/exit angle of the DNA (3) andthus, together with other constraints, such as length andrigidity of the linker DNA, contribute to the formation ofirregular chromatin fibers, even at low ionic strength (4, 5).The participation of the linker histones in chromatin folding athigher ionic strength is also well recognized (1, 2). There is alsoevidence that the linker histones participate in transcriptionalregulation (reviewed in refs. 6 and 7). Exactly how the linkerhistones fulfill all these functions is not clear.The belief that the linker histones interact primarily with the

DNA in chromatin has led to numerous studies of theircomplexes with DNA (reviewed in refs. 8 and 9). The inter-action is a complex phenomenon dependent upon the ionicconditions and the protein to DNA ratio. In addition to thenonspecific, electrostatically determined affinity of Hi mole-cules for DNA, there are also other, more specific types ofinteractions. Some of these are DNA structure specific, like thepreferential binding to crossovers of double-stranded DNAs(10-12), for which the synthetic four-way junction is a goodmodel (13, 14). Others may involve interactions between Hiand specific DNA sequences (ref. 15; for a review of the earlierliterature, see ref. 9).

In a continuation of our previous studies on interactions oflinker histones with DNA, we have made the unexpectedobservation that histone Hi from chicken erythrocytes bindswith high affinity to a small subset of a broad population ofrestriction fragments from plasmids pBR322 and pUC19.Although these high-affinity fragments exhibit intrinsic cur-vature and the presence of curvature appears to facilitate

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binding, curvature alone is not sufficient to account for thestrong binding observed.

MATERIALS AND METHODS

Isolation of Histone HI. Chicken histone Hi was isolatedfrom erythrocytes as described (16) and checked for purity bySDS/PAGE (17). The concentration of Hi was determinedspectrophotometrically, by using an extinction coefficient of1.85 ml cm-1 mg-1 at 230 nm (18).

Purification and Manipulation of DNA. Plasmid DNA wasprepared with Qiagen purification kits (Chatsworth, CA).Isolation of DNA fragments from agarose gels was performedwith QIAEX gel extraction kits (Qiagen). Restriction diges-tions were carried out with enzymes and buffers from NewEngland Biolabs. The reactions were stopped by addition ofEDTA, and the samples were extracted with phenol/chloro-form (1:1) and precipitated with ethanol. The pellets weredissolved in 10 mM Tris-HCl, pH 7.5/0.1 mM EDTA at aconcentration of 0.1 mg/ml. The concentration of DNA wasdetermined spectrophotometrically by using an extinction co-efficient of 20 ml cm-1 mg-1 at 260 nm.

Construction of the Circular Permutation Clone pGBP-kDNA. A 43-bp, double-stranded oligonucleotide sequence(5'-GATCCAAAAAATGTCAAAAAATAGGCAAAAA-ATGCCAAAAATC-3') located at the "bending center" ofkinetoplast DNA (19) was inserted into the Bgl II restrictionsite of plasmid pCY-7, constructed to study DNA bending bythe circular-permutation assay (20, 21). When our new con-struct (pGBP-kDNA) is digested with either BamHI orEcoRV, two 445-bp fragments are created which differ only bythe position of the bend within the fragments (21). TheBamHI-generated DNA fragment places the bend near oneend of the molecule, while the EcoRV-generated fragmentplaces the bend near its center.

Analysis of Histone Hi-DNA Interaction. Histone Hi wasincubated with DNA in 15 ,ul of 50 mM Tris HCl, pH 7.5/20mM NaCl/1 mM EDTA/0.1% Triton X-100 for 40 min atroom temperature. The mixture was routinely electrophoresedthrough 2.2% agarose gels at 8-10 V/cm at room temperaturein Tris acetate/EDTA (TAE) buffer (40 mM Tris acetate, pH7.5/1 mM EDTA). Where indicated, electrophoresis was alsoperformed on preelectrophoresed 6% polyacrylamide gels(acrylamide to bisacrylamide ratio of 29:1; 10 V/cm, 4°C) ineither TAE or E buffer (40 mM Tris HCl, pH 7.2/20 mMsodium acetate/i mM EDTA). Gels were stained with 0.1 ,jgof ethidium bromide per ml for 30 min, briefly destained, andphotographed on either Polaroid 55 or 667 (Polaroid) film.The apparent lengths of DNA fragments on polyacrylamidegels were determined by using the 123-bp DNA ladder(GIBCO/BRL), which has been shown to be a useful, nonbentmolecular mass standard (22).

