DNA folding in the nucleosome

J. Mol. Bid.

(1977) 116, 49-71

DNA Folding in the Nucleosome MARKUS NOLL Biocenter of the University, Klingelbergstrasse

70, CH-4056

Basel,

Switzerland (Received 21 December 1976, and in revised form 14 June 1977) Digestion of chromatin with a number of nucleases shows that the DNA is regularly folded in the nucleosome. Particularly cleavage by pancreatic DNase (DNase 1) in the 140 base-pair nucleosome has been examined. This nuolease nicks the DNA every ten ba.ses on each strand as demonstrated by labeling the 5’-ends of the 140 base-pair nucleosome. Cleavage sites on opposite st’rands are st,aggered by two bases. This proves that the DNA is arranged on the outside of the histone core in a regular way. The probabilit)y distribution of nicking might indicate a 2-fold symmetry of the 140 base-pair nucleosome. In particular it is shown that the predominant band of 80 bases is derived from several regions nit,hin t,he 140 base-pairs and suggested to reflect) the pitch of the DNA superhcllix surrounding the histone core of the nucleosome. Its possible significance w.it,h respect to chromatin structure is discussrd.

1. Introduction The quantitative features of the subunit model of chromatin structure (Kornberg, 1974) appear now to be well-established. Nuclease digestions of chromatin reveal protected sites alternating with exposed DNA regions at a regular interval (Hewish $ Burgoyne, 1973: Noll, 1974a). Thus the chromatin subunit (nucleosome) is obtained free in solution as the unit size product of a partial micrococcal nuclease digestion of chromatin and may be purified by sucrose gradient fractionation (Noll, 1974a). It, has been shown that at least 90% of the chromatin is based on this subunit (Noll, 1974a), which consists of about 200 base-pairs of DNA (Noll, 1974a; Burgoyne et al., 1974: Oudet et al., 1975; Senior et al., 1975; Axel, 1975; Sollner-Webb & Felsenfeld, 1975; Shaw et al., 1976; No11 & Kornberg, 1977) and a histone octamer of H2A, H2B, H3, and H4 (Thomas & Kornberg, 1975). In the electron microscope chromatin fibers have been observed to consist of spheroid units (Olins $ Olins, 1974; Woodcock, 1973; Oudet et al., 1975), which have been demonstrated to correspond to the biochemically defined chromatin subunits (Griffith, 1975; Finch et al., 1975). In some lower eukaryotes the DNA size per nucleosome is found to be only 170 basepairs (Noll, 1976a; Morris, 1976; Thomas & Furber, 1976). This variation is due to a structural change of the link of adjacent subunits and may reflect differences in histones Hl (Noll, 1976a,1977; Morris, 1976). This would also be consistent with the interaction of Hl with the linker region (No11 & Kornberg, 1977; Noll, 19763,1977). The postulate that the DNA is on the outside of the chromatin subunit (Kornberg, 1974) receives support from digestions of chromatin with pancreatic DNase (DNase I). This nuclease cleaves both strands in nucleosomes at regularly spaced sit#eswhich are 49 .I

M. NOLL

50

multiples of ten bases apart, (Nell, 19746). This is compatible with the same regular arrangement of the DNA on the outside of the histone octamer in all nucleosomes. The intervals between adjacent DNase I-sensitive sites on the same &and have been proposed to reflect the pitch of the double helix (Noll, 19746). The folding of the DNA around the histone octamer might occur by kinking (Crick & Klug, 1975; Sobell et al., 1976) or bending the DNA. Although the previously reported digestions of chromatin with pancreatic DNase suggest that the DNA is accessible to a considerable extent in the nucleosome, these studies do not exclude the possibility that there are extensive protected regions on the DNA strands. Therefore, a proof that the DNA is on the outside of the chromatin subunit depends on the demonstration of nicks every ten bases on each strand. Experiments to answer this question and to further characterize the folding of the DNA in the nucleosome are the subject of this paper.

2. Materials and Methods (a) Nuclease digestions Rat liver nucleiwere prepared and suspended in 0.34 M-SUCrOSA, 15 mnr-Tris.HC!I (pH 7*4), 15 mM-NaCl, 60 mM-KCl, 15 mM-2-mercaptoethanol, 0.5 mm-spermidine, 0.15 mM spermine (0.44 ml/g of liver, resulting in about 1.5 x lOa nuclei/ml) according to Hewish & Burgoyne (1973). For incubation with nucleascs, Ca2+ and/or Mg2+ were added as indicated in the Figure legends. Acid-soluble DNA was determined as described by No11 & Kornberg (1977). (b) Sucrose gradient analysis Isokinetic sucrose gradients (Noll, 1967) of 12 ml and 36 ml wore prepared as described (Noll, 1974a, with V, = 9.2 ml, and Finch et aE., 1975) using the automatic gradient maker of Molecular Instruments Company. After centrifugation the gradients were monitored for absorbancy by passing the effluent from the bottom of the tube through a turbulence-free flow cell (Molecular Instruments Co., PO Box 1652, Evanston, Ill. 60201, USA). (c) X1 n&ease

digestion

Double-stranded DNA obtained from chromatin digested with DNase I was incubated in 20 ~1 of 30 mM-sodium acetate (pH 4.5), 10 mm-NaCl, 0.3 mM-ZnSO, with 4 units of S1 nuclease/pg DNA for 30 min at 37°C. One unit of S1 nuclease (Miles Laboratories Ltd) liberates 160 pmol nucleotides/min at 37°C. (d) Labeling of 140 bme-pair nucleosornee Nucleosomes with a reduced DNA size of 140 base-pairs were produced by degradation from the ends with micrococcal nuclease as described by No11 Q Kornberg (1977). The nucleosomes (0.79 Azao units) were incubated in 0.40 ml of 50 mar-Tris*HCl (pH 8-l), 10 mM-MgCl,, 2 mM-dithiothreitol with 110 nmol [y-32P]ATP (145 Ci/mmol) and polynucleotide kinase (both were generous gifts from Dr V. Pirrotta) for 1 h at 37°C to label the 5’-ends. Labeled nucleosomes were separated from unreacted [y-32P]ATP on a B-ml Sephadex GSO column equilibrated in 5 mM-Tris.HCl (pH 7.4); 70% of the 5’-ends were labeled. Peak fractions eluted at the void volume were pooled, loaded together with 0.25 Aas,-, units of unlabeled 140 base-pair nucleosomes on 11-ml isokinetic sucrose gradients, and centrifuged at 4°C for 15 h at 40,000 revs/min in a Beckman SW41 rotor. This 11 S peak was collected and used for digestion with pancreatic DNase. Figure 12 shows that all the radioactivity of this material sediments as 11 S subunits. (e) Preparation

of labeled

DNA

fragments

of muEtiplea

of 10 nucleotides

The DNA of 11 S subunits of a pancreatic DNase digest was isola,ted and analyzed as described in the legend to Fig. 7. The 140 base-pair band was cut out of the gel, and the

