Cell,

Vol. 14. 611-627,

July 1978,

Copyright

0 1978 by MIT

DNAase I, DNAase II and Staphylococcal Nuclease Cut at Different, Yet Symmetrically Located, Sites in the Nucleosome Core Barbara Sollner-Webb,* William Melchior, and Gary Felsenfeld Laboratory of Molecular Biology National Institute of Arthritis, Metabolism and Digestive Diseases Bethesda, Maryland 20014

Jr.t

Summary We have determined the relative location of pancreatic DNAase (DNAase I), spleen acid DNAase (DNAase II) and staphylococcal nuclease cleavage sites in the nucleosome core. Each of these three enzymes cleaves the DNA of chromatin at 10.n nucleotide intervals (n integer); this specificity presumably reflects the internal structure of the nucleosome. We have already reported that DNAase I cleaves nucleosomal DNA so that nearest adjacent cuts on opposite strands are staggered by 2 nucleotides, 3’ end extending (SollnerWebb and Felsenfeld, 1977). Here we show that the nearest cuts made by DNAase II in nucleosomal DNA are staggered by 4 nucleotides, 3’ end extending, while cuts made by staphylococcal nuclease have a stagger of 2 nucleotides, 5’ end extending. The cutting sites of the three enzymes thus do not coincide. Each pair of staggered cuts, however, is symmetrically located about a common axis-that is, the “dyad axes” that bisect nearest pairs of cutting sites coincide for all three enzymes. This result is consistent with the presence of a true dyad axis in the nucleosome core. Our results support the conclusion that a structural feature of the nucleosome, having a 10 nucleotide periodicity, is the common recognition site for all three nucleases. The position of the cut is determined, however, by the individual characteristics of each enzyme. Sites potentially available to nuclease cleavage span a region of 4 nucleotides out of this 10 nucleotide repeat, and a large fraction of these sites are actually cut. Thus much of the nucleosomal DNA must in some sense be accessible to the environment. Introduction Nucleases have been structure of chromatin. (Hewish and Burgoyne, clease (Nell, 1974a), 1974a) and spleen acid ier and Rill, 1975) all * Present address: tion of Washington, t Present address: Jefferson, Arkansas,

widely used to study the Endogenous endonuclease 1973), staphylococcal nupancreatic DNAase I (Nell, DNAase II (Oosterhof, Hozpreferentially cut between

Department of Embryology, Carnegie InstituBaltimore, Maryland 21210. National Center for Toxicological Research, 72079.

nucleosomes and thus generate the familiar nucleosomal DNA repeat pattern. This presumably results from the fact that the DNA in the 140 base pair (bp) “core” of the nucleosome (Sollner-Webb and Felsenfeld, 1975; Axel, 1975) is tightly associated with the four core histones (Honda, Baillie and Candido, 1975; Shaw et al., 1976; Sollner-Webb, 1976), and is thus less accessible to nuclease than is the connecting “intercore” DNA. The nucleosome core may also be cleaved internally by nucleases in a highly defined manner. DNAase I (Noll, 1974b), staphylococcal nuclease (Axe1 et al., 1974; Camerini-Otero, Sollner-Webb and Felsenfeld, 1976), DNAase II (Sollner-Webb, Camerini-Otero and Felsenfeld, 1976) and endogenous endonuclease (Simpson and Whitlock, 1976) all have cutting sites nominally separated by integral multiples of 10 nucleotides along the nucleosomal DNA (see Discussion); the resulting DNA fragments are 10.n nucleotides in length (n integer). An understanding of the structural basis for this 10.n nucleotide preferential cleavage pattern should aid our understanding of the internal organization of the nucleosome. In an earlier paper (Sollner-Webb and Felsenfeld, 1977), we described the mode of action of a DNAase I (pancreatic DNAase) on the nucleosome, and showed that this enzyme cleaves the DNA in such a way that cuts on opposite strands are separated or staggered by 8 nucleotides (3’ OH recessed) and 2 nucleotides (5’ P recessed). We suggested that such a pattern could arise if the recognition site of the enzyme did not coincide with its cutting site. Similar results and conclusions have recently been reported by Lutter (1977). This paper examines the action of spleen acid DNAase (a DNAase II) and staphylococcal nuclease on nucleosomes. To relate these results to the earlier ones obtained with DNAase I, it is necessary to determine not only the nature of the staggered cutting pattern for each enzyme, but also the positions of the cutting sites for all three enzymes relative to one another. We show that each enzyme has a different set of cutting sites on the nucleosome, but that these sites are symmetrically related: if nearest cutting sites on opposing DNA strands are thought of as having a dyad symmetry axis, then the dyad axes for all three enzymes coincide. It seems probable that this symmetry reflects a repeating structural feature, spaced at 10 nucleotide intervals along each chain of the nucleosome, which is the recognition site of all three enzymes. Although all the enzymes share this recognition site, their cuts are displaced from it along the chains in a direction and by a number of nucleotides determined by the characteristics of the individual enzyme.

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Results Stagger of DNAase I and DNAase II Cuts When chromatin is cleaved by DNAase I or DNAase II, a striking DNA fragment pattern is produced. Most of the phospodiester bonds are protected against cleavage by these enzymes; the vast majority of the sites available to nucleases are separated by integral multiples of 10 bases along each DNA strand. The resultant digest DNA, when denatured and fractionated on polyacrylamide gels, forms a series of bands 10.n nucleotides in length (n integer). Although there are slight intensity differences between DNAase I (Nell, 1947b) and DNAase II (Sollner-Webb et al., 1976) digest bands, the patterns are very similar (Figure 1A). As the extent of digestion increases, the average size of the 10-n nucleotide fragments decreases until after extensive digestion with DNAase I (Sollner-Webb and

A.

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Duck erythrocyte nuclei were digested with DNAase I or DNAase II, and the isolated DNA was electrophoresed on 10% acrylamide gels. A densitometer tracing of a photographic negative is shown. (A) DNA was denatured prior to electrophoresis; the number of nucleotides corresponding to each band is indicated. DNAase I digest 14% acid-soluble: DNAase II digest 23% acid-soluble. Digests of nucleosome cores are virtually identical. (8) DNA was not denatured prior to electrophoresis; the largest and most abundant single-stranded component of each native band is indicated. DNAase I digest 34% acid-soluble; DNAase II digest 43% acid-soluble.