Distamycin A Treatment of DNA and Hi-DNA Complexes.In some experiments, histone Hi binding was also performedin the presence of the DNA-binding drug distamycin A (Sig-ma). The DNA was preincubated with the drug for 20 min atroom temperature, or.the drug was added to the mixture justbefore histone Hi.

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Proc. Natl. Acad. Sci. USA 92 (1995) 7061

Computer Modeling of DNA Curvature. DNA curvature inrestriction fragments was modeled on a DEC Microvax II GPXworkstation using the BIOCAD program of P. Shing Ho (21).Briefly, the program allows building of models of polynucle-otides of defined sequence, helical repeats, and "wedge"angles, assuming a helical repeat of 10.5 bp and the wedgeangles defined in Bolshoy et al. (23).

RESULTSHistone Hi Selectively Binds Certain Restriction Frag-

ments from Plasmids pBR322 and pUC19. Earlier experi-ments have suggested the possibility that histone Hi maypreferentially bind to certain DNA structures and/or DNAsequences (see the Introduction). As part of a general searchfor such strong binding sites, different populations of restric-tion fragments from plasmid pBR322 were titrated with in-creasing amounts of histone Hi, and the binding was moni-tored by agarose gel electrophoresis.The titration of most mixtures of restriction fragments from

pBR322 with Hi showed no selectivity in binding: all frag-ments bound Hi equally well, as increasing the amount of Hiled to the disappearance of all the DNA bands from the gelsat about the same concentration of added HI. This phenom-enon, observed repeatedly, presumably results because thebound DNA is converted to insoluble complexes which aretrapped in the sample wells. However, a unique result wasobserved with a Dra I/BstNI double digest of pBR322. In thiscase, a 550-bp fragment was reproducibly titrated out at muchlower Hi ratios than the other fragments (Fig. 14). At a weightratio of Hi to total DNA of 0.2 (1 mol of Hi per -150 bp), thequantity of the 550-bp band in the gel was significantlyreduced, and at a ratio of 0.7, it was totally lost. In some cases,a smeared band of much lower electrophoretic mobility wasobserved (see Fig. 1A), but in most experiments, the DNAband simply disappeared from the gel altogether. The remain-ing bands showed no change in intensity until much higher Hito DNA ratios. A quantitatively similar result was obtainedwith plasmid pUC19 (Fig. IB). The 766-bp fragment from theDra I/BstNI double digest of pUC19 was preferentially boundby Hi, as it disappeared from the gel before the other frag-ments were diminished in intensity.

It is well known that the linker histones generally preferbinding to longer over shorter DNA molecules (8). UsingDNAfragments that are successive multiples of the same sequence(the 123-bp ladder), we have shown that this preference is validin the range of fragment lengths studied (results not shown).Despite this general rule, it is obvious that the peculiar be-

O! r-~0 (' U0 I- 0 c LO r- 0 c(4 rN- 0o o o .- ca H1:DNA

FIG. 1. Titration of Dra I/BstNI restriction enzyme digests ofpBR322 (A) and pUCi9 (B) with increasing amounts of histone Hl on

agarose gels. The protein to DNA ratios (wt/wt) are designated belowthe lanes. The arrows denote the specific fragments from bothplasmids that are preferentially bound by histone Hl (see text).

havior of the Dra I-BstNI pBR322 fragment cannot be ex-plained in this way, since there are four DNA fragments whichare larger than the 550-bp fragment selectively bound by Hi.The pUC19 fragment is only marginally larger than fragmentsthat are quite unaffected.

