Proc. Natl. Acad. Sci. USA Vol. 76, No. 5, pp. 2133-2137, May 1979 Biochemistry
Reaction of nucleosome DNA with dimethyl sulfate (chromatin/methylation)
JAMES D. MCGHEE AND GARY FELSENFELD Laboratory of Molecular Biology, National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014
Contributed by Gary Felsenfeld, January 29, 1979
ABSTRACT We have measured the effect of the histones in the nucleosome core particle on methylation of purines in nucleosome DNA by dimethyl sulfate. By using 32P terminally labeled nucleosome cores, we have examine the pattern of strand cleavage at methylated sites in the nucleosome DNA and compared it to the pattern observed in histone-free DNA. We are unable to detect any significant difference between the reactivity of N7 of guanines in nucleosome DNA and of that in naked DNA, with the exception of a single site of enhanced reactivity at approximately nucleotide 62 from the 5' end of the nucleosome. Contrary to our expectation, there is no detectable periodic modulation of reactivity corresponding to the twist of the DNA on the nucleosome surface. We are able to place a low upper limit on the extent to which the histones of the nucleosome can protect N7 of guanine in the large groove. With somewhat less precision, we also conclude that the N3 of adenine in the small groove is largely unprotected. These results indicate that in nucleosome DNA the bases are nearly as accessible to solvent as they are in DNA free of protein.
Considerable evidence indicates that, in the nucleosome core particle, DNA is wrapped around the outside of the histone octamer (for reviews, see refs. 1-3). More detailed questions can now be asked about the spatial relations between the histones and the DNA. It has recently been suggested (4) that as many as 3 out of every 10 nucleotides on each strand of nucleosome DNA are accessible to various nucleases. In the present paper, we use the much smaller chemical probe, dimethyl sulfate, to examine the accessibility of the grooves of nucleosome DNA. The experiment is similar in design to experiments used to determine the contact points between operator DNA and the lac and X repressor molecules (5, 6) except that in the present experiment all genomic sequences, rather than only a single specific sequence, are exposed to the probe. Our principal findings are that, for a distance up to 60 nucleotides from the 5' end of the nucleosome DNA, little if any protection of guanine N7 in the major groove can be detected; with less certainty, the same statement can be made about the lack of protection of adenine N3 in the DNA minor groove. At 60-65 nucleotides from the 5' end of the nucleosome DNA, there is a region of enhanced reactivity in both the major and the minor groove. By using nucleosomes labeled at their 3' ends, we find that the remaining half of the nucleosome DNA (i.e., from about 70 to 140 nucleotides from the 5' end) also appears to be unhindered in its reaction with dimethyl sulfate. These results suggest that DNA on the nucleosome surface is remarkably accessible to solvent.
MATERIALS AND METHODS Rooster erythrocyte nuclei were digested to 12-15% acid solubility with micrococcal nuclease (Worthington) and disrupted by dialysis at low ionic strength. Nucleoprotein particles conThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 2133
taining H1, H5, or both were precipitated (7), and core particles were purified on an isokinetic sucrose gradient containing 0.4 M NaCl. Core particles were labeled at their 5' termini, using T4 polynucleotide kinase and [32P]ATP (New England Nuclear; 2000-3000 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels), by a modification of the original procedure of Simpson and Whitlock (8), and then reisolated on isokinetic sucrose gradients. The detailed conditions for the dimethyl sulfate reaction will be given in the text. Dimethyl sulfate-treated core particles were reisolated on isokinetic sucrose gradients and deproteinized by proteinase K digestion, repeated phenol extractions, and ethanol precipitations. The overall recovery of radioactivity was 80-100%. Following the procedure of Maxam and Gilbert (9), we cleaved the DNA backbone exclusively at the 7-methylguanine adducts by heating in 1 M piperidine at 90'C for 30 min and then repeatedly lyophilizing from water. To measure protection in the minor groove, treated DNA was depurinated at 0C in 0.1 M HCI, and the backbone was then cleaved with alkali
(9).