Felsenfeld, 1977) or DNAase II (data not shown), the vast majority of the original DNA is in fragments 10 nucleotides long. Only when about half the original DNA is reduced to fragments 10 nucleotides in length by DNAase I or DNAase II does the nucleoprotein precipitate. The preponderance of 10 nucleotide fragments in extensive digests suggests that sites accessible to these nucleases are spaced 10 nucleotides apart over much of the DNA, although the rates of attack at different sites vary considerably. This denatured DNA digest pattern gives no information about the location of the cuts introduced into one DNA strand relative to those in the other strand. The double-stranded (nondenatured) DNA fragments resulting from DNAase I or DNAase II digestion of chromatin do, however, contain information about the stagger of the cuts in the two DNA strands. When such duplex fragments are resolved on acrylamide gels, it is found that the pattern generated by DNAase I and DNAase lj are somewhat different from one another and more complex than the denatured patterns (Figure 16). The lengths of the single-stranded components present in each of the duplex bands of the DNAase II digest were determined by two-dimensional electrophoresis. As was found for the DNAase I bands (Sollner-Webb and Felsenfeld, 1977), each doublestranded DNAase II gel band, when denatured, gives rise to several single-stranded fragments (data not shown). In all cases for a given doublestranded fragment, the longest single-stranded fragment is also the most abundant. This predominant component is indicated in Figure 1B. It has been noted that each double-stranded gel band does not constitute one pure molecular species, but rather is made up of several classes of very similarly migrating molecules (Sollner-Webb and Felsenfeld, 1977). This accounts, at least in part, for their lack of resolution. That the DNAase I duplex gel bands are not completely base-paired molecules, but have termini with single-stranded tails, has been demonstrated by Sl nuclease treatment (Sollner-Webb and Felsenfeld, 1977). When DNAase II duplex fragments are similarly treated with Sl nuclease, the fragments decrease in size consistent with the removal of single-stranded tails (data not shown). These results suggest that the difference between the double-stranded gel patterns of the DNAase I and DNAase II digests arises from a difference in the size of the tails formed by the pairing of singlestranded fragments of a given size. This must reflect a difference in the stagger of the cuts produced by the two enzymes. To measure directly the stagger of the DNAase I and DNAase II cuts, the repair activity of E. coli DNA polymerase II was used. This DNA-dependent

Cutting 613

Sites

of Nucleases

in the Nucleosome

Core

DNA polymerase adds nucleotides to recessed 3’ OH ends to fill in single-stranded regions of partially duplex DNA templates (Wickner et al., 1972a, 1972b). We have shown that when DNA cut by Eco Rl or Hind III restriction enzymes is used in a DNA polymerase II reaction, the only detectable activity is faithful and complete repair (to form doublestranded DNA) of the 4 nucleotide long singlestranded regions which terminate each end (Sollner-Webb and Felsenfeld, 1977). Thus when a partly duplex molecule with a recessed 3’ OH terminus is treated with DNA polymerase II, the primer strand should increase in length by the number of nucleotides by which it is recessed. This number of nucleotides is a measure of the stagger in the cuts on the two DNA strands. We reported earlier that when nucleosomal DNA, previously cut by DNAase I, is treated with DNA polymerase II and radioactive deoxynucleoside triphosphates, the resultant radioactive DNA frag00

A

71)

60

50

ments have a single-stranded size of 10. n + 8 nucleotides. DNAase I thus creates ends in which 3’ termini are recessed by 8 nucleotides and correspondingly where 5’ termini are recessed by 2 nucleotides (Sollner-Webb and Felsenfeld, 1977; Figure 2A). In contrast, we now find that when this same experiment is performed on nucleosomal DNA cut by DNAase II, the radioactive fragments that form are 10.1-1 + 6 nucleotides long (Figure 28). (Since DNAase II leaves 3’ P termini, the DNAase II digest DNA must be phosphatase-treated prior to the polymerase reaction: marker DNA was also phosphatase-treated to assure valid comparisons of mobility.) DNAase II thus cleaves nucleosomal DNA with a stagger of 6 nucleotides, with 3’ ends recessed. When these single-stranded regions are filled in by DNA polymerase II, DNA molecules with an original single-stranded length of 10.1-1 nucleotides are extended by 6 nucleotides, to generate

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Duck erythrocyte nuclei were digested with DNAase I or DNAase II, to 25 and 43% acid solubility, respectively. The isolated native DNA was treated with E. coli DNA polymerase II (0.5 units per 50 ng DNA) in the presence of **P-deoxynucleotide triphosphates after phosphatase treatment of the DNAase II digest. The resultant DNA was mixed with excess unlabeled marker DNA, denatured and electrophoresed on 30 cm 10% acrylamide gels. Marker DNA was a DNAase I nuclear digest for the DNAase I sizings (a) and a phosphatase-treated DNAase I nuclear digest for the DNAase II sizings (b). The gel was ethidium bromide-stained, approximate band positions were marked with radioactive ink and a photograph was taken. An autoradiogram was then made of the gel. Densitometer scans of the autoradiogram (top, solid line) and the marker DNA-stained photographic negative (top, broken line), were aligned by the location of adjoining radioactive ink dots as described (Sollner-Webb and Felsenfeld. 1977). A semilog plot of migration against size was calibrated for each sample using the 10.n nucleotide long markers. From the relative migration of the radioactive fragments, their sizes were determined as indicated (middle). A diagram of the fragment ends which were filled in by the polymerase is shown (below); neighboring potential cutting sites are marked by arrows. The cut staggers determined by this method are independent of both the extent of the original DNAase digestion and the extent of the polymerase treatment. Reactions with saturating amounts of polymerase are shown.

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molecules 10.n + 6 nucleotide long. Since potential DNAase II cutting sites are located every 10 nucleotides over much of the DNA, cuts must also be created with a stagger of 4 nucleotides, with 5’ ends recessed. Thus the stagger of the cuts created in nucleosomal DNA by DNAase I is 8 and 2 nucleotides, while that of DNAase II is 6 and 4 nucleotides (Figure 2). DNAase I and DNAase II Cut Locations and Dyad Axes The above results show that although DNAase I and DNAase II both cut at intervals of 10.n nucleotides on each strand of nucleosomal DNA, the stagger of the cuts produced by the two enzymes is different. Thus the cutting sites of these two enzymes cannot be identical. To determine the relative position and the absolute location of the DNAase I and DNAase II cutting sites, a reference point was created, and the distance from the DNAase cutting sites to this reference was measured. Nucleosome cores, the 140 bp evolutionarily conserved elements of the nucleosome, were radioactively labeled at the 5’ termini with polynucleotide kinase (Simpson and Whitlock, 1976) and then treated with DNAase I or DNAase II. The resultant DNA was purified, denatured and fractionated on long acrylamide gels (Figure 3). Neighboring gel wells contained kinase-labeled 10.n nucleotide fragments as a marker. [Since DNAase I leaves 3’ OH termini and DNAase II leaves 3’ P termini, markers were appropriately constructed to have termini identical to the fragments being sized (Figure 3).] Nucleosome cores were kinased to about 70% of complete labeling, thus insuring detection of cuts created in both strands of the DNA. Knowing the distance from the 5’ termini of the nucleosome core to the DNAase cuts and the stagger of these cuts (Figure 2), one can measure precisely the locations of the DNAase I and DNAase II cleavage sites within the nucleosome core. Such an experiment is shown in Figure 3. DNAase I creates cuts 10.n + 2 nucleotides from the 5’ terminus of the nucleosome core; DNAase II creates cuts 10.n + 3 nucleotides from the 5’ terminus (Figure 3). While not all potential sites are cut with equal efficiency, virtually all the sites are cleaved at a detectable level. Thus DNAase II cutting sites always occur one nucleotide to the 3’ side of DNAase I cutting sites (Figure 3). In five separate series of digestion experiments using three different nucleosome core preparations and two different sets of markers, these sizings varied by G l/2 nucleotide. In all the experiments with increasing extent of DNAase I or DNAase II digestion, fragments remained 10.n + 2 and 10.n + 3 nucleotides long, respectively, but