In an attempt to find the minimal subfragment that wouldstill be capable of this kind of selective binding by Hi,additional titration experiments were performed with restric-tion fragment populations containing shorter or longer frag-ments centered around the preferred Dra I-BstNI fragment.Trimming the fragment from either the 5' or the 3' end orusing fragments extending beyond the original fragment in onedirection with the other end trimmed led to a loss of theselectivity of binding (Fig. 2).The Preferred DNA Fragments Are Retarded on Polyacryl-

amide Gels. In a search for structural features that maydetermine the preference of Hi binding, we analyzed theelectrophoretic behavior of various restriction digests ofpBR322 and pUC19 in polyacrylamide gels. It is known thatanomalously slow migration in polyacrylamide gels is a featurecharacteristic of intrinsically curved DNA molecules (19, 25-28). Analysis of the electrophoretic behavior of 12 pBR322 andpUC19 restriction fragments indicated that only 3 of themdisplayed the mobility expected on the basis of their length(Table 1), while 7 migrated slightly faster than expected. Theremaining two fragments ran significantly more slowly thanexpected, suggesting the presence of intrinsically curved DNAstructures in these molecules. These two curved DNA frag-ments were the two molecules which were selectively bound byhistone Hi (see Fig. 1 A and B).Computer Modeling of the Hl-Selected DNA Fragments.

The results of the electrophoretic analysis strongly suggestedthe presence of intrinsic curvature in the DNA moleculeswhich were selectively bound by histone Hi. Accordingly, acomputer program was used to model the three-dimensionaltrajectory of these molecules. The program uses the 16"wedge" angles developed by Trifonov and colleagues (23) and

3607 3757 3941 4168 4341 29 130 229 375 523

a) X)CL I

789

DZ0 E0 0u u U 0

Cl olI m z m L

4340VZ21 92

Dral BstN

Dral E2:]SspI

Dral EcoR1Dral Hindlil

SspI .Hn IHIndlSspI BstN

Sspi NhelSspI BamHI

EcoOl109 r .]EcoOlQ9lEcoRI 3 BstNIEcoRI NhelHindlll -:- BamHI

PstIl _ a SspI

FIG. 2. Restriction map of pBR322 encompassing the restrictionfragments tested for preferential histone HI binding by the assayshown in Fig. 1. The positions of restriction cleavage are designatedabove the map. The fragment which is preferred by histone Hl isshown by a black bar, whereas the other fragments tested are shownby dotted bars. In all cases, Hl titration was conducted on the entirepopulation of fragments obtained after digestion. The hatched barrepresents the fragment described as selected by histone Hl byWellman et al. (24). It should be noted, however, that in this case, Hltitration was conducted on the isolated fragment, in the absence of anyother competing fragments.

I-1

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(1

Proc. Natl. Acad. Sci. USA 92 (1995)

Table 1. Electrophoretic behavior of restriction fragments from plasmids pBR322 (R) and pUC19(UC) on polyacrylamide gels: Comparison between actual and apparent length

Fragment

Dra I-EcoRI (R)BstNI-BstNI (R)BstNI-BstNI (R)Dra I-BstNI (UC)Dra I-Dra I (R)Dra I-BstNI (R)Dra I-BstNI (R)Dra I-EcoRI (R)BstNI-BstNI (R)Dra I-BstNI (UC)BstNI-BstNI (UC)BstNI-BstNI (R)

Position inplasmid*

4359-32301442-2500130-1059

2274-3543249-39412634-32303941-1303941-43591059-1442545-833354-545

2500-2621

Actuallength, bp

32321058929766692596550418383288191121

Apparentlength, bp

275010908401000540540690430290220160120

Apparentlength/actual

length

0.851.030.901.300.780.911.261.030.760.780.841.00

Plasmids pBR322 and pUC19 were digested with the restriction endonucleases shown, and the digestswere separated on 6% polyacrylamide gels. The apparent length (bp) was determined by using a standardcurve of migration versus logarithm of length constructed on the basis of the electrophoretic behavior ofthe 123-bp ladder (see Materials and Methods). The actual length was determined from the knownsequence of the two fragments. The fragments which show retarded mobility on the gels are underlined.Note that these are the fragments selectively bound by HI in Fig. 1.*The nucleotide numbering for pBR322 starts at the unique EcoRI restriction site and that for pUC19 startsat the first T in the sequence ... TCGCGCGTTT ... and proceeds clockwise around the molecule.