The DNA cleavage products were separated at 50'C on 40-cm-long 6% or 10% polyacrylamide gels, containing 7 M urea, 50 mM Tris borate, and 1 mM EDTA at pH 8.3 (10). Gels were autoradiographed at -20'C with Kodak SB5 film. To analyze these gels quantitatively, we photoreduced the autoradiographs about 1:3, made them into positive transparencies (Air-Survey, Inc., Bethesda, MD), and scanned them at 100-,gm resolution with an Optronics high-speed digital microdensitometer. To estimate the degree of histone methylation, we treated unlabeled nucleosomes with [3H]dimethyl sulfate and then adsorbed them to hydroxylapatite. Excess dimethyl sulfate was removed by washing with 10 mM phosphate (pH 6.9), treated histones were released with 2 M NaCI/5 M urea in 10 mM phosphate (11), and finally DNA was released by 2 M NaCI/5 M urea/0.5 M phosphate, pH 6.9. Histones were examined by fluorography after sodium dodecyl sulfate gel electrophoresis.
RESULTS The reaction of dimethyl sulfate with guanine in the major groove of nucleosome DNA The basic experiment in this study involved the reaction of 5' terminally labeled nucleosome core particles with dimethyl sulfate. This results in the methylation of guanines at the N7 position in the major groove and adenines at the N3 position in the minor groove (see refs. 12 and 13 for more details of the reaction). The dimethyl sulfate-treated core particles were reisolated as 11S particles on a sucrose gradient, proteins were removed, and the backbone was cleaved exclusively at the position of the original 7-methylguanine derivatives. When the single-stranded cleavage products were electrophoresed on a denaturing polyacrylamide gel and the gel was autoradiographed, the length of each observed fragment measured the
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distance between the 5' end of the nucleosome DNA and the original site of methylation in the nucleosome core particle. It is our expectation that if the N7 site of guanine were involved in a bond with histones, it would not be available for reaction, the DNA backbone would then not be cleaved at this point, and hence a blank area would appear on the gel at a position corresponding to the distance of the protected area from the 5' end of the DNA. Fig. 1 shows the gel autoradiograph from such an experiment. Lanes a, b, and c represent core particles treated with 50 niM dimethyl sulfate for 0, 10, or 30 min at 200C, respectively. Lianes d, e, and f represent the identical experiment but with protein-free DNA-i.e., DNA isolated from labeled nucleosWmes prior to the dimethyl sulfate reaction. In both cases, inc&easing reaction times led to increasing degradation of the DNA. (We estimate 1 or 2 and 3 or 4 methyl groups per DNA stand in lanes b and c, respectively.) However, the most striking observation is that little difference can be detected between the iraction pattern of DNA in nucleosomes and that of DNA free in solution. The gel resolved individual DNA fragment sizes up td about 50 nucleotides long; in the treated nucleosome, every oke of these bands appeared on the gel. The upper region of the gel (where sequence-dependent migration lowers resolution) cfih be expanded by using gels of lower aprylamide concentthtion or a longer running time, but in neither case was a diffetence between the nucleosome and free DNA reaction pattetns detected. There are, however, two minor features of the gels in Fig. 1 that reproducibly differentiate the dimethyl sulfate reaction with the nucleosome from that with free DNA: (i) judging from the relative concentration of intact unreacted DNA remaining, the guanines in the nucleosome reacted about 20-30% faster than in the isolated DNA; (ii) a reasonably intense band about 62 nucleotides from the 5' end appeared in the reaction with nucleosomes but not in the reaction with free DNA. a
b
c.
d
C.