the relative abundance of the small ‘radioactive fragments increased (the average value of n decreased). The extent of DNAase I and DNAase II digestion ranged from a point at which >80% of the terminal label remained >120 nucleotides long, to a point at which >50% of the terminal label was ~20 nucleotides long. (When -50% of the 5’ terminal label is -10 nucleotides long, the bulk of the nucleosomal DNA remains a40 nucleotides long.) In all these digests, the 10.n + 2 and 10.n + 3 cutting specificity was maintained, indicating that the structural feature of the nucleosome that confers this digestion specificity is itself fairly resistant to nuclease digestion. The data of Figures 2 (stagger of cuts) and 3 (distance of cuts from the 5’ termini) allow us to relate the location of the DNAase I and DNAase II cutting sites, as shown at the bottom of Figure 3. We consider a simple geometrical model of DNA and for each enzyme draw a “dyad axis,” defining the symmetric point halfway between the nearest pairs of DNAase I or DNAase II cutting sites. Thus the DNAase I dyad axis is 1 nucleotide to the 5’ side of its cutting site, while the DNAase II dyad axis is 2 nucleotides to the 5’ side of its cutting site. (In Figure 3, the dots represent the dyad axes of these cutting sites. Neighboring similar dyad axes are separated by 10 bp.) Since DNAase I cuts occur 10.n + 2 nucleotides from the 5’ end of the nucleosome core, the DNAase I dyad axes are 10.n + I nucleotides from the 5’ end of the nucleosome core. Similarly, since DNAase II cuts are 10.n + 3 nucleotides from the 5’ end of the core, DNAase II dyad axes also occur 10.n + 1 nucleotides from the 5’ end of the nucleosome core (Figure 3, bottom). Thus the DNAase I and II dyad axes coincide. As is discussed below, these results are consistent with the presence of a common recognition site for both enzymes and an overall dyad axis of the nucleosome. Effect of Ionic Conditions on Cutting Locations The noncoincidence of the DNAase I and DNAase II cutting sites does not arise simply from a difference in digestion conditions. When DNAase I and DNAase II digestions were both performed in identical solvents [lo mM Na acetate (pH 6), 0.5 mM MgCI,], the lengths of the radioactive fragments remained 10.n + 2 (data not shown) and 10.n + 3 nucleotides (Figure 4a), respectively. It was noted, however, that the efficiency with which DNAase II cleaves at its various potential (10.n + 3 nucleotide) cutting sites is a strong function of the ionic conditions. Whether digested in a cacodylate buffer (Figure 3) or acetate buffer (data not shown) each containing 0.5 mM EDTA, the preferred cleavage sites are the same, forming

Cutting 615

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Duck erythrocyte nuclei were digested with staphylococcal nuclease to 15% acid solubility. The nucleosome cores were isolated and labeled at the 5’ ends with polynucleotide kinase at -70% efficiency. Kinase-labeled nucleosome cores were next treated with DNAase I [in 10 mM Tris-HCI (pH 8), 1 mM MgCI,, 0.1 mM EDTA] to 24% bulk DNA acid solubility or DNAase II [in 10 mM sodium cacodylate (pH 5.5), 0.5 mM EDTA] to 31% bulk DNA acid solubility, and the resultant DNA fragments were isolated. All samples were mixed with excess equal amounts of unlabeled carrier DNAase I nuclear digest DNA, denatured and electrophoresed on 30 cm 10% acrylamide gels. Markers were run in wells flanking the experimental samples: ethidium bromide staining demonstrated that all gel wells ran synchronously. An autoradiograph of each gel was scanned with a microdensitometer (top); solid lines represent the experimental samples; broken lines represent the flanking size standard marker DNA. The experimental radioactive fragments all have a 5 ’ J2P (kinased) terminus. Their 3’ terminus is 3’ OH after DNAase I digestion or 3’ P after DNAase II digestion. The markers were appropriately constructed: nuclear DNAase I digest (5’ P, 3’ OH) was phosphatased and then kinased (5’ 32P, 3’ OH) for the DNAase I experiments; nuclear DNAase II digest (5’ OH, 3’ P) was kinased (5’ J2P, 3’ P) for the DNAase II experiments. (The additional terminal phosphate changes the migration by somewhat less than the removal of one nucleoside monophosphate.) A semilog plot of migration against size was calibrated for each sample using the 10.n nucleotide long markers. From the relative migration of the experimental fragments, their sizes were determined as indicated (middle). Combining these results with the cutting Staggers determined in Figure 2, a diagram of the DNAase cutting sites (arrows) in the nucleosome core is shown (bottom), The dot represents the dyad axis of the indicated neighboring cutting sites.

a more or less symmetrical pattern with respect to the two ends of the DNA chain-that is, cuts about the pairs of dyads at 10.n and 140-10-n nucleotides occur at similar frequencies (Figure 3). In contrast, when similar DNAse II digestions are performed in acetate buffer with 0.5 mM MgCI,, the pattern of preferred cutting sites is strikingly unsymmetrical (Figure 4a). The sites 103, 123 and 133 nucleotides from the 5’ terminus are by far the most frequently cleaved. From Figure 4a, one can calculate that the probability of DNAase II cleavage occurring at sites

283 nucleotides from the 5’ terminus (83, 93, 103, 113, 123 or 133) is -77%, while the probability of a cut being between 23 and 73 nucleotides from the 5’ end is only -12% in the MgCI,-containing buffer. In contrast, when the DNAase II digestion is carried out in EDTA-containing buffer (Figure 3B), the probabilities of cleaving at 83-133 nucleotides and 23-73 nucleotides from the 5’ end are both -38% (Table 1). We do not know whether this ionic effect results from differences in nucleosome conformation or differences in nuclease specificity.