a helical repeat of 10.5 bp per turn. The modeling resultssuggest that the curvature is located in one half of the 550-bpDra I-BstNI pBR322 fragment (Fig. 3A) and that a similar kindof structure is present in homologous regions from the 766-bpDra I-BstNI pUC19 fragment (Fig. 3B). The two fragmentsshare about 410 bp of common sequence located at the 5'end/promoter region of the ampicillin-resistance (f3-lacta-mase) gene. Thus, our results indicate that the region near thepromoter of the ampicillin-resistance gene is strongly curvedin both pBR322 and pUC19, which is consistent with previousresults of others (29-31). Furthermore, modeling of the DraI-BstNI pBR322 fragment by the CURVATUR program of R.Harrington yields an almost identical structure (R. Harrington,personal communication).The 550-bp pBR322 fragment was imaged by using the

scanning force microscope operated in the tapping mode (32).The images (data not shown) were very similar to the structurepredicted on the basis of the computer modeling. Thus, on thebasis of the anomalous electrophoretic mobility of the Hi-selected fragments, the computer analysis, and the scanningforce microscopy imaging, we can assert with a reasonabledegree of certainty that these fragments possess significantintrinsic curvature.

ApBR322550 bp

BpUC19766 bp

The Selectivity of Hi Binding Is Lost in the Presence ofDistamycin. The antibiotic distamycin A is known to bindpreferentially to oligo(dA.dT) tracts (33, 34). Binding ofdistamycinA to curvedDNA tends to "straighten" the intrinsiccurvature associated with the phased tracts ofA residues (19,35). To test the extent that preferential Hi binding to thesequences in question may be determined by the DNA cur-vature, the Hi titration experiments were repeated in thepresence of distamycin A. The concentration of the drug to beused was determined by titrating Dra I/BstNI double digestswith increasing amounts of distamycin A and following thechanges in the electrophoretic mobility of the majority of thefragments in polyacrylamide gels at 4°C: above a certaindistamycin concentration the electrophoretic mobility stabi-lized and showed no additional changes upon further increasesin the concentration of the drug.

In the presence of saturating amounts of distamycin A, theselectivity of Hi binding was lost (Fig. 4). The same was true

+ + + + Distamycincn cn LO Hi :DNA

EcoR I

Qsp

FIG. 3. Computer modeling of the fragments selected frompBR322 and pUC19 byhistone Hi (see Fig. lA andB). (A) The 550-bpDra I-BstNI fragment from pBR322. (B) The 766-bp Dra I-BstNIfragment from pUC19. The positions of certain restriction sites shownin Fig. 2 are indicated; they occur at the same positions in bothfragments.

FIG. 4. Agarose gel electrophoresis of a Dra I/BstNI double digestof pBR322 titrated with histone Hi in the absence or presence of 10mM distamycin A. Note that in the presence of distamycin A, there isno selectivity of interaction of histone Hi with any of the fragments,even at high Hi to DNA ratios; on the other hand, in the absence ofdistamycin, the 550-bp fragment disappears from the gel even at an Hito DNA ratio as low as 0.3.

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Proc. Natl. Acad. Sci. USA 92 (1995) 7063

when the population of DNA fragments was preincubated withthe drug before being titrated with Hi. These results suggestedthat the curvature in the DNA trajectory may be an importantdeterminant in the selectivity of Hi binding.