DNA

FOLDING

IN

THE

NUCLEOSOME

51

DNA was eluted and concentrated. After incubation with bacterial alkaline phosphatase (Worthington Biochemical Corp.) for 1 h at 30°C to remove 5’-phosphates (Weiss et al., 1968). the DNA was extracted 4 times each with phenol and ether. Traces of ether were removed by a st,ream of air. Thr DNA was then labeled at the 5’-ends and purified on a Sephatlex G50 column equilibrated in 10 mm-NH,HCO, as described above. The denat~urrd DNA shows labeled fragments that are exact multiples of 10 nucleotides usc~l as refclencc in i,he autoradiograph of the go1 shown in Fig. 13. (f) Patw-eatic

DNase digestion of 110 base-pair the 5’-ends

nucleosomea

labeled at

140 base-pair Unlabeled (0.80 Azeo units) and labeled (4 x IO6 cts/min) of the same preparation, as described in section (d) above, were incubated 1.4 mM-CaCl,, 1 mM-EDTA, 5 m&r-Tris-acetate (pH 7.8), l-4 mM-MgCl,,

with Id or

60 units of pancreatic DNase by thta addit,ion of 0.05 ml of 0.1 M-EDTA

ext,ensivcly

against

1 mM-NH4HC03,

nucleosomes

in

0.55

12%

ml of sucrose

at 0°C for various t’imes. Reactions were stopped

(pH 7), and t,ha DNA and lyophilized.

was ext,ra.cted,

dialyzed

3. Results (a) Polynucleosomee

of a pancreatic

DNase digest

Previous experiments have shown that the DNA in the nucleosome can be divided into two topologically distinct classes (Noll, 1976a,1977). The DNA of the first class which is not accessible to the primary attack by micrococcal nuclease consists of 140 base-pairs tightly bound to t’he histone core and forming, with the histone octamer, a structurally conserved core particle. The DNA of the second class is part of the link of adjacent subunits and varies in size, perhaps according to variations in the structure of histone Hl. This linker DNA contains the site exposed to the primary attack by micrococcal nuclease (primary cutting site) and a fragment protected by histone Hl against micrococcal digestion (Varshavsky et al., 1976; Whitlock & Simpson, 1976; Shaw et al., 1976; No11 & Kornberg, 1977; Nell, 19763,1977). In contrast to micrococcal nuclease, DNase I attacks the DNA of both classes (Nell, 19746). Since the DNA of the first class is associated more tightly with the histone core, one would expect that nicks in this region do not grossly change the structure of the core particle, whereas digestion of the linker DNA results in disruption of the chromatin fiber. Therefore, chromatin fragments produced by DNase I digestion should sediment at the same rates as monosomes, disomes, trisomes, etc. generated by micrococcal nuclease digestion (Nell, 1974a). This prediction is confirmed by the experiment in Figure 1, which shows a sucrose gradient analysis of chromatin fragments obtained after incubation of nuclei with DNase I. With progressing digestion polynucleosomes are converted t’o single nucleosomes. The sedimentat’ion coefficient of these single nucleosomes is the same as that of intact 11 S chromatin subunits, although their DNA is extensively nicked, as evident from Figure 2. Similarly, it was shown that the DNA of disomes (Fig. 3) and of larger polynucleosomes is nicked to a similar extent, and thus is also exposed to DNase I in the nucleosomes of larger chromatin fragments. It is important t*o emphasize that the distribution of single-stranded DNA fragments shows relatively few deca- and eicosanucleotides irrespective of the length of digestion with DNase I when this DNA has been isolated from the surviving intact 11 S nucleosomes (Fig. 2). Conditions necessary to convert DNA completely to fragments of 10 and 20 bases, on the other hand, cause precipitation and possible denaturation of ahromat,in. As long as the preservation of the native

M.

NOLL

Effluent volume (ml)

FIG. 1. Time-course of pancreatic DNase digestion: sedimentation analysis of chromatin fragments. Rat liver nuclei (2 ml) were incubated at 0°C with 1500 units of pancreatic DNase (Sigma Chemical Co) in 10 miw-MgCl, for the time indicated. Digestions were stopped by the addition of 0.5 ml of 0.1 M-EDTA (pH 7). After centrifugation (5 min at 4000 g) the nuclear pellets were lysed in 1 mM-EDTA (pH 7). The supernatants of a second centrifugation step were layered on 3t3-ml isokinetic sucrose gradients and centrifuged at 4°C for 21.5 h at 27,000 revs/min in a Beckman SW27 rotor.

structure is not verified by some independent means, the production of the eicosa- and decanucleotide fragments under these conditions is not a valid argument for cleavage sites every 10 or 20 nucleotides on each strand over most of the nucleosomal DNA, as claimed by Sollner-Webb & Felsenfeld (1977). The digestion with DNase I illustrated in Figures 1 and 2 was carried out at 0°C in the absence of Ca2+ . At 37°C the rate of digestion is increased 50-fold, whereas the addition of Ca2+ accelerates the rate five-fold at both 0°C and 37°C. The distribution of nicks (as judged by analysis of the DNA in a denaturing gel (Fig. 2)) is not influenced by the elevation of the incubation temperature or the addition of Ca2+ if equivalent stages of digestion are compared (as judged by the sucrose gradient profiles (Fig, 1)). However, chromatin prepared by lysis of the nuclei precipitates at 0°C during a much later stage of digestion than at 37°C (60% precipitates between 80 min and 160 min of digestion at 0°C (Fig. l), whereas at 37°C 90% has precipitated at a stage equivalent to 80 min of digestion at O’C). A possible explanation would be that small DNAfragments that remain bound to the nucleosomes at 0°C dissociate at 37°C and are turned acid-soluble. Such an interpretation is supported by the observation that the release of acid-soluble DNA is retarded at 0°C whereas at both 0°C and 37°C precipitation occurs when 20% to 30% of the DNA has leaked out of the nuclei as acid-soluble products. A similar effect, a tighter binding of the DNA ends of nucleosomes to the histone core at O”C, has been suggested (Noll, 19763) to account for the inhibition of degradation from the ends by micrococcal nuclease at this temperature (No11 & Kornberg, 1977). The regular pattern of bands in Figure 2 has been shown to be due to the nicking of the DNA strands in nucleosomes by DNase I at sites multiples of ten bases apart

DNA

FOLDING

IN

THE

N[‘(“T,EOSOME

53

SO-

5

IO

20 Digestion

80

40 tdme (mln

160

1

FIQ. 2. Time-course of pancreatic DNLtse digest,ion: polyacrylamide gel electrophoresis of DNA fragments from 11 R subunits. DNA was extracted from the 11 S monosome fractions indicated in Fig. 1, precipitated with 2 vol. ethanol at -.- 20°C overnight, and ardyzed under denaturing conditions in 7 &I-urea in a 12% polyarrylamitle gel (hlaniatis PI ctl., 1975). Thr DNA was stained with rthidium bromide ae described by Ncrll (1971b).

hi.