When the above experiment was repeated with nucleosomes labeled with the enzyme terminal deoxynucleotidyl transferase at the 3' end of the DNA, gel patterns were obtained that were essentially identical to those of Fig. 1, except that the region of enhanced reactivity occurred at 85-90 nucleotides from the labeled end, as expected (data not shown). To analyze the reaction patterns in a more detailed and quantitative fashion, film intensities of the gel autoradiograms were digitized, and corrections were made for film background, for different amounts of total radioactivity loaded, and for small differences in migration distances among the individual gel lanes. Examples of such corrected traces are shown in Fig. 2a (corresponding to lanes c and f of Fig. 1). Overall, the two traces were extremely similar. The lower concentration of intact unreacted DNA as well as the region of heightened reactivity around 62 nucleotides was apparent in the nucleosome trace. In the gel region below 60 nucleotides, there was some indication of a slight modulation of the film intensity; however, this same modulation also appeared in the naked DNA control and can undoubtedly be traced to the presence of a low level of micrococcal nuclease nicks in the starting material (see Fig. 1, lanes a and d). Although the gels lost resolution above 60 nucleotides, the nucleosome trace definitely had less intensity in this size region than did the DNA control. Since, as mentioned above, 3'-endlabeled nucleosomes also exhibited little groove protection, this intensity difference was probably due to depletion of the larger
g
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b
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FIG. 1. Nucleosome
core
particles (5' end labeled) were treated at 20'C with 50 mM dimethyl sulfate for 0 min (lane a),
.cne
10 min (lane b), and 30 min (lane c). The control (protein-free DNA) was run in parallel for 0 min (lane d), 10 min (lane e), and 30 min (lane f0. After the reaction was quenched with excess 2mercaptoethanol, nucleosomes were reisolated on isokinetic sucrose
AM
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IP
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and cleaved at the position of 7-methylguanine residues. The cleavage products were then electrophoresed on 10% polyacrylamide/7 M urea denaturing gels. Lane g is a set of sequenced restriction fragments used as size standards.
0
cr
0.5
_0
lU
50 N ucleotides
FIG. 2. (a) Densitometer scans of lanes c and f of Fig. 1. -, Nucleosome; ---, protein-free DNA. Film backgrounds were subtracted and each tracing was normalized to the total intensity in the gel lane to correct for slightly different amounts of radioactivity applied. The traces were aligned at the peak of intact DNA and then one trace was numerically contracted so that a particular low molecular weight band (usually 10-12 nucleotides long) matched in the two traces (this corrects for slight differences in migration in different wells, a correction of the order of 0.1-0.2 %). (b) Ratio of corrected film intensity in nucleosome reaction to that in DNA reaction, plotted against fragment size in nucleotides.
Biochemistry: McGhee and Felsenfeld DNA fragments as a result of multiple reactions (see curve b of Fig. 3). To compare densitometer traces more directly and to display them in a manner better designed to reveal reaction periodicity, we calculated the ratio of the film intensity for the nucleosome to that of the naked DNA at each point in the scans of Fig. 2a and then plotted this ratio vs. the length of the corresponding DNA fragment (using a set of sequenced restriction fragments and equation 4 of ref. 14 to transform gel migration distances into DNA fragment length). Such a plot is shown in Fig. 2b. In the region from 0 to 60 nucleotides from the 5' end, where the gel resolved individual bands, this intensity ratio was slightly greater than one. More importantly, there is no evidence for any significant periodicity in the reaction. The region of heightened reactivity around 62 nucleotides is apparent and supports our contention (see Fig. 3) that any other region of significant protection or enhancement would have been detected. In a later section we will calculate the expected reaction patterns for various hypothetical degrees of groove protection, and compare them to the results shown in Fig. 2b. Similar reaction patterns were obtained with four different batches of nucleosomes. Two sets of reaction conditions were used: (i) 0-50 mM dimethyl sulfate at 20'C for 0-60 min in 100 mM sodium cacodylate/I mM EDTA at pH 8.0; or (ii) 0-10 mM dimethyl sulfate for 20 hr at 00C in 25 mM sodium cacodylate/0.25 mM EDTA at pH 8.0. (Under both sets of conditions, the final pH never dropped below 6.7.) The same apparent lack of large groove protection was observed under both sets of buffer conditions, with or without the addition of 5 mM MgCl2, whether or not the reaction was quenched with excess sulfhydryl reagent, whether or not the nucleosomes were reisolated on sucrose gradients after reaction, and at extents of reaction varying from 10 methylguanines formed per nucleosome. We also did not detect regions of preferential protection when unlabeled nucleosomes were compared with unlabeled DNA, using either ethidium bromide fluorescence or "Stains-All" to detect the DNA cleavage products. In preliminary experiments, no significant degree of protection was detected by using the larger probe, diethyl sulfate. The above results suggest an almost complete lack of protection of the major groove of the nucleosome DNA, but there are two obvious ways in which this interpretation could be in error. The first possibility is that during the reaction the nucleosome structure was disrupted in such a way that the DNA became exposed to solvent. The second possibility is that, for reasons such as length heterogeneity of the original nucleosome DNA, the gel patterns are deceptive and are an insensitive measure of protection. In the following two sections, we eliminate these two possible sources of error.