Curvature Itself Is not a Sufficient Condition for Strong HiBinding. The fact that only strongly curved fragments areselected for strong Hi binding might suggest that such curva-ture is a sufficient condition for selectivity. One kind ofevidence to indicate that this is not the case is contained inFigs. 2 and 3. Note that several subfragments which contain themost highly curved portion of the strongly bound fragment (forexample, Ssp I-BstNI and Ssp I-Nhe I) do not exhibit strongbinding to Hi. For another test of the curvature hypothesis, wemade use of the properties of phased tracts of A residues,which are known to be a strong determinant in DNA curvature(19). A 43-bp, double-stranded oligonucleotide containing the"bending center" of kinetoplast DNA (19) was cloned into aplasmid vector designed to study DNA curvature by use of thecircular-permutation assay (20, 21). This plasmid can bedigested by restriction enzymes to yield two types of 445-bpfragments: one in which the bend is located near the middle ofthe molecule and one in which the bend is located near the end.The results of modeling these two molecules are shown in Fig.SA. When these two fragments were mixed in equal amountsand then titrated with Hi, neither of the fragments waspreferred (Fig. SB). Similarly, no preference was obvious whenthe titration was conducted in the presence of competingunrelated restriction fragments from the plasmid "body" (Fig.SC). Finally, when the 445-bp fragment with the curvature inthe middle was present together with the hyperreactive 550-bpfragment, the 445-bp fragment was not selected, whereas the550-bp fragment was (Fig. SD). Thus, the presence of staticcurvature toward the middle of a fragment, although evidentlycontributing to the selectivity of Hi binding to the fragments,is not in itself sufficient to cause binding.

DISCUSSIONThe results reported here show that histone Hi may have astrong preference for certain DNA fragments. The electro-phoretic assay that demonstrates such preference depends onthe reduction in the amount of a specific DNA restrictionfragment that remains at its original position in the gel. Thisreduction is due to the formation of complexes, often so largeas not to enter the gel. Thus, the assay reflects not only theinitial, highly selective binding to a high affinity site(s) on theDNA but also the cooperative binding of additional histonemolecules to the same DNA fragment leading to the formationof high molecular mass, often insoluble complexes. This occursin the apparent absence of binding to other fragments, asevidenced by the lack of either mobility shift or decrease inband intensity.The extremely high cooperativity that is obvious from our

electrophoretic gels is impressive, especially in view of the factthat the salt concentration in the binding buffer was only 20mM. However, Watanabe (36) has demonstrated cooperativityat salt concentrations this low. It is still not clear whether anyhistone Hi binds to the other restriction fragments in themixture; even at relatively high Hi to total DNA ratios, thereare no signs of retardation, which is to be expected uponformation of soluble Hi-DNA complexes.There are at least two independent DNA structural param-

eters that contribute to the preferred Hi binding. One of themis related to the stable intrinsic curvature present in the pre-

ferred fragments. Both fragments recognized here as strongHi binders are curved. This conclusion comes from theelectrophoretic analysis in polyacrylamide gels (Table 1), fromcomputer modeling (Fig. 3), and from direct scanning forcemicroscopy imaging (data not shown). Previously published

A

Fragment A Fragment B

A-B-

H1:DNA

C

in U~ r-- a a_ 6a HtDNA

n

A-

o-o ci Hl:DNA

FIG. 5. Curved kinetoplast DNA fragments are not selected byhistone Hi. (A) Computer analysis of the curvature of the twopermuted DNA fragments (A and B) containing 43 bp of kinetoplastDNA at a different position within the fragment (see Materials andMethods). (B) Titration of the two isolated permuted fragments A andB with increasing amounts of histone Hi, as designated below thelanes. Marker I (MI) is the 123-bp DNA ladder and marker II (MII)is pBR322 digested with Hinfl. (C) Titration of the two permutedfragmentsA and B from plasmid pGBP-kDNA in the presence of otherrestriction fragments from the plasmid. MI is the 123-bp DNA ladder.(D) Interaction of histone Hi with a Dra I/BstNI double digest of therecombinant plasmid containing the kinetoplast DNA insert. Thearrowhead indicates the position of the 550-bp fragment andA denotesthe position of the 445-bp fragment containing the kinetoplast insertwith the curvature positioned at the middle of the fragment. Note inB and C that neither of the kinetoplast permuted fragments ispreferred, either in the presence or in the absence of competing DNAfragments. Note in D that in the presence of histone Hi (right lane)the 550-bp fragment disappears, whereas the 445-bp fragment remainsunchanged.