54

NOLL

0

-

200 160

-

Monomer

Polyac1 pancreatic DNase DNA fn 3m mor pancreatic DNase brief diges t is sho from the lt :ft . FIG.

3.

Oimcr

-ylamide gel analysis of DNA fragments from mono - and dinucleosomes : of digests. losomes (monomer) and disomes (dimer) of a brief (10 min) and long (40 n lin) digest at 0°C was analyzed as described in the legend tc ) Fig. 2. The DNA of the an in the 1st and 3rd lane, the DNA of the long digest in the 2nd and 4th 1anr

DNA

FOLDING

IN

THE

55

NVCLEOSOME

1974b). These bands are broader than t,hose of defined DNA fragments which could be explained by a heterogeneity of size or sequence. However, the breadth of the bands which could correspond to a size h&erogeneity of & 2 bases appears too large to be entirely accounted for by sequence heterogeneity, since we are dealing main1.v with DNA fragments of average rather t’han extremely different base compositions. Thus, it is possible that the regularly spaced sites susceptible to clcavage by DNase I do not consist simply of single sites but include adjacent sites. onra on each side. After brief digestion with pancreatic DNase, discrete DNA fragments of single nucleosomes that are larger than 140 bases and mult,iples of ten bases are apparent,, although they are obscured by a high background (Figs 2 and 3). This suggests that, the DNA of the linker region is also preferentially attacked at regularly spaced sites by DNase I but is cleaved at many additional sites (secondary sites). These sites might be always accessible or become exposed only after cleavage of the linker DNA at, the regularly spaced sites followed by disruption of the chromat,in fiber. This lat,ter possibility receives some support from t’he observation that the background is reduced in DNA isolated from dinucleosomes of a DNase I digest’ (Fig. 3). Alt,ernatively, the background might be explained by a size heterogeneity of thtb linker DNA which contains regularly exposed sites. Interestingly, at early digestions a sharp band of a.bout 190 bases, which is close to t,he DNA size per nucleosome, is observed (5’ to 20’ in Fig. 2). An explanation of t’his finding might, be that DNA of the full length has a higher affinity for the histone core and hence is more resistant to cleavage at secondary sites within the linker DNA. (Nell,

(b) Digestion with streptodornasv

or Ca 2+, Mg2 C-clependent

endonuclease

DNase I is not the only nuclease that, nicks the DNA in chromatin at sites spaced by multiples of ten bases. Similar patterns are produced by digestion with strependonuclease (Fig. 4; and Simpson & todornase (Fig. 4) or Ca 2+, Mg2+-dependent 2 + , Mg2 +-dependent endonuclease (Hewish Whitlock, 1976a). However, both the Ca 6 Burgoyne, 1973) and streptodornase cut predominantly in the linker region, generating a distribution of double-stranded DNA fragments similar to that of a partial micrococcal digest (Fig. 5). On the other hand, pancreatic DNase only slightly prefers t)o attack the linker DNA as evident from the high background between multiples of 200 base-pairs (Fig. 6). As a result of this lower probabilit’y of cleavage within the 140 base-pair nucleosome by streptodornase or Ca2+, Mg2 +-dependent endonuclease, the distribution of single-stranded DNA fragments is expected to be skewed towards larger sizes as compared to the same stage of pancreatic DBase digestion (same fraction of single nucleosomes at stages where they comprise a relatively small fraction). This is confirmed by the analysis of the DNA of single nucleothe distribution of fragments somes in a denaturing gel (Fig. 4). Particularly, produced by streptodornase exhibits much stronger bands of 150 and 160 bases. This might reflect a steric hindrance which prevents this enzyme from cutting at secondary sites within the 20 base-pairs of the linker region adjacent to the 140 base-pairs of the core particle. It is consistent with the view that the DNA in the linker region is also mainly exposed at regularly spaced sit,es. The 20 base-pairs adjacent t)o the 140 base-pairs of t,he core part,icle havra also been implicated as site of strong interaction with histone Hl (No11 & Gornberg. 1955: Varshavsky Pt al.. 1976; Whitlock & Simpson, 1976; Shaw ft al., 1976: Noll. 1976h.197i).

.\I.

DNose

I

NOLL

Co2+,Mg2+dep. endonucleose

FIG. 4. I’olyacrylamide gel electrophorwis of DNA fragments from I 1 S subunits of pancreatic DNase, Ca2 + , Mg2 + -dependent endonucloase, and streptodornase digests. 1 miwCaC1, for 270 min at 37°C (Caz+, Mg2+ Rat liver nuclei were incubated in 10 mwMgCl,, dependent endonuclease) or in I mM-MgCl,, 1 mix-CaCl, with 1250 units streptodornase/ml (Lederle) for 5 min at 37°C. The DNA from 11 8 subunits of equivalent stages of digestion (as judged by sucrose gradient analysis) was isolated and analyzed as described in the legend to Fig. 2. At the left DNA from 11 S subunits of a DNase I digest (10 min in Fig. 2) is shown.

DNA

FOLDING

IN

THE

Dhlars I

Straptodornora

125

625 hnits)

67

NUCLEEOSOME

2500

30

60

Ref.

270

(min)

F’ro. 5. l’olyacrylamide gel electrophoresis of DNA from microrwcal nuclease, strrptodomasr, (‘a2’, 31g*+ -depenclent endonuclease, and pancreatic DNaw digests of’ ohromatin. Rat liver nuclei (0.5 ml) were incubated in 1 mM-MgCl,, 1 mu-CaCl, with 125, 625 and 25(JO units of streptodornase for 5 min at 37°C or in 10 miw-MgCl,, 1 mwCaC1, without exogenous nuclease for 30 min, 60 min and 270 min at 37°C. The DNA was extracted and analyzed in a 2.5% polyacrylamide slab gel according to Loening (1967). The 2nd lane from the right shows DNA from a pancreatic DNase digest (10 min at 0°C as described in the legend to Fig. l), whereas in lanes at t,he far right, and left DNA of a partial microcorcal digest of rat liver nuclei at 2°C (No11 & T~ombwp, 1977) is shown as refercnre (Ref.).