Control experiments By all available criteria, the nucleosome core particles remain intact and the DNA conformation remains unchanged both during and after the reaction with dimethyl sulfate. In all experiments, the core particles after reaction still sedimented as 11S particles on sucrose gradients, even when there were as many as 20 DNA methyl groups per nucleosome. Moreover, these reacted core particles still gave a narrow discrete band when subjected to electrophoresis (data not shown). Even in the presence of 50 mM dimethyl sulfate, the circular dichroic spectrum of reacted nucleosomes still retained its characteristic low ellipticity relative to free DNA at wavelengths greater than 260 nm. This rules out the possibility that the nucleosome was unfolded during the reaction but refolded during the subsequent sucrose gradient sedimentation. When end-labeled
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nucleosomes were digested with pancreatic DNase under the same solvent conditions used for the dimethyl sulfate reaction and even in the presence of dimethyl sulfate, the usual distinctive DNase I cleavage pattern (8) was obtained at all extents of digestion. This rules out the possibility that the nucleosome DNA is able to slide back and forth on the nucleosome surface during the reaction, thus exposing each base to solvent a certain fraction of the time and thereby giving the appearance of a uniform lack of protection. We can also rule out the possibility that dimethyl sulfate reacts only with a small fraction of the DNA that is rapidly exchanging between the nucleosome surface and free solution because, when a trace amount of labeled DNA was added to the reaction of unlabeled nucleosomes with dimethyl sulfate, no significant incorporation of label into an IIS particle was observed. Under the most extreme reaction conditions used, the core particle histones remain intact as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The histones do however react with dimethyl sulfate and the degree of methylation can be estimated. Under several reaction conditions, for each methyl group bound to DNA, 0.7-1 methyl group was bound per histone octamer, distributed about equally among the four histone types. It seems unlikely that such a low level of protein modification would grossly perturb the DNA behaviour. Since the apparent lack of groove protection was observed even at very low levels of reaction (less than 0.1 methyl group per DNA) most of the observed DNA reaction must have come from nucleosomes carrying unreacted histones, provided that the protein and DNA reactions occurred independently. The base specificity both of the initial dimethyl sulfate reaction and of the piperidine cleavage reaction remained unchanged in the presence of histones, because, when the entire reaction sequence was conducted with a sequenced, end-labeled, 140-base pair-long restriction fragment, either added to the reaction with unlabeled nucleosomes or reconstituted into an 1IS particle (unpublished results), cleavage was observed only at the known positions of guanine residues. Finally, it seems unlikely that there is any significant intermolecular transfer of methyl groups during the deproteinization steps when the DNA becomes exposed to solvent, because we could observe no significant degradation of a labeled restriction fragment that was added to the pooled sucrose gradient fractions of unlabeled treated nucleosomes and then processed as usual. Simulation of the gel patterns It is important to analyze further the gel patterns shown in Figs. 1 and 2 in order to estimate the degree of groove protection that, if it existed, could have been detected. Probably the most obvious limitation in the overall approach (at least for regions in the nucleosome DNA where individual gel bands can be resolved) arises from heterogeneity in the length of the initial nucleosome DNA, because this would have the effect of smearing any protection pattern. To examine the effect of heterogeneity and of varying extents of protection, we simulated plots such as Fig. 2b by using a simple model for the reaction in which a specified number of contiguous bases out of every 10 bases were assumed to be blocked to a specified extent. This is described in more detail in the legend to Fig. 3. The length heterogeneity of the starting DNA then is superimposed on these calculations. The actual distribution of DNA lengths was obtained experimentally from a DNase I digest of the same batch of nucleosomes by analyzing the intensity of the individually resolved bands in the gel region around 20 nucleotides. If the DNase I cutting site is unique, this distribution should be an accurate measure of the heterogeneity at the 5' end of the