data have recognized this region of pBR322 as significantlycurved (29-31).That DNA curvature may be important in Hi binding is

suggested from the distamycin A experiments. This drug isknown to straighten curved DNA upon binding (19). When Hititration was done in the presence of distamycin A, all selec-tivity of binding was lost (Fig. 4). An alternative explanationmay be that distamycin simply competes with histone Hi forbinding to oligo(dA-dT) tracts. This interpretation was used toexplain the effects of distamycin on the preferential binding ofHi to scaffold attachment region (SAR) sequences (37).Interestingly, SARs also contain intrinsically curved regions,so it is possible that those results, like these, could be inter-preted in both ways. Additional experiments would be requiredto distinguish between these two possibilities.

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7064 Biochemistry: Yaneva et al.

The presence of intrinsic curvature seems to be a necessarybut not sufficient condition for the selectivity of Hi binding.First, among the numerous restriction fragments tested thereare also others that exhibit curved trajectories but do not bindHi preferentially (see Results). Moreover, in these fragments,the curvature is situated with respect to the ends in a similarway as in the reactive Dra I-BstNI fragment. Further evidencecomes from the behavior of the kinetoplast fragments. Thesecontain significant curvature, situated either in the middle ofthe fragment or near its end (see Fig. 5A), but neither of themshows preferred Hi binding. These fragments are similar inlength to the hyperreactive fragments but cannot competeagainst them.The data in Fig. 2 indicate that preferential binding requires

some specific DNA sequences flanking the curved sequence.One may envisage that the hyperreactive fragments possesstwo sequence-specific, high-affinity binding sites, to which onesingle molecule of Hi can bind initially by virtue of the twoDNA-binding sites present in the globular domain (38). If thebinding affinity of HI to just one DNA site is in the relativelyweak range of Kd i0-4 M, then simultaneous binding to asecond site of similar affinity may increase the total affinity to..10-8 M, which is the affinity observed for the two-sitebinding ofHi to four-way junctionDNA (13, 14). The presenceof intrinsic curvature may facilitate bringing these two sitesinto spatial proximity, such that Hi binding to both of them isfacilitated. Loss of either of the higher affinity binding sites,even if the curvature is still there, would lead to a loss of theobserved selectivity. How and why this initial high-affinitybinding may trigger the cooperative formation of the largeHi-DNA complexes remains unknown.

Existing in a prokaryotic plasmid, the Hi-binding site wehave identified cannot have direct physiological significance.Nevertheless, it carries features which remarkably resemblethose thought to determine chromatosome positions in eu-karyotic DNA. It has been long recognized that curved DNAsegments provide preferred sites for binding of the histoneoctamer (39). More recently, Muyldermans and Travers (40)have conducted a survey of sequence features of a large set ofchromatosome sites in chicken DNA. These exhibit a statisticalpreference for certain DNA sequence elements at the ends,which may provide preferential interaction with linker his-tones. Sites similar to the one we have identified could providelocations for very strongly fixed chromatosomes, perhapssetting the phasing of nucleosomes in a local chromatin region.

The authors thank Dr. R. Harrington (University of Nevada, Reno,NV) for his critical and constructive comments on the manuscript andfor independently analyzing the pBR322 fragment by using the pro-gram CURVATUR. We also thank Valerie Stanick for skillful technicalassistance and Emily Ray and Ivan Dimitrov for help with purificationof histone HI. Dr. Sanford Leuba (University of Oregon, Eugene) isacknowledged for the scanning force microscopy imaging of DNAfragments. G.P.S. is supported by a postdoctoral fellowship from theAmerican Cancer Society. This work was supported in part by NationalInstitutes of Health Grant GM50276 to K.E.v.H.

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Proc. Natl. Acad Sci. USA 92 (1995)