113. NOLL

5s

(c) Pancreatic

I)Nase generates

staggered

cuts

The DNA of mononucleosomes of a pancreatic DNase digest may also be analyzed in its double-stranded form in a non-denaturing polyacrylamide gel. As illustrated in Figure 6, this might yield valuable information on the relative position of the potential cleavage sites on opposite strands. In the following we assume that cleavage sites occur every ten bases on each strand. Basically we have to discriminate between two possibilities; (1) that these sites are exactly opposite each other on the two strands

(I ) No stogqer (a)

Singlets II nl/N

Cb’ II I’[Ii 1:; N/N

N/W-IO)

Triplets

N/m/-20,

( 2 ) Stogger Doublets for n=5

N/N N/N

N/(N-IO)

Fra. 6. Schematic illustration of double-stranded DNA fragn-ents in the nucleosome generated by cleavage with pancreatic DNase. Double-stranded DNA is represented by 2 solid straight lines. For simplicity, internal nicks in the 2 strands are not shown. The strand lengths of double-stranded DNA are denoted by N/N’, where N and N are multiples of 10 bases. The size of protruding single-strands at staggered ends is indicated by the number of bases 12. See the text for a detailed explanation.

or (2) that they are staggered. Assuming that ten base-pairs are sufficient to prevent melting of the two strands, in the first case a pattern of double-stranded DNA fragments that are multiples of ten base-pairs is expected (l(a) in Fig. 6). If ten basepairs are not sufficient to keep the two strands from melting, each single band splits into triplets of the kind shown under l(b) of Figure 6 (in order to derive all possible cases it is useful to keep the length of one strand constant). Similarly, in the second case where cleavage occurs at staggers different from five bases, triplets or degenerate singlets are also predicted depending on the stability of overlaps larger than five base-pairs. In the particular case of a stagger of five bases, the triplets degenerate into doublet,s (2 in Fig. 6). The triplets in the case of no stagger are expected to be clearly resolved in a gel due to their difference in t’he length of one strand. On the other hand, it may not be possible to separate the two DNB fragments with strands of the same length of the triplets if cleavage occurs at a stagger. Thus, observation of doublets would indicate that cleavage sites are staggered, although the size of the stagger would not have to be five bases, since one band might be degenerate. As evident from Figure 7, analysis of the DNA fragments of DNase I monosomes in a non-denaturing gel reveals doublets which ma.y be resolved up t,o a8size of 100 basepairs (in the gel on t,he right in Fig. 7). This suggests that cleavage occurs at a stagger.

DNA

FOLDING

IN

THE

59

NITCLEOAORIE

90s c-pairs

Bore-pairs

-

152

60-

70 6049 -44 40-

-

60

-

70

-

60

30-

40

FIG. 7. Polyacrylamide gel electrophoresis of native DNA fragments from 11 S subunits of a pancreatic DNase digest. DNA from the 11 S monosome fraction of an 80 min pancreatic DNase digest at 0°C (Fig. 1) was extracted as described in the legend to Fig. 2 and analyzed in 6% polyacrylemide slab gels in T&-borate (Peacock & Dingman, 1967). The double-stranded fragments with strand lengths of 20/20 and 20/10 bases ortn be detected only by overloading the gel or after extensive digestion. The gels were run at 200V for 2-O h (gel at the left) and 3.5 h (gel at the right). Numbers in the middle refer to the number of base-pairs of markers of known lengths (Ha&II fragments of PM2 DNA: Noll, 1976a) running at the positions indicated.

hr.

60

NOLL

Again the bands are broader than those of defined DNA fragments, which probably reflects nicking at t#hree adjacent, s&es rat’her t,han a single site. Tf bhese doublets are of the type illustrat,cd in Figure 6, analysis of t,he bands of a doublet under denaturing conditions should cbxhibit, single-stranded fragments of multiples of t,cn bases up t,o the same size N corresponding to t,he lengths of the double-stranded fragments N/N and N/(N ~ 10). In addition, the largest single-stranded fragments of neighboring doublets should differ by ten bases. Both these features are verified in Figure S(a). It may also be observed in Figure 8(a) that the ratio of single st,rands of the full length N t,o those of the size N - 10 is slightly smaller for the lower than for the upper band of a doublet. This effect, expected to decrease with increasing N, is consistent with the interpretation of doublets according to Figure 6. Separate analysis of both bands of a doublet which showed no detectable cross-contamination (Fig. S(b)) was actually carried out up t,o a size N equal t(o 90 bases (shown only for both bands up to N equal to 60 bases). The sizes of the double-stranded fragments measured in this way were confirmed by an independent calibration with HaeIII fragments of YM2 DNA of known lengths (Noll, 1976a), as indicated in Figure 7. A plot of the mobility of the double-stranded DNA fragments as a function of the logarithm of their size (taken from Fig. 7) yields a smooth curve for either the upper or the lower bands of the doublets (Fig. 9). This suggests that all the fragments of the lower bands (or those of the upper bands) are of the same type. It might) be expected that fragments with staggered ends of five bases of the type N/(N ~ 10) migrate as fragments (N - 5)/(N - 5). A H evident from Figure 9, this is not’ the case, suggesting that the stagger is different from five bases.

0 ases

-

140

-

00

-

40

r

50

60

70

(a) Frn. 8.

00

90

r

100 110 120 140

DNA

FOLDING

IN

THE

NUCLEOSOME

60

r

Base -pairs

604030-

30

r

40

50

70 60 90 100 II0 120 140 r

(b) FIG. 8.(a) Single-strand composition of double-stranded DNA fragments of a pancreatic DNase digest. Bands of a preparative slab gel, as shown in Fig. 7, were cut out and the DNA was eluted, concentrated, and subjected to electrophoresis in a denaturing gel as described in the legend to Fig. d. Lanes, from left to right, show the analysis of DNA of decreasing mobility in the non-denaturing gel (Fig. 7) and are labeled at the bottom by the number of base-pairs of the double-stranded fragment. Analysis of individual bands of tho doublets in Fig. 7 is shown only up to a size of 60 basepairs. The lanes labeled r contain total DNA from monosomes prepared as described in the legend to Fig. 7. (b) I’olyacrylamide gel electrophoresis of purified double-stranded DNA fragments of a pancreatic DNase digest. Portions of the samples shown in (a) were analyzed in a non-denaturing gel as described in thrs legend to Fig. 7. Lanes are labeled as in (a).

1 5

I

I

I

I Dlstonce

I IO mlgroted

I

I

,

I 15

(cm)

Pm. 9. Mobility of DNA fragments in the polyarrylamitk gel shown in Fig. 7 as a function of lengths of DNA strands. Distances of migration were measured from the origin to the midpoint of the bands of individual doublets in Fig. 7. Sizes of DNA strands are given in Figs 7 awl 8(a). The notation of strand lengths of double-stranded DNA is as in Fig. 0.

62

11.