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2136
starting DNA (i.e., the heterogeneity needed for the calculation). If the DNase I cutting site is itself nonunique, then the length distribution estimated in this fashion is an overestimate of the true breadth of the distribution. Fig. 3 shows the calculated ratio of film intensities of nucleosome to DNA as a function of DNA size. The different sections of Fig. 3 represent models in which (i) a block of 5 bases out of every 10 is 30% protected; (ii) 1 base out of every 10 is completely protected; and (iii) 1 base out of every 10 is 50% protected. The actual degrees of methylation are chosen to correspond to those of Fig. 2. In curve b of Fig. 3, the simulated pattern is superimposed on the experimental data taken from Fig. 2b and shows that a large part of the overall decline in intensity towards the 3' end can be ascribed to multiple methylation events on each DNA strand. From a number of these simulations in which reaction extent and degree of protection were varied, we believe that the protection of as few as 1 nucleotide in 10 could have been detected, at least in the gel region below 60 nucleotides. If several bases in every 10 were appreciably protected, we feel certain that it would have been detected.
1.5
b
.0
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-
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FIG. 3. Simulation of gel patterns, such as those in Fig. 2b, using the following simple model for the reaction. (i) There is a probability of 0.215 that any position in the DNA is occupied by a guanine residue (i.e., chicken DNA is 43% GC). (ii) Guanines react independently of each other. (iii) The probability of each guanine reacting can be calculated from the amount of intact, unreacted DNA remaining under each set of reaction conditions. Thus, for free DNA, the concentration of a particular length of DNA fragment, i nucleotides long, appearing on the gel, can be calculated as 0.215a (1 - 0.215a)( -1), where a is the fraction of guanine residues that reacted. The nucleosome reactivity is computed in a similar fashion but allowing for a region of n bases out of every 10 bases to be blocked by a specified factor. For the calculations shown, a = 0.12 for the nucleosome trace and 0.10 for the free DNA control, corresponding to the conditions of lanes c and f of Fig. 1. As described in the text, the length heterogeneity of the starting DNA is superimposed on these calculations. The figure shows the calculated ratio of film intensities for the nucleosome to that of DNA plotted as a function of DNA size for the cases in which (curve a) a block of 5 bases out of every 10 was 30% protected; (curve b) 1 base out of every 10 was completely protected; and (curve c) 1 base out of every 10 was 50% protected. On graph b is superimposed the experimental data from Fig. 2b.
,
2)
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0.
.Y..-. %.
,C 1.0 E
..
._
0
.2
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FIG. 4. Nucleosomes (5' terminally labeled) and protein-free DNA were treated with 10 mM dimethyl sulfate at 0C for 20 hr. After reisolation on a sucrose gradient and deproteinization, the reacted DNAs were treated under "adenine-cleavage conditions" (9). Gel autoradiograms were scanned and processed as described in the legend to Fig. 2. The ratio of film intensity at each point in the nucleosome trace to that of the free DNA control is plotted vs. the corresponding DNA fragment length.