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Table 1. Probability Nucleosome Core

Digestion Buffer MgCI,

EDTA

Figure 4. Determination of the DNAase II and Staphylococcal Nuclease Cleavage Sites within Nucleosome Cores (a) The kinase-labeled nucleosome cores of Figure 3 were digested with DNAase II in 10 mM NaOAc (pfi 6), 0.5 mM MgCI,, 0.1 mM EDTA, to approximately 12% bulk DNA acid solubility. Digest DNA (solid line) was analyzed as described in Figure 3 using the marker constructed for the DNAase II digest (broken line). Undigested nucleosome core DNA is also shown (dotted line). (b) The kinase-labeled nucleosome cores of Figure 3 were digested to the limit (29% bulk DNA acid solubility) with staphylococcal nuclease in 1 mM Tris-HCI (pti 8) 0.2 mM CaCI,, 0.1 mM EDTA, and analyzed as described in Figure 3 (solid line) using the appropriate (DNAase II) marker (broken line). The largest size radioactive peak corresponds to -160 nucleotide long fragments which contaminate the nucleosome core preparations and which were not digested under these conditions. (c)The nucleosome cores described in Figure 3 (unlabeled) were digested to the limit (31% acid solubility) with staphylococcal nuclease in 1 mM Tris-HCI (pH E), 0.2 mM CaCI,, 0.1 mM EDTA (solid line), or with DNAase I in 10 mM Tris-HCI (pH 8), 1 mM MgCI,. 0.1 mM EDTA to 19% acid solubility (broken line). DNA was isolated, fractionated on a 10% acrylamide gel and visualized by Stains-all staining (solid line).

Staphylococcal Nuclease Cut Stagger We wished to measure the cutting stagger of staphylococcal nuclease to determine also the location of this enzyme’s cutting sites and dyad axes. The measurement was made using single-strand-specific nucleases, and hence required a number of controls concerned with enzyme specificity (described below). The conclusion of these studies is that staphylococcal nuclease produces cuts with staggered ends; the 5’ terminus protrudes by 2 nucleotides. We have shown elsewhere that at early times in the staphylococcal nuclease digestion of chroma-

of DNAase

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13 23-63 73 a3 93 103 113 123 133 2140

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0.18 0.134 0.070 0.136 0.158 0.333 0.125 0.245 0.282

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Kinase-labeled nucleosome cores were digested in various buffers with DNAase Il. A densitometer tracing of the resulting fragment pattern (Figures 38 and 4a) was resolved into gaussian curves, and the approximate number fraction of labeled molecules represented in each 10.n + 3 nucleotide size class was measured (f,,). A labeled molecule of size 10.n + 3 means that a cut occurred at 10.n + 3 nucleotides from the 5’ terminus, but no cuts occurred at sites nearer the labeled 5’ terminus (no cuts at sites 10.m + 3, m < n). At a given time, the probability that site 10.n + 3 has been cut, C,,, may be determined using the relationship flon = Con [I - C,,.-,,I [l - C,,,-,,I . (1 - C,,,) with fIo = C,,. The relative probability of cutting at site 10.n + 3, R,,, is determined from: R,,, = C,,,,,/~]~ C,,.. The implicit assumption in this calculation-that cuts occur independently at the various sites-appears valid, since R,, is relatively independent of the extent of digestion (at least for ~50% of the label appearing as the 13 nucleotide fragment; data for other digestion times not shown). See Lutter (1978) for a detailed study of this general kind for the DNAase I cleavage sites.

tin (~30% acid-soluble), double-stranded DNA fragments appear which, when denatured, yield fragments exactly 10.n nucleotides long (CameriniOtero et al., 1976; Figure 5). When these duplex DNA fragments are treated with Sl nuclease under conditions known to remove single-stranded “tails” (Sollner-Webb and Felsenfeld, 1977), the fragments decrease in size, indicating the presence of staggered termini. (To make accurate size measurements, all samples were treated with phosphatase prior to electrophoresis. To rule out gel migration artifacts further, all samples were then phenolextracted and combined with trace amounts of a radioactive, 105 nucleotide long, restriction enzyme fragment. Minor migration differences, which could have been detected in an autoradiograph of this very sharp band, were not observed.) From many such experiments with Sl nuclease, two examples of which are shown in Figure 5, the fragments are found to decrease in length by -2.2 nucleotides, with a range between 1.8 and 2.6 nucleotides. Thus Sl nuclease removes a single-

Cutting 617

Sites

of Nucleases

in the Nucleosome

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All samples were denatured in 0.1 M NaOH prior to loading onto preelectrophoresed 10% acrylamide gels. (a) 16% acid-soluble staphylococcal nuclease digest of chromatin; (b) 23% acid-soluble DNAase I digest of nuclei. (c-f) Reactions contained 60 pg of a 22% acid-soluble staphylococcal nuclease digest of chromatin in 250 ~1 of 50 mM Na acetate (pH 4.75) 150 mM NaCI, 0.5 mM ZnSO,. These were incubated for 1 hr at 37°C with (c and f) no Si ; (d) 15 units Sl; (e) 21 units Si. All samples and (g), a 26% acidsoluble DNAase I digest of nuclei, were phenol-extracted and ethanol-precipitated. They were then treated with 0.3 units of Worthington alkaline phosphatase (BAPF) in 50 mM Tris-HCI (pH 6), 0.5 mM EDTA for 1 hr at 37°C and again phenol-extracted. Trace amounts of a 105 nucleotide long restriction enzyme fragment labeled at one end with polynucleotide kinase were added, and after ethanol precipitation, the material was denatured and electrophoresed. (h-j) Reactions contained 60 pg of a 26% acidsoluble staphylococcal nuclease digest of chromatin, and were Sl- and phosphatase-treated and prepared for electrophoresis as described above. (h) No Sl; (i) 9 units Sl; (j) 15 units Sl. These samples show well the action of Sl on the 50, 60 and 70 nucleotide fragments.