NOLL

Staggered cleavage sites were first proposed on the basis of doublets as shown in Figure ‘7 at the Dahlem conference (Nell, 1976b). Unless certain conditions are met, these doublets cannot be resolved into discrete bands. Thus it is important (1) to carry out the digestion at low temperature (O’C) in order to prevent dissociation of the DNA ends from the histone core (No11 & Kornberg, 1977), (2) to isolate the DNA from purified 11 S nucleosomes after digestion to prevent contamination with degradation products not specific for nucleosome structure, and (3) to concentrate the DNA after extraction at sufficiently high ionic strength to prevent dissociation of the shorter DNA strands. It is not surprising that these doublets cannot be seen in experiments reported more recently by Sollner-Webb & Felsenfeld (1977), although these authors

E3arcr

-

I20

-

.80

-

.60

-

r

30

40

r

70

80

r

140

,

conditions of A,-treated FIG. 10. Polyacrylamide gel electrophoresis under denaturing doublets of a DNase I digest. The lower and upper native DNA bands of the 30, 40, 70 and 80 base-pair doublets were isolated from a preparative gel as shown in Fig. ‘7. After digestion with S, nuclease as described in Materials and Methods, the products were analyzed under denaturing conditions in a 7 M-urea gel as shown in Fig. 2. The lanes labeled r show a DNase I digest for reference of multiples of 10 nucleotides. The remaining lanes contain from left to right: the lower (left) and upper (right) bands of the 30, 40, 70 and 80 base-pair doublets. The lane labeled 140 contains an S1 nuclease digest of the total DNA isolated from 11 S nucleosomes of a DNase I digest.

DNA

FOLDING

IN

THE

63

NVCLEOSOME

believe them to be visible (Fig. 1B of Sollner-Webb & Felsenfeld, 1977). For it is obvious from their description of their experimental conditions, as well as from the absence in their supposed doublets of single-stranded fragments shorter than 40 bases that none of the above conditions was met,. (d) DNase I cleavage sites on opposite strands nre staggered hy two bases In order to determine the size of the stagger, the DNA of individual bands of tjhe doublets shown in Figure 7 was treated with S, nuclease, which trims down the single-stranded tails to flush ends. The stagger is measured by t)he reduction in size of the trimmed DNA fragments in a denaturing gel. The analysis of such an experiment is shown in Figure 10. It is clear that the largest single strands of the lower bands of t)he doublets (shown in the left lanes) are reduced by about ten, whereas those of the upper bands (shown in the right lanes) only by about two nucleotides after S1 nuclease digestion (compare with Fig. 8(a)). This is consistent with t,he interpretation of the doublets shown in Figure 6, assuming a stagger of two nucleotides. Furthermore, it confirms that upper and lower bands of t.he doublets can be separated at, least up to the 80 doublet’. It, remains unclear, however, why double-stranded fragments of the type N/N with a stagger of two but not of eight nucleotides are detected in Figure 10. Such fragments would result in single-strand lengths of 1On + 2 bases after treatment with S, nuclease. These DNA fragments could have been located at positions different from the bands isolated for further analysis from t,he gel shown in Figure 7. This was ruled out b,y digestion of the entire DNA input in Figure 7 with S, nuclease followed by analysis under denaturing conditions. The results show single strands of 10n f 8 (Fig. 11) and 10~ nucleotides but no fragments of 10% + 2 bases (lane labeled 140 in Fig. 10). Alternatively, one could explain the absence of these fragments if a stretch of eight base-pairs was stable. However, t.his argument is not convincing because it is inconsistent with the observation of fragments of the type N/(N - 10) in t’he lower bands of the doublet,s as evident from Figure 10. A stagger of eight (and two) bases has been reported recently by Sollner-Webb & Felsenfeld (1977) who measured a size change of the DNA fragments after filling-in the single-stranded ends with polymerase I.1 followed by analysis of the DNA

IO

I 5

, IO

‘1

..

Distance migrated km)

FIG. 11. Size calibration of &-treated double-stranded DNA of a DNase I digest. Distances migrated of the single-stranded fragments shown in the lane labeled 140 in Fig. 10 are plotted and the corresponding fragment lengths calibrated with respect to the references consisting of exact multiples of 10 nucleotides shown in the lanes labeled r in Fig. 10.

64

31.

NOLL

in a denaturing gel. This size change may be determined from the gel shown by these authors (Fig. 8 in Sollner-Webb & Felsenfeld (1977)). After careful calibration of the shifted bands an average shift, of 6.8 & 0.14 nucleotides is obtained, which corresponds to a stagger of seven (and three) bases and not to a stagger of eight (and two) nucleotides. The discrepancy between their results and those presented here might be explained by incomplebe filling-in of the single-stranded ends with polymerase II or incomplete trimming by S, nuclease. Whereas the latter explanation appears unlikely, since the results shown in Figure 10 remain unchanged over a tenfold concentration range of S, nuclease in t’he reaction mixture, incomplete filling-in by polymerase I1 cannot be excluded since no attempt has been made to study the kinetics of the reaction. On the other hand, the results of careful experiments to determine the stagger by a different approach agree with a stagger of two (and eight) nucleotides (Lutter, 1977). (e) DNA

on the outside of the chromatin subunit

So far we have assumed that the DNA strands of the nucleosome are exposed to pancreatic DNase every ten bases. To test this hypothesis it is sufficient to analyze after digestion with DNase I the size distribution of single-stranded fragments that have one end at a fixed location, for example at the end of the tightly bound 140 base-pairs. Selection of fragments derived from the same end is achieved by 32P-labeling of the 5’-ends with polynucleotide kinase. Thus 11 S subunits are prepared by prolonged digestion with micrococcal nuclease to yield nucleosomes with a reduced DNA size of 140 base-pairs (No11 & Kornberg, 1977). The 5’-ends of the nucleosomal DNA are labeled and the 11 S subunits digested with pancreatic DNase. This approach provides meaningful information on the distribution of sites exposed to DNase I in the 140 base-pair nucleosome only if the labeling of the nucleosomes does not alter their structure. Indeed, as evident from Figure 12, labeled nucleosomea still sediment at 11 S, indicating that at least no gross structural change has occurred. IIS 0.3

14 *b

12

;

10

G 2 2 a 5 t 0” 0 E

5 0.2

3

0

z

6

2 x $ 2

0-I

4 2

0

2 4 6 8 IO Effluent volume (ml)

0

FIG. 12. Sedimentation analysis of 140 base-pair nucleosomes labeled at the 5’-ends. The 11 S peak of 3aP-labeled 140 base-pair nucleosomes was collected (see Materials and Methods) and a portion of it was analyzed together with 0.27 A 260 units of unlabeled 140 basepair subunits in an 1 l-ml isokinetic sucrose gradient. Centrifugation was at 4°C for 12 h at Fractions (0.5 ml) were counted by Cerenkov40,000 revs/min in a Beckman SW41 rotor. counting in a Packard Tri-Carb scintillation counter.-O-o-, a2P radioactivity; , absorbance at 200 nm.