Reaction of N3 of adenine in the minor groove The same kind of experiment can be used to examine protection in the DNA minor groove, utilizing the reaction of dimethyl sulfate with the N3 of adenine. Such an experiment is necessarily less definitive than that with guanine, because conditions have not yet been found that cause the DNA backbone to be cleaved exclusively at 3-methyladenine. When the cold acid depurination procedure of Maxam and Gilbert (9) was used, about half of the intensity at any position in the gel came from the cleavage at 3-methyladenine and the remaining half, from cleavage at guanine. However, there is the possibility that a striking pattern of adenine protection, if it existed, might be discerned above the background of guanine cleavage, which was shown above to be quite featureless. Nucleosomes were treated with dimethyl sulfate, reisolated on sucrose gradients, deproteinized, and treated under conditions most favorable for cleavage at 3-methyladenine. The cleavage products were subjected to electrophoresis, and autoradiograms were processed as described above. Fig. 4 shows the point-to-point ratio of the film intensity in the densitometer scan of the nucleosome autoradiogram to that of the free DNA control, plotted vs. DNA fragment size. Throughout the best resolving region of the gel, this ratio is close to unity, just as in the analysis of the major groove reaction discussed above. There is, however an indication of a region of enhanced reactivity at about 45 nucleotides from the 5' end as well as a region of strongly enhanced reactivity at about 65 nucleotides. DISCUSSION The reaction of dimethyl sulfate with chromatin has been investigated by Mirzabekov et al. (13) by comparing the rate of reaction of chromatin with that of free DNA. They concluded that the major groove was 10-20% blocked, but they had no means of determining whether this small kinetic difference was due to reaction within the core particle or within the linker region. Furthermore, it is difficult to interpret overall kinetic effects when there are local regions of enhanced reactivity as described above. The method used in the present paper allowed us to examine directly the protection of individual groups in the core particle DNA; our conclusion is that in most of the nucleosome DNA no protection can be detected, certainly none with a pattern repeating at 10-base intervals. Our results, combined with re-
Biochemistry: McGhee and Felsenfeld cent nuclease digestion studies (4), suggest that the DNA in the core particle is remarkably accessible to solvent in both the major and the minor groove. Goodwin and Brahms (15) have also concluded, from Raman spectroscopy, that the N7 of guanine is unbonded in the core particle. At some point in each 10-base pair interval, each DNA groove must face inward towards the central histone core. Our results thus imply that, at these points, there must be some groove or cleft in the histone core that still allows free solvent access to the DNA. Such a cleft need not be very large as illustrated by the example of lac or X repressor's protection of their respective operators from the dimethyl sulfate reaction (5, 6). In these cases, it is clear that protected and unprotected guanines can be adjacent to one another, and thus protection against dimethyl sulfate can be a very local phenomenon. The lac or X repressor systems also give a precedent for the enhanced reactivity of certain bases. It is a present limitation in the use of dimethyl sulfate as a probe that there is no detailed chemical knowledge of the causes of reaction inhibition or enhancement. The obvious explanations of the enhanced reactivities of certain nucleosome regions are that, at about 62 nucleotides from the 5' end in the major groove and at about 65 nucleotides from the 5' end in the minor groove, there is some altered DNA conformation or some protein constellation that enhances reaction. It is of interest that the DNase I cutting site falls exactly between these two reaction sites (data not