stranded “tail” of 2 nucleotides from the termini produced by staphylococcal nuclease. To conclude from this experiment that staphylococcal by 2 nuclease generates termini that are staggered nucleotides, it must be shown that under the conditions used, Sl nuclease produces absolutely flush ends. This was done starting with DNA cleaved by Hinf restriction enzyme; the terminus has the general structure 5’pApNpTpC.... After light kinase-labelG . .. ing, the material was subjected to Sl nuclease digestion until ~80% of the terminal label was removed. The resultant 5’ terminal nucleotide was then determined. The DNA was phosphatasetreated and kinase-labeled after denaturation. Next it was purified and digested to mononucleotides, and the labeled residue was determined by paper electrophoresis in acid (Brownlee, 1972). 74% of the label co-migrated with dCMP, 9% with dTMP, 12% with dAMP and -5% with dGMP. The simplest explanation of this result is that Sl nuclease fully removed the single-stranded tails (pApNpT) to generate flush ends @‘PC...) in about three fourths of G .. . the molecules. We therefore feel justified in con-

cluding that staphylococcal nuclease generates termini with a stagger of 2 nucleotides. The nucleosome cores which we used in these studies came from early (lo-17% acid-soluble) digests, so they, like the subnucleosome fragments, may also be expected to have ends staggered by 2 nucleotides. Accordingly, when DNA from the 140 bp nucleosome core is treated with Sl, the molecules decrease in size, consistent with a loss of 2 nucleotides from each single-stranded fragment (data not shown). In contrast, when double-stranded DNA subnucleosome fragments from a late (>35% acid-soluble) chromatin digest, which has a single-stranded length of 10-n-2 nucleotides (Camerini-Otero et al., 1976), are similarly treated with Sl, the fragments do not decrease in size (data not shown). This result is consistent with the presence of flush ends on DNA from chromatin extensively digested with staphylococcal nuclease. To determine the direction of the 2 nucleotide stagger generated by staphylococcal nuclease, we again used Sl nuclease, this time measuring digestion kinetics of 5’ (kinase)-labeled staphylococcal nuclease digest DNA. Control experiments with Sl nuclease were performed using DNA with a 2 nucleotide (Hpa II cut) or 3 nucleotide (Hinf cut) protruding single stranded 5’ terminus. The rate at which the kinase-labeled 5’ termini of these molecules was rendered acid-soluble by Sl nuclease was the same as the rate of digestion of singlestranded DNA (Figure 6A). In contrast, the kinase label of DNA with flush ends (Sma I cut) requires nearly 10 times more Sl for removal (Figure 6A). There is every reason to believe that recessed 5’ termini would be attacked at least equally slowly by Sl nuclease. When DNA of a 22% acid-soluble staphylococcal nuclease digest of chromatin is kinase-labeled and Sl nuclease-treated, it is digested at the same rate as single-stranded DNA (Figure 6A). Kinase-labeled DNA from 9 and 18% digests of chromatin and from nucleosome cores (17% acid-soluble digest) all digested at rates virtually identical to that of single-stranded DNA (variation ~8% at any one point from the indicated line; no indication of biphasic digestion kinetics for any sample; data not shown.) Furthermore, when 32P solubilized by Sl nuclease from nucleosome cores and from a 22% acid-soluble digest of chromatin was assayed, >90% was found as organic phosphate (see Experimental Procedures), indicating that it arose from a true Sl nuclease action rather than a contaminating phosphatase. Thus DNA from staphylococcal nuclease digests of chromatin (~30% acid-soluble) and from nucleosome cores behaves as if its 5’ termini have a single-stranded character-that is, it has protruding 5’ termini.

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616

40 s Figure

6. Single-Strand-Specific

Nuclease

Digestion

160 MO ““l,J staph Kinetics

(A) Sl: reactions contained 25 fig denatured calf thymus DNA and -5000 cpm of (high specific activity) kinase-labeled test DNA and the indicated amounts of Si nuclease in 250 1.11of 50 mM Na acetate (pH 4.75) 150 mM NaCI, 0.5 mM ZnSO,. After a 1 hr incubation at 3PC, reactions were made 100 pglml in BSA and brought to 0.8 M Nacl, 0.8 M perchloric acid, chilled to 0°C for 10 min and centrifuged. The fraction of material rendered acidsoluble was monitored by kso and Cerenkov counting. For all the parallel reactions, the fraction of the single-strand DNA digested at each time point varied by ~5%; an average value is presented. (x) single-stranded DNA: (0) kinase-labeled Sma cut DNA , flush termini; (0) kinase-labeled Hpa II cut DNA, 5’terminus protruding by 2 nucleotides; (A) kinase-labeled Hinf cut DNA, 5’ terminus protruding by 3 nucleotides; (0) kinase-labeled 22% acid-soluble staphylocccal nuclease digest of chromatin. (B) Staphylococcal nuclease: reactions contained 50 fig of a 9% acid-soluble (low salt) staphylococcal nuclease digest of chromatin and -5000 cpm of (high specific activity) kinase-labeled test DNA and the indicated amount of phosphatase-free staphylococcal nuclease in 300 ~1 of 0.4 M NaCI, 10 mM MgCI,, 10 mM TrisHCI (pH 6), 0.1 mM CaCI,. A parallel reaction contained 50 pg of denatured duck reticulocyte DNA and the indicated amount of enzyme in the same buffer. After incubation for 30 min at 37”C, reactionswere made 100 pglml BSA, 0.8 M NaCI, 0.8 M perchloric acid, chilled at 0°C for 10 min and centrifuged. The fraction rendered acid-soluble was monitored by AZ80 or Cerenkov counting. Sample designations as in (a).

The data in Figure 5 make it most probable that the 5’ terminus protrudes by 2 nucleotides (values averaged 2.2 nucleotides). That this stagger was actually 2 nucleotides, rather than 3, was demonstrated using another property of staphylococcal nuclease. Under high salt conditions, this enzyme acts as a single-strand-specific nuclease (Kacian and Spiegelman, 1974), and we determined its action on short protruding single-stranded regions (Figure 6B). The enzyme attacks flush ends (kinaselabeled DNA cut by Sma I) far more slowly than it does single-stranded DNA. Termini with the 5’ end protruding by 2 nucleotides (kinase-labeled DNA cut by Hpa II) are attacked at the same rate as flush termini, but termini with the 5’ end protruding by 3 nucleotides (kinase-labeled DNA cut by Hinf) are digested at a faster rate, intermediate between that of flush termini and single-stranded DNA (Figure 68). The rate of single-strand-specific digestion of termini generated by staphylococcal nuclease acting on chromatin under normal, low salt conditions (kinase-labeled DNA from a 22% acid-soluble di-

gest of chromatin) is like that of DNA with the 5’ end protruding by O-2 nucleotides. It is unlike the rate of digestion of DNA with a 3 nucleotide long single-stranded 5’ terminus (Figure 6B). Thus the stagger of 2.2 nucleotides determined in Figure 5 is clearly 2 rather than 3 nucleotides. The cutting stagger which we have determined for staphylococcal nuclease cannot be verified using the DNA polymerase II repair technique. Control experiments demonstrate that a cut stagger of 2 nucleotides, with the 3’ end recessed, does not provide a terminus which is suitable as a substrate for this polymerase. DNA cleaved by the restriction enzyme Hpa II, which has termini of the form