DNA

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66

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Analysis of the DNA fragments produced by DNase I digestion of the labeled 140 base-pair nucleosomes is shown in Figure 13. It demonstrates that the bands of singlestranded fragments derived from the two 5’-ends of the 140 base-pairs are shifted with respect to the pattern of fragments that are multiples of ten nucleotides. Close examination of Figure 13 reveals that these bands are also broader and actually consist of doublets. These may be resolved for fragments smaller than 50 bases, and calibration shou-s that t’hey fall into two size classes of fragments of 10n + 1 and IOn + 4 (n = 1, 2, 3,. . .) bases (the fragment sizes may be read off directly from Fig. 13 since DNase I creates 5’-phosphates and the 5’-OH ends produced by micrococcal nuclease have been phosphorylated with [32P]ATP and polynucleotide kinase). The most plausible interpretation of this result is that the two classes are derived from the 5’-ends of opposite st,rands. Since cleavage sites on opposite strands arc st,aggcrrd bv tjwo nucleotides as shown above. it follows t,hat the ends of the 140 Ethidium

bromide Eases

-40

-20

-

0

2

5

Ref

15

45 Time

(0)

IO

(min) (b)

13. Time-course of pancreatic DNase digestion of 140 base-pair nucleosomes labeled at the B/-ends: polyacrylamide gel electrophoresis of single-stranded DNA fragments. Labeled 140 base-pair nucleosomes were digested with 60 units of pancreatic DNase at 0°C for the t,ime indicated, and the DNA was extracted, concentrated, and subjected to electrophoresis in a denaturing slab gel as described in Materials and Methods and in the legend to Fig. 2. (a) An autoradiograph of the gel, (b) the gel after staining with ethidium bromide. For comparison 140 base-pair DNA of 11 S subunits of a pancreatic DNase digest (see section (e) of Materials and Methods) is analyzed in the lane labeled Ref. Its input is too low to be deteoted by staining with ethidium bromide. The input of the zero time control is 12%, the input of the 45 min digest is 80% of that of the remaining lanes. 6 FIG.

citi

M.

NOLL

base-pairs associated more tightly with the histone core are also staggered. The exact nature of these staggered ends cannot be deduced from the S, nuclease experiment because it does not discriminate between 3’ and 5’-ends. One possible structure of the ends of the 140 base-pairs is illustrated in Figure 14 and consists of 5’-ends protruding by three nucleotides.

Fm. 14. Probability distribution of nicks at sites exposed to pancreatic DNase in the 140 base-pair nucleosome. The accessibility of the DNA in the 140 base-pair nucleosome as reflected by the nicking probabilities P, is illustrated schematically by the lengths of arrows. The location of the sites exposed to pancreatic DNase is indicated by their distance (number of bases) from the 5’.ends of the 140 base-pairs. Values for P, are taken from Table 1. It is assumed that P, at the sites 19, 124 and 9, 134 (broken arrows) are the came a8 at the sites 119, 24 and 129, 14.

A quantitative analysis of the distribution of nicks should not only clarify the question whether they occur every ten bases on each strand but also yield information on the accessibility of the DNA along its entire length in the nucleosome. This requires a study of the change of the distribution of the labeled single-stranded fragments with digestion time as shown by the following derivation. Let N1 be the number of single-stranded fragments at the time t extending from the %-end of the 140 bases to the site i (the sites are numbered with increasing distance from the 5’ towards t)he 3’-end) created by a nick, and let us assume that the substrate concentration is much larger than the nuclease concentration, then N, changes during the time interval dt according to

in which pi denotes the probability of nicking at the site i during dt. If we assume that pi is independent of time, integration over the finite but short time interval At yields for the change AN, of Ni during At AN, = pi At Z fl, - fl, zZ p, At, (2) kci k>i where the bars indicate time averages during At. Thus we obtain AN, +fl, pr At = -----

Z pkAt -.--,

kc-i

33,

(3)

k>i

from which pi At may be calculated by reiteration and the probability nicking at the site i by normalization P, =piAtwithZp,,At = 1 . k

pi of

The kinetics of pancreatic DNase digestion of the 140 base-pair subunits are shown in Figure 13 and Table 1. The probabilities of nicking P, given in Table 1 were calculated according to equation (3) for the shortest digestion time, between zero time and 025 minute, at the lower nuclease concentration. Larger time intervals

DNA

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67

NIJCLEOSOME

1

Time-course and nicking probabilities Pi of pancreatic DNase digestion of 140 base-pair nucleosomes labeled at the 5’-ends Fraction of counts (%) Time of digestion (min) Size of DNA (bases) I2(IL I60 I lO-Il.5

100-105 9OL95

x0- 85

70.-75 60-65 50-55 40-45 30-35 20-25 IO---l5

/

10

0

0.25 (15 IT)

0.25 (60 U)

2

IO

40

681 4.8 3.6 3.6 2.4 2.4 2.2

63 4.6 3.8 3.9 2.5 2.6 2.3 6.7 4.1 1.96 1.62 2.14 0.36

50 4.6 3.5 3.6 2.5 3.1 2.7 6.3

30 4.4 2.5

20 1.7 1.li

1.1 0.25

3.2

0.2 1

64

5.9

3,3 1,63 0.81 0.75 0.14

5.0

2.6 1.X

3.3 2.1 6.0 6. I

4.1 4.5

4.8

8.8

14.5

2.1

5.7

6.9

1 .I)

1.3 2.9 2.0 5.8

3.6 7.2 11.9 24.4 Il.6

0.33 0.33 0.70 0.68 2.0 4.0 6.3 15.4 27.4 42

P,(%)

7.1

2.9 4.0 3.1 11.3 9.3 3.9

8.7 14.3

Lab&d 140 base-pair nucleosomes were digested with 60 units of DNase I at 0°C for the time indicated. Digestion for 0.25 min was also carried out with only 15 units of the DNase. The DNA was processed and analyzed in a denaturing slab gel as described in the legend to Fig. 13. DNA bands of sizes shown in the column on the left were cut out of the gel and counted by cerenkovcounting. The columns give the fraction of counts in each band. From t,his the probabilities of nicking P, were computed as described in the text. P, for nicking at sites closer than 10 bases to the 5’-ends is not shown, since t,hese values most likely would reflect the affinity of pancreatic DNase for DNA ends rather than the accessibility of the DNA. t About 800/, of this is in a sharp band of 140 bases (lane labeled 0 min in ethidium stained gel of Fig. 13).

bromide-

would reduce errors in AN, at the expense of an increase in the error of mi. The computation of P, is based on three assumptions. First, pi was taken to be independent of time, i.e. the probability of nicking a,t a site i is not altered by nicks at other sites. Since the values for Y, were calculated during a very short time interval at the beginning of digestion, during which most of the 140 base-pairs remain intact, this assumption would not influence initial values of Pi if false. Second, the distribution of fragments consisting of 120 to 160 base-pairs at zero time was ignored and assumed to cant ain only 140 base-pair fragments (actually about 8004 is in the 140 base-pair band as evident from the 0 minute control of the ethidium bromide-stained gel in Fig. 13). Finally, nicks at sites on opposite stra,nds giving rise to a doublet were taken to occur with equal probabilities, since the doublets cannot be resolved for fragments larger than 50 bases. A quantitative analysis as described under Table 1 showed that’ t’his assumption is correct for the resolved doublets within limits of the error of P,. There are two or possibly three sources for errors in Pi. The major error results from the low resolution of fragments larger than 80 bases (Fig. IS), whereas another error due t’o small differences AN, is not serious. As evident from Table 1, there is a low background of fragments smaller than 140 bases at zero time. It, is assumed that this background is generated by a small number of 140 base-pair subunits that, contain internal breaks in their DNA which do not, change the nicking probabilities Pi. Should this assumption be wrong, an additional relatively small error would