shown) and that this is a low-frequency cutting site (8). Mirzabekov et al. (16) have crosslinked histones to DNA by first treating nucleosome core particles with dimethyl sulfate, depurinating in situ, and then reducing any Schiff base crosslinks formed. The ability to crosslink with dimethyl sulfate is not necessarily in conflict with the present results, because crosslinking (and hence backbone cleavage) may occur at only a small fraction of all methylpurine residues or may trap conformations too infrequent to be recorded as reaction protection. Alternatively, the sugar aldehyde created by depurination may be close to histone reactive groups or be capable of migrating some (not necessarily large) distance prior to its reaction. We mention two further corroborations of the suggestion that nucleosome DNA is quite accessible to solvent (unpublished results). (i) We have repeated the dimethyl sulfate reaction with nucleosomes reconstituted onto a unique restriction fragment 140 base pairs long and have seen no significant degree of protection. This experiment avoids all the problems of DNA sequence and length heterogeneity but depends upon establishing that reconstitution is correct. (ii) We have been able to reconstitute histones onto T4 DNA and obtain complexes showing properties of nucleosomes. This DNA has every cytosine replaced by glucosylated hydroxymethylcytosine, with
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the glucose residue protruding into the large groove. Our results point to a model for the nucleosome core in which both grooves of DNA are almost completely accessible to solvent, and suggest that contacts with the histone core must be predominantly localized to the DNA phosphodiester backbone. The volume occupied by the histone core appears to be considerably less than the total volume available in the region circumscribed by the coiled DNA (see for example the calculations in ref. 17). This suggests that DNA might be held suspended on the surface of the nucleosome in a manner designed to maximize exposure to the surroundings. The accessibility revealed by these experiments suggests that the histones of the nucleosome may be so designed that they can render the DNA more compact while interfering as little as possible with its biological and chemical activity. We thank Dr. Manuel Navia who very kindly provided the digitized densitometer scans of the gel autoradiographs and Drs. D. CameriniOtero, M. Gellert, and M. Zasloff for reading the manuscript. 1. Kornberg, R. D. (1977) Annu. Rev. Biochem. 46,931-954. 2. Felsenfeld, G. (1978) Nature (London) 271, 115-122. 3. Cold Spring Harbor Laboratory (1978) Chromatin, Cold Spring Harbor Symposia on Quantitative Biology (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), Vol. 42. 4. Sollner-Webb, B., Melchior, W., Jr. & Felsenfeld, G. (1978) Cell 14,611-627. 5. Gilbert, W., Maxam, A. & Mirzabekov, A. D. (1976) in Control of Ribosome Synthesis, The Alfred Benzon Symposium IX, eds. Kjeldgaard, N. C. & Maalle, 0. (Munksgaard, Copenhagen), pp. 139-148. 6. Humayun, Z., Kleid, D. & Ptashne, M. (1977) Nucleic Acids Res. 4, 1595-1607. 7. Olins, A. L., Carlson, R. D., Wright, E. B. & Olins, D. E. (1976) Nuicleic Acids Res. 3, 3271-3291. 8. Simpson, R. & Whitlock, J. P., Jr. (1976) Cell 9, 347-353. 9. Maxam, A. & Gilbert, W. (1977) Proc. Natl. Acad. Sci. USA 74, 560-564. 10. Maniatis, T., Jeffrey, A. & van deSande, H. (1976) Biochemistry 14,3787-3794. 11. Bloom, K. S. & Anderson, J. N. (1978) J. Biol. Chem. 253, 4446-4450. 12. Singer, B. (1975) Prog. Nucleic Res. Mol. Biol. 15, 219-284. 13. Mirzabekov, A. D., San'ko, D. F., Kolchinsky, A. M. & Melnikova, A. F. (1977) Eur. J. Biochem. 75, 379-389. 14. Kovacic, R. T. & van Holde, K. E. (1977) Biochemistry 16,
1490-1498. 15. Goodwin, D. C. & Brahms, J. (1978) Nucleic Acids Res. 5, 835-850. 16. Mirzabekov, A. D., Shick, V. V., Belyavsky, A. V. & Bavykin, S. G. (1978) Proc. Natl. Acad. Sci. USA 75,4184-4188. 17. Voordouw, G. & Eisenberg, H. (1978) Nature (London) 273, 446-448.