5’*4 3’**G-G-Cp, was shown to be only 5% as efficient a template (per repairable nucleotide) as DNA cleaved by Hind III, which terminates in a singlestranded tetranucleotide (see Experimental Procedures). In agreement, DNA extracted from nucleosome cores or from a 15% acid-soluble chromatin digest is a very inefficient DNA polymerase II substrate. After exhaustive phosphatase treatment, these DNA preparations were treated with the polymerase as described in Figure 2. At saturation 90% of which are smaller than 130 nucleotides in length. Thus even the small amount of incorporation which was observed involved minor undefined species rather than the 140 nucleotide fragments. From these polymerase II experiments, we can only conclude that the staphylococcal nuclease cutting stagger is 70% of the DNA acid-soluble), the DNA fragments remain 10.n nucleotides in length (n = 1 and 2 mainly). In addition, Lutter (1978) has demonstrated that until late in the digestion process, the frequency of DNAase I attack at the various potential cutting sites remains unchanged, and we have noted a similar stability in the relative cutting probabilities for DNAase II. Second, the entire nucleosomal histone complement is not required to generate such restricted cleavage sites. The arginine-rich histones are sufficient to restrict staphylococcal nuclease, DNAase I and DNAase II cleavages to sites separated by 10-n nucleotides (Camerini-Otero et al., 1976; Sollner-Webb et al., 1976). Furthermore, the 10.n nucleotide sites on the two DNA strands of an arginine-rich histone-DNA complex appear to have the same relative location (stagger) as they do in whole chromatin, for their staphylococcal nuclease limit digest band size is also 10.n - 2 nucleotides in length. Third, the structure which determines the 10.n nucleotide restriction extends beyond the nucleosome core. Staphylococcal nuclease digestion of soluble chromatin produces DNA fragments 150 and 160 nucleotides long in high yield (Axe1 et al., 1974; Camerini-Otero et al., 1976). In staphylococcal nuclease digestion of gently lysed nuclei, a metastable DNA intermediate, 160 bp in length, is formed, rather analogous to the 140 nucleotide long nucleosome core from digests of intact nuclei (Sollner-Webb, 1976). Thus at least two sites in the “intercore” region may be expected to manifest the same cutting restriction as has been shown for sites within the nucleosome core. In addition, since DNAase I digestion of nuclei produces regular 10.n nucleotide long bands, well beyond 200 nucleo-

Cutting 625

Sites

of Nucleases

in the Nucleosome

Core

tides in length, there appears to be a mechanism whereby, at least in some instances, nucleosome cores may be phased along the DNA (Lohr, Tatchell and Van Holde, 1977). It should be obvious from our data that the sizing of the DNA fragments reported here are made relative to the sizes of the DNAase I digest fragments cleaved from nuclei. Thus the results given in Figure 2 show that when the duplex DNA from a DNAase I or DNAase II digest is treated with DNA polymerase II, chains are elongated by 8 or 6 nucleotides relative to the corresponding untreated DNA digestion fragment. Similarly, radioactive fragments cleaved from kinase-labeled nucleosome cores by DNAase I or DNAase II are 2 or 3 nucleotides longer, respectively, than the corresponding DNAase I or DNAase II digestion fragments obtained from unlabeled nuclei (Figures 3 and 4a). Finally, the DNA of the nucleosome core co-migrates with the fourteenth band of this DNAase I digest. While we have assumed that the DNAase I digest fragments used as standards are exact integral multiples of 10 nucleotides in length (Nell, 1974b), it may be that some deviation from this regularity will be detected using more recent and precise methods of chain length determination. For example, there is evidence that the DNA of the nucleosome core may be about 145 bp long (T. Kovacic and K. Van Holde, personal communication). It is obvious that for most simple models which one could propose to account for this possible increase of -3% in nucleosome core size, the relative local cutting sites determined in this study are essentially unaffected. The fragments resulting from digestion with any of the three nucleases form somewhat broader bands on gels than might be expected for species of homogeneous length. In principle, this could arise either from variation of mobility with base composition for chains of identical length, or from a real variation in length. The latter appears to be the correct explanation, at least for fragments shorter than -40 nucleotides in length. In that range, it is possible, under appropriate conditions, to resolve numerous sharp bands in a digest of chromatin or protein-free DNA, which are spaced at about 1 nucleotide intervals (Jackson and Chalkley, 1975; B. Sollner-Webb and R. D. CameriniOtero, unpublished data). Thus sequence heterogeneity does not give rise to all of the broadening of the gel bands. In some cases, the broad gel bands of the DNAase I nuclear digest pattern can be resolved into groups of discrete bands (Lutter, 1977; J. McGhee, personal communication). We therefore attribute the breadth of at least some of the chromatin digest bands to length heterogeneity. Since a given band in a total digest pattern may contain components excised from various regions

of the nucleosome, the heterogeneity may reflect small differences among these regions in the placement of cutting sites. Alternatively the recognition sites may be regularly spaced (either integrally or nonintegrally), but the nuclease may sometimes cut 1 nucleotide to either side of these sites. The results presented in this paper thus refer to the mean positions of cutting sites. In summary, the cuts made by DNAase I, DNAase II and staphylococcal nuclease are located at different sites, but are symmetrically clustered about common dyad axes occurring every 10 bp on nucleosomal DNA. These three nucleases probably recognize a common periodic structural feature of the nucleosome, but individual nuclease specificity dictates that each enzyme cleave a different number of nucleotides away from the recognition site. While not every nuclease cleaves about each dyad axis, virtually all axes are recognized by two, and some by all three, nucleases. Thus over much of the nucleosome, there are regions comprising 4 in every IO nucleotides which are potentially cleavable, and so in some sense accessible to the external environment. This suggests the existence of a somewhat more uncovered structure of the nucleosomal DNA than may have previously been envisioned. ExPerimental