08

M. NOLL

arise. Analysis of several experiments showed that the total error of P, is less than 20% for sites closer than 80 bases to the 5’-ends and less than 50% for the remaining sites. A schematic representation of the distribution of sites exposed to pancreatic DNase in the 140 base-pair nucleosome and of the corresponding nicking probabilities is shown in Figure 14. It is consistent with a qualitative analysis published recently (Simpson & Whitlock, 19763) and demonstrates that nicks may indeed occur every ten bases on each strand, although with widely different probabilities. The distribution of nicking probabilities appears to reflect a Z-fold symmetry of the nucleosome, although caution due to relatively large errors at sites close to the 3’-ends is indicated. Sites that are about 10, 20, 40 and 50 base-pairs from the ends are most, susceptible to pancreatic DNase, whereas a 40 base-pair region in the middle of the 140 base-pairs and sites around 30 base-pairs from the ends exhibit a much lower accessibility (Simpson & Whitlock, 1976b). It must be emphasized, however, that such a distribution holds for the 140 base-pair subunit and may not be the same for intact nucleosomes or polynucleosomes. Since each strand of the 140 base-pairs is exposed every ten bases and cleavage sites on opposite strands are staggered by two nucleotides, the DNA must be arranged regularly on the outside of a histone core. This is consistent with recent dat
4. Discussion Nuclease digestions of chromatin represent a powerful tool for the analysis of the structural organization of chromatin at various levels (Hewish & Burgoyne, 1973; Noll, 1974a,b). Two classes of nucleases acting at different levels of chromatin structure are known. One class consists of nucleases that cut predominantly in the linker region of adjacent nucleosomes. Nucleases of this class as, for example, micrococcal nuclease may be used to test the spacing of nucleosomes (Nell, 1974a). Thus it has been shown that shearing forces destroy the native structure of chromatin by partly pulling the DNA off the histone cores, which results in enlarged sites exposed to the primary attack by micrococcal nuclease (No11 et al., 1975). The other class consists of nucleases that attack extensively the DNA of the remaining part of the nucleosome. Nuclease digestions of this type (e.g. by DNase I) yield valuable information on the DNA-histone interactions in the nucleosome and thus represent, critical tests for the internal structure of nucleosomes (Nell, 19743). Some nucleases, represented by streptodornase and Ca2 + , Mg2 + -dependent endonuclease, belong to both classes since they preferentially cut the linker DNA but also attack sites in the 140 base-pair subunit (Figs 4 and 5). It is important to emphasize that the primary attack of micrococcal nuclease occurs only in the linker region. After such primary cleavage resulting in breaks of the chromatin fiber, micrococcal nuclease may degrade the remaining DNA from the ends (No11 et al., 1975) to a limiting size of 140 base-pairs (Sollner-Webb 8z Felsenfeld, 1975; Shaw et al., 1976; No11 & Kornberg, 1977). During further digestion sites within t’he 140 base-pairs are attacked (Axe1 et al., 1974; Weintraub & Van Lente, 1974; Camerini-Otero et al., 1976). However, conclusions from these results with regard to the native structure of nucleosomes have to be considered with caution, since the chromatin forms a precipitate (No11 $ Kornberg, 1977; Noll, 19766,1977). Hence, a better test for

DNA

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69

the native arrangement of the DNA in nucleosomes represents digestion with pancreatic DNase which generates 11 S subunits nicked at specific sites (Figs 1, 2 and 3). It has been shown that the DNA of the nucleosome falls into two topologically distinct classes; (1) the DNA of the linker region containing the primary cutting site and (2) the DNA of the conserved 140 base-pair nucleosome (Noll, 1976a,1977). Digestion with pancreatic DNase which cuts the DNA of both classes yields valuable information on the folding of the DNA in the nucleosome. Although this nuclease preferentially attacks the more accessible DNA of the primary cutting site (Fig. 5), it also nicks the DNA of the 140 base-pair core particle. After brief digestion, discrete single-stranded fragments up to a size corresponding to the DNA length per nucleosome are isolated from 11 S subunits (Fig. 2). This suggests that the DNA is associated with histones along its entire length in the nucleosome and supports the idea of closely spaced chromatin subunits (Finch et al.? 1975). In addition, it indicates that the DNA of the linker region is also regularly folded. Whereas streptodornase and Ca 2 +, Mg2 +-dependent endonuclease also appear to cut in t,he linker region predominantly at regularly spaced sites (Fig. 4), it is not clear whether this holds for micrococcal nuclease as well but is obscured by secondary cleavage breaking the DNA down from the ends (No11 & Kornberg, 1977). From these results it is apparent that the DNA of the linker region must be on the outside of the nucleosome, since it is exposed to nucleases at least every ten nucleotides. For a demonstration that cleavage sites occur every ten nucleotides on each strand it is crucial to be able to discriminate between labeled fragments derived from opposite strands of the 140 base-pairs. Whereas this was not possible in a previous report (Simpson & Whitlock, 197633, t,he observation of labeled doublets in the experiments shown here (Fig. 13) allows such a distinction between fragments derived from opposite DNA st,rands. Furthermore, in the present study a quantitative rather than qualitative analysis of nicking probabilities has been carried out (Fig. 14). It demonstrates that cleavage does not occur at sites that are spaced at exact multiples of ten ba.ses from t,he 5’-ends. Moreover, the distances of the cleavage sites from the 5’-ends are not the same on opposite strands, as indicated by the presence of doublets (Fig. 13). The observation of doublets and of labeled fragments spaced at ten-nucleotide intervals (Fig. 13) further suggests that the DNA is nicked on each st*rand every ten bases. In addition, it is shown that cleavage sites on opposite strands are staggered by two nucleotides (Figs 10 and 11). This proves that the DNA is on the outside of the histone core (probably running in a groove) and folded in a regular way. This may be achieved, as suggested previously (Nell, 19746), by a continuous bending of the DNA resulting in a slight unstacking of the bases or by kinks in the DNA alternating with straight regions of multiples of ten base-pairs (Crick & Klug, 1975; Sobell et al., 1976). In either case the presence of cleavage sites every ten bases on each strand is attributed to the pitch of the doubleihelix. The observation that cleavage sites on opposite strands are staggered by two nucleotides does not exclude kinks in the DNA4 nor does it rule out a model in which DNase I binds t,o sites most exposed to the surroundings, since cleavage may not occur at t,he kinks or at the binding sites for DNase I. The probability distribution of cleavage by pancreatic DNase (Fig. 14) is consistent with a S-fold symmetry. However, unambiguous proof has not been obtained due to unresolved doublets larger than 50 bases (Fig. 13). Nevertheless, as easily verified from Figure 14, the distribution of sites most! frequently nicked is consistent,

70

ill.