Reaction of nucleosome DNA with dimethyl sulfate (chromatin/methylation)
JAMES D. MCGHEE AND GARY FELSENFELD Laboratory of Molecular Biology, National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014
Contributed by Gary Felsenfeld, January 29, 1979
ABSTRACT We have measured the effect of the histones in the nucleosome core particle on methylation of purines in nucleosome DNA by dimethyl sulfate. By using 32P terminally labeled nucleosome cores, we have examine the pattern of strand cleavage at methylated sites in the nucleosome DNA and compared it to the pattern observed in histone-free DNA. We are unable to detect any significant difference between the reactivity of N7 of guanines in nucleosome DNA and of that in naked DNA, with the exception of a single site of enhanced reactivity at approximately nucleotide 62 from the 5' end of the nucleosome. Contrary to our expectation, there is no detectable periodic modulation of reactivity corresponding to the twist of the DNA on the nucleosome surface. We are able to place a low upper limit on the extent to which the histones of the nucleosome can protect N7 of guanine in the large groove. With somewhat less precision, we also conclude that the N3 of adenine in the small groove is largely unprotected. These results indicate that in nucleosome DNA the bases are nearly as accessible to solvent as they are in DNA free of protein.
Considerable evidence indicates that, in the nucleosome core particle, DNA is wrapped around the outside of the histone octamer (for reviews, see refs. 1-3). More detailed questions can now be asked about the spatial relations between the histones and the DNA. It has recently been suggested (4) that as many as 3 out of every 10 nucleotides on each strand of nucleosome DNA are accessible to various nucleases. In the present paper, we use the much smaller chemical probe, dimethyl sulfate, to examine the accessibility of the grooves of nucleosome DNA. The experiment is similar in design to experiments used to determine the contact points between operator DNA and the lac and X repressor molecules (5, 6) except that in the present experiment all genomic sequences, rather than only a single specific sequence, are exposed to the probe. Our principal findings are that, for a distance up to 60 nucleotides from the 5' end of the nucleosome DNA, little if any protection of guanine N7 in the major groove can be detected; with less certainty, the same statement can be made about the lack of protection of adenine N3 in the DNA minor groove. At 60-65 nucleotides from the 5' end of the nucleosome DNA, there is a region of enhanced reactivity in both the major and the minor groove. By using nucleosomes labeled at their 3' ends, we find that the remaining half of the nucleosome DNA (i.e., from about 70 to 140 nucleotides from the 5' end) also appears to be unhindered in its reaction with dimethyl sulfate. These results suggest that DNA on the nucleosome surface is remarkably accessible to solvent.
MATERIALS AND METHODS Rooster erythrocyte nuclei were digested to 12-15% acid solubility with micrococcal nuclease (Worthington) and disrupted by dialysis at low ionic strength. Nucleoprotein particles conThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 2133
taining H1, H5, or both were precipitated (7), and core particles were purified on an isokinetic sucrose gradient containing 0.4 M NaCl. Core particles were labeled at their 5' termini, using T4 polynucleotide kinase and [32P]ATP (New England Nuclear; 2000-3000 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels), by a modification of the original procedure of Simpson and Whitlock (8), and then reisolated on isokinetic sucrose gradients. The detailed conditions for the dimethyl sulfate reaction will be given in the text. Dimethyl sulfate-treated core particles were reisolated on isokinetic sucrose gradients and deproteinized by proteinase K digestion, repeated phenol extractions, and ethanol precipitations. The overall recovery of radioactivity was 80-100%. Following the procedure of Maxam and Gilbert (9), we cleaved the DNA backbone exclusively at the 7-methylguanine adducts by heating in 1 M piperidine at 90'C for 30 min and then repeatedly lyophilizing from water. To measure protection in the minor groove, treated DNA was depurinated at 0C in 0.1 M HCI, and the backbone was then cleaved with alkali
(9).
The DNA cleavage products were separated at 50'C on 40-cm-long 6% or 10% polyacrylamide gels, containing 7 M urea, 50 mM Tris borate, and 1 mM EDTA at pH 8.3 (10). Gels were autoradiographed at -20'C with Kodak SB5 film. To analyze these gels quantitatively, we photoreduced the autoradiographs about 1:3, made them into positive transparencies (Air-Survey, Inc., Bethesda, MD), and scanned them at 100-,gm resolution with an Optronics high-speed digital microdensitometer. To estimate the degree of histone methylation, we treated unlabeled nucleosomes with [3H]dimethyl sulfate and then adsorbed