Procedures

lsolatlon of Nuclei Chromatin and Nucleosome Cores Nuclei were prepared from frozen duck enthrocytes by several successive washings in 10 mM Tris-HCI (pH 8), 1 mM MgCI,, 0.25 M sucrose as described previously (Sollner-Webb and Felsenfeld, 1975). The addition of 0.25% Triton X-100 in an early wash had no effect on the digestion results. Nuclei were used the day on which they were prepared. Chromatin was isolated from salt-washed nuclei as described previously (Camerini-Otero et al., 1976). Nucleosomes were prepared from nuclei washed in 1 mM TrisHCI (pH 8), 0.5 mM CaCI,, 0.25 M sucrose, and resuspended in 1 mM Tris-HCI (pH 8), 0.1 mM CaCI,, 0.08 M sucrose at l-4 mg DNA/ml. Enough phosphatase-free staphylococcal nuclease was added for the reaction to reach -15% acid-solubility in about 30 min, and it was then stopped with 0.5 mM NaEDTA (pH 8). Digests were dialyzed for 2 hr against 1 mM Tris-HCI (pH 8), 0.1 mM EDTA, and fractionated on 5-20% sucrose gradients in the same buffer at 4°C (SW27 rotor, 25,000 rpm, 14 hr). Peak monomer fractions were pooled and sucrose was removed by dialysis, essentially as described (Sollner-Webb and Felsenfeld, 1975). Over 50% of the starting DNA is routinely recovered as monomer nucleosomes. Nucleosomes containing lysine-rich histones were precipitated by dialysis into 0.1 M KCI, 1 mM Tris-HCI (pH 6), 0.1 mM EDTA. and centrifuged (2000 x g, 10 min) out of solution as described (Dlins et al., 1976). Nucleosomes without lysine-rich histones remain in solution; they are largely (>80%) nucleosome cores, containing 140 bp of DNA. The contaminant is almost entirely a nucleosome species with -180 bp DNA, which lacks the lysinerich histone. Very few nucleosome cores are internally cleaved in these preparations. With our 15% acid-soluble nuclease digests, -75% of the monomer is recovered in the nucleosome core fraction. Kinase, Kinase

Nuclease, reactions

Polymerase were carried

and Phosphatase Reactions out using a modification of the

Cell 626

procedure of Simpson and Whitlock (1976), in 50 mM Tris-HCl (pH 8), 5 mM MgCI,, 50 mM DTT, 20 PM~-~*P-ATP (New England Nuclear) with chromatin at -25 Kg DNA per ml and -0.2 IJ polynucleotide kinase (PL Laboratories) per pmole termini. After incubation at 37” for 1 hr, 20-70% of theoretical labeling was obtained. Reactions were stopped with EDTA, dialyzed for several hours against 1 mM EDTA and purified from unreacted triphosphate on 5-20% sucrose gradients as described above for monomer isolation. Isolated DNA fragments were similarly labeled; unreacted triphosphates were removed by two precipitations with 2 vol of ethanol. DNAase I (pancreatic DNAase; Worthington) was used to digest nucleosome cores (-0.1 mg DNA per ml) in 10 mM Tris-HCI (pH 8). 1 mM MgC12. 0.1 mM EDTA, and nuclei (l-3 mg DNA per ml) in 10 mM Tris-HCI (pH 8), 1 mM MgCI,, 0.25 M sucrose at 37°C. Enzyme concentration was adjusted to give the desired amount of acid solubilization as described (Sollner-Webb et al., 1976). Reactions were terminated by the addition of 5 mM EDTA. Alternatively, nucleosome cores were digested with DNAase I, as with DNAase II, in IO mM NaOAc (pH 6), 0.5 mM MgCI,, 0.1 mM EDTA at 37°C. Controls omitting enzyme were unchanged on incubation. DNAase II (spleen acid DNAase; Worthington) was used to digest nucleosome cores (-0.1 mg DNA per ml) in 10 mM sodium cacodylate (pH 5.5), 0.5 mM EDTA or 10 mM NaOAc (pH 6), 0.5 mM EDTA; nuclei (1-3 mg DNA per ml) were digested in the former buffer with 0.25 M sucrose. Time of reaction at 37°C was adjusted to give the desired amount of acid solubilization. Reactions were terminated with 1 M NaCI, transfer to 0°C and rapid DNA extraction or with 1 M NaCI, 0.1% SDS. Alternatively, nucleosome cores were digested with DNAase II, as with DNAase I, in 10 mM NaOAc (pH 6) 0.5 mM MgCI,, 0.1 mM EDTA at 37°C. Controls omitting enzyme were unchanged upon incubation. Staphylococcal nuclease (Worthington) was used to digest nucleosome cores (-0.1 mg DNA/ml) in 1 mM Tris-HCI (pH 8). 0.2 mM CaCI,, 0.1 mM EDTA at 37°C. Time of reaction was adjusted to give the desired amount of acid solubilization. Alternatively, no additional nuclease was used, and the reaction was performed by activation, with 0.1 mM CaCI, final concentration, of the staphylococcal nuclease contaminating the nucleosome cores. Reactions were terminated by the addition of 1 mM EDTA. Digestions of nuclei were performed as described above. Staphylococcal nuclease preparations used in these studies were freed of phosphatase contamination by the method of Fuchs, Cuatrecasas and Anfinsen (1967). Following purification, the acid-soluble 32P released by staphylococcal nuclease digestion of kinase-labeled nucleosome cores was shown to be >90% organic material. Staphylococcal nuclease was also used as a single-strandspecific nuclease as described by Kacian and Spiagelman (1974). These three nucleases were demonstrated to be free of detectable protease by digesting chromatin or nucleosomes under the conditions described above for nucleosome core digestions, but using 5 times as much nuclease. Digests and untreated controls were run on SDS stacking gels, and protein was visualized by Coomassie blue staining as described (Sollner-Webb et al., 1976). Certain DNAase II lots were protease-contaminated and were discarded; other DNAase II lots, and all the DNAase I and staphylococcal nuclease lots used, had no detectable protease. Sl nuclease digestions of digest DNA (-50 pg/ml) were carried out in 50 mM NaOAc (pH 4.75), 150 mM NaCI, 0.5 mM ZnSO, at 37°C for 1 hr as described (Sollner-Webb and Felsenfeld, 1977). Kinetics of digestion were measured by mixing trace amounts of kinase-labeled test DNA with excess denatured calf thymus DNA and adding varying amounts of enzyme under the above conditions. After 30 min, the reactions were terminated, and the extent of digestion of the radioactively labeled end and the bulk DNA were determined for each sample using 32P and optical absorbance, respectively. To determine whether flush-ended termini are produced by Sl nuclease, 0.1 pg of Hinf cut DNA (-6 pmole of termini) was treated with Sl until >60% of the kinase-labeled termini were