NOLL

with the characteristic intensities of the single-stranded fragments produced during brief digestions (see e.g. Fig. 2). For example, the strong band of 80 bases is derived mainly from four regions t,erminated by sites of frequent attack, whereas t,he weak bands of 60 and 100 nucleotides originate from only one such region in each strand (sites 39 to 99, and 19 to 119, respectively). If one assumes that the DNA is arranged helically on the outside of the histone core, one turn would comprise about 80 base-pairs. In this case, sites most accessible to pancreatic DNase would be located next to each other separated only by the pitch of the DNA helix. Thus, as nicks every ten bases are the consequence of the pitch of the double helix, the predominant band of 80 bases would be created by the pitch of the DNA helix in the nucleosome. Two such groups of sites would be present in the 140 base-pair nucleosome (sites 9, 19, 89 and 99, and 39, 49, 119 and 129 plus corresponding sites on t,he opposit’e strand in Fig. 14) separated by three-eighths of a turn. This might reflect an important feature of the nucleosome which could play a crucial part, as sites of DNA-protein interaction in higher orders of folding of the chromatin fiber (Noll, 1977) or as binding sites of regulatory proteins. I thank Dr W. Gehring for laboratory space and the use of equipment, Dr V. Pirrotta for gifts of [Y-~~P]ATP, polynuclootide kinase, and bacterial alkaline phosphatase, and Drs P. Schedl, V. Pirrotta, and H. No11 for helpful discussions. Part of this work was supported by grant No. 3.719.76 of t’he Swiss National Science Foundation. The results presented in Figs 1 to 9 and 12 to 14 of this paper have been reported previously at the Annual Meeting of Swiss Societies of Experimental Biology in Fribourg, Switzerland, on April 9, 1976 (Experientia, 32, 806) and at the Dahlem Konferenzen on Organization and Expression of Chromosomes in Berlin (May 17-22, 1976).

REFERENCES Axel, Axel,

R. (1975). Biochemistry, 14, 2921-2925. R., Melchior, W. Jr, Sollner-Webb, B. & Felsenfeld, G. (1974). Proc. Nat. Acad. xci., U.S.A. 71, 4101-4105. Burgoyne, L. A., Hewish, D. R. & Mobbs, J. (1974). Biochem. J. 143, 67-72. Camerini-Otero, R. D., Sollner-Webb, B. & Felsenfeld, G. (1976). Cell, 8, 333-347. Crick, F. H. C. & Klug, A. (1975). Nature (London), 255, 530-533. Finch, J. T., Noll, M. & Kornberg, R. D. (1975). Proc. Nat. Acad. Sci., U.S.A. 72, 33203322. Griffith, J. D. (1975). Science, 187, 1202-1203. Hewish, D. R. & Burgoyne, L. A. (1973). Biochem. Biophys. Res. Commun. 52, 5OP510. Hjelm, R. P., Kneale, G. G., Suau, P., Baldwin, J. P., Bradbury, E. M. & Ibel, K. (1977). Cell, 10, 139-151. Kornberg, R. D. (1974). Science, 184, 868-871. Loening, U. E. (1967). Biochem. J. 102, 251-257. Lutter, L. (1977). Cold Spring Harbor Symp. @ant. BioZ. In the press. Maniatis, T., Jeffrey, A. & van desande, H. (1975). Biochemidry, 14, 3787.-3794. Morris, N. R. (1976). Cell, 8, 357-363. Noll, H. (1967). Nature (London), 215, 360-363. Noll, M. (1974a). Nature (London), 251, 249-251. Noll, M. (1974b). Nucleic Acids Res. 1, 1573-1578. Nell, M. (1976a). Cell, 8, 349-355. Noll, M. (1976b). In Organization and Expression of Chrcnnosomes (Allfrey, V. G., Bautz, E. K. F., McCarthy, B. J., Schimke, R. T. & T&i&es, A., eds), pp. 239252, Dahlem Konferenzen 1976, Berlin. Noll, M. (1977). In Nucleic Acid-Protein Recognition (Vogel, H., ed.), pp. 139.-150, P & S Biomedical Sciences Symposium 1976, New York.

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Noll, M, & Kornberg, R. D. (1977). J. Mol. Biol. 109, 393-404. Noll, M., Thomas, J. 0. & Kornberg, R. D. (1975). Science, 187, 1203-1206. Olins, A. L. & Olins, D. E. (1974). Science, 183, 330-332. Oudet, P., Gross-Bellard, M. & Chambon, P. (1975). Cell, 4, 281-300. Pardon, J. F., Worcester, D. L., Wooley, J. C., Tatchell, K., T’an Holde, K. E. &. Richards, B. M. (1975). Nucleic Acida Res. 2, 2163-2176. Peacock, A. C. & Dingman, C. W. (1967). Biochemistry, 6, 1818-1827. Senior, M. B., Olins, A. L. & Olins, D. E. (1975). Science, 187, 173-175. Shaw, B. R., Herman, T. M., Kovaric, R. T., Beaudrean, (4. S. & Van Holde, K. E. (1976). Proc. Nat. Acad. Sri., 1T.S.A. 73, 505-509. Simpson, R. T. & Whitlock, J. P. Jr (1976~). hTucZeic Acids Res. 3, 117-127. Simpson, R. T. & Whitlock, J. P. Jr (1976h). Cell, 9, 347-353. Sobell, H. M., Tsai, C., Gilbert, S. G., Jain, S. C. & Sakorc, ‘I’. D. (1976). Proc. Nat. Acad. Sci., U.S.A. 73, 3068-3072. Sollnw-Webb, B. & Felsenfeld, G. (1975). Biochemistry, 14, 2915-2920. Sollner-Webb, B. & Felsenfeld, G. (1977). Cell, 10, 537-547. Thomas, J. 0. & Furber, V. (1976). FEBS Letters, 66, 274--280. Thomas, J. 0. & Kornberg, R. D. (1975). Proc. Na,t. Acad. Std., U.S.A. 72,2626-2630. Varshavsky, A, J., Bakayev, V. V. & Georgiev, G. P. (1976). iVucZe?:c A&da. Res 3,477-492. Weint,ra,ub, H. & Van Lente, F. (1974). Proc. Nat. Acad. Scci., IJ.S.A. 71, 4249-4253. Weiss, B., Live, T. R. & Richardson, C. C. (1968). ,J. BioZ. Chem. 243, 4530-4542. Whitlock, J. P. Jr & Simpson, R. T. (1976). Biochemistry, 15, 3307-3314. Woodcock, C. L. F. (1973). J. Cell BioZ. 59, 368a.