rendered acid-soluble. The material was phosphatased, kinaselabeled immediately after denaturation by heat and purified on a Biogel P60 column. After the addition of 0.5 +g of carrier plasmid DNA (pxlr 14 a), it was digested with 0.1 pg DNAase I (pancreatic DNAase; Worthington) in a 5 ~1 reaction in 5 mM Tris-HCI (pH a), 5 mM MgCI, for 1 hr at 37°C. It was then brought to 50 mM TrisHCl (pH 9), 10 mM MgCI, in a final volume of 10 1.11,and digested with 1 fig spleen phosphodiesterase for 1 hr at 37°C. Finally it was made 20 mM in EDTA and loaded onto Whatman 52 paper (21 in. long). Markers were an alkaline hydrolysate of &2P-rRNA, provided by Dr. Ted Maden (rXMPs run a few percentage points faster than dXMPs). After electrophoresis at 4000 V for 45 min in pyridine, 5% HAc (pH 3.5) 1 mM EDTA (Brownlee, 1972) the paper was dried and an autoradiogram was made. Spots corresponding to the four XMPs were cut out and counted for quantitation using Cerenkov radiation. Plasmid pxlrl4a DNA, provided by Dr. Ronald Reeder, was cleaved by the following restriction enzymes for use in singlestrand-specific nuclease assays: Sma I (CCC L GGG) in 30 mM Tris-HCI (pH 9), 15 mM KCI, 3 mM MgCI,; Hpa II (C 1 CGG) in 20 mM Tris-HCI (pH 7.4) 10 mM MgCI,, 500 pg/ml BSA, 10 mM pmercaptoethanol; Hinf I (G 1 ANTC) in 20 mM Tris-HCI (pH 7.3), IO mM MgC12, 500 pg/ml BSA, IO mM p-mercaptoethanol. After incubation at 37”C, the DNA was assayed for complete cutting on agarose or acrylamide gels. szP released from kinase-labeled DNA by action of the above nucleases was assayed for organic and inorganic components by the method of Conway and Lipmann (1964). E. coli DNA polymerase II reactions were performed in polymerase excess as described previously (Sollner-Webb and Felsenfeld, 1977). 2 units of Hpa II endonuclease (New England Biolabs) were used to cleave 1.5 pg of SV40 DNA (a gift from Dr. Maria Persico-DiLauro) in IO mM Tris-HCI (pH 7.4) IO mM MgCI,, 6 mM KCI, 1 mM DTT. Reactions were incubated at 37°C for 1 hr and monitored by agarose gel electrophoresis. a-32P-dCTP was used for the comparison of DNA polymerase II activity on Hpa II (5’ C 1 pCpGpG 3’) and Hind Ill (5’ A 1 pApGpCpTpT .3’) cleaved termini. Bacterial alkaline phosphatase (Worthington, BAPF) reactions were performed in 10 mM Tris-HCI (pH 8). 0.1 mM EDTA, 50 mM NaCl at 37°C. The extent of reaction was monitored by following the release of 3*P from added kinase-labeled termini, previously prepared from an aliquot of the material to be digested. To digest 90% of the termini of a DNAase I, DNAase II or staphylococcal nuclease nuclear digest, 1 ~1 of enzyme per 100 pg DNA incubated for 1 hr at 37°C was sufficient. 10 times this amount of enzyme, however, was used to insure removal of possibly less accessible phosphate termini. (In order for incorporation to proceed, it is necessary to phosphatase-treat DNAase I digest DNA prior to kinase labeling, and to treat DNAase II and staphylococcal nuclease digest DNA prior to DNA polymerase II treatment.) DNA Isolation and Gel Electrophoresis Duplex DNA was extracted after terminating digestions by making the reaction mixture 1 M in NaCl and treating with 0.1 mg/ml Proteinase K (E. Merck) at 37°C for 1 hr, followed by extraction with chloroform-isoamyl alcohol-phenol (24:1:24) and ethanol precipitation as described (Sollner-Webb and Felsenfeld, 1975). We have noted that nucleosomal DNA, nicked by DNAase I or DNAase II, has a tendency to denature as the histones are removed. We can reproducibly recover significant amounts of duplex DNA from extensive DNAase I or DNAase II digests (>50% acid-soluble) if we use the method described above. Alternate methods of extraction omitting the protease and using only phenol or chloroform-isoamyl alcohol, with or without added salt, result in virtually totally denatured DNA fragments (as reported by Oliver and Chalkley. 1974). Once the native digest DNA is purified, however, it is then totally stable to phenol or chloroform extraction (under the conditions which would have caused its denaturation when complexed with the nucleosomal histones). DNA samples to be denatured prior to electrophoresis were

Cutting 627

Sites

of Nucleases

in the Nucleosome

Core

isolated as described above, but omitting the Proteinase K treatment. To retain small fragments, DNA was concentrated by dialysis against polyethylene glycol 6000 (Baker) as described (Sollner-Webb and Felsenfeld, 1977). Precipitation by 3 vol of EtOH at -70°C was used alternatively to increase small fragment recovery. Electrophoresis of DNA fragments on 10% polyacrylamide slab gels was performed as described previously (Sollner-Webb and Felsenfeld, 1977). Allowing polymerization to proceed for 12 hr before use is essential for good resolution of single-stranded DNA fragments. Gels were stained with Stains-all (Eastman), destained in water and photographed (Axe1 et al., 1974). Negatives were scanned in a Joyce-Loebl microdensitometer. For the polymerase and the nucleosome core redigestion experiments, where very high resolution is necessary, 30 cm long (1 mm thick) slab gels were used. DNA was visualized by staining in ethidium bromide (0.5 pg/ml in H,O for 30 min) and photographed under an ultraviolet light with a Wratten 23A filter. DNA bands were marked, and gels were then “fixed” with 20% TCA (30 min) and autoradiographed. Electrophoresis of restriction enzyme-cut DNA fragments was carried out in 1.6% agarose disc gels, as described above for the acrylamide system, but without aging of the gels. DNA was visualized with ethidium bromide. Acknowledgments We thank Dr. S. Wickner for her gift of E. coli DNA polymerase II. We are also grateful to Drs. M. Zasloff and P. Williamson for Sl nuclease, and to Dr. Ft. Reeder for encouragement and facilities to complete these studies. Finally, we especially thank Drs. J. McGhee, P. Williamson, D. Camerini-Otero. L. Korn and T. Maden for many helpful discussions and suggestions. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

January

9. 1978;

revised

April

24, 1978

Jackson, V. and Chalkley, R. (1975). The effect of urea on staphylococcal nuclease digestion of chromatin. Biochem. Biophys. Res. Commun. 67, 1391-1400. Kacian, D. L. and Spiegelman, S. (1974). Use of micrococcal nuclease to monitor hybridization reactions with DNA. Anal. Biochem. 58, 534-540. Lohr, D.. occurrence 836.

Tatchell, K. and Van Holde, K. E. (1977). On the of nucleosome phasing in chromatin. Cell 72, 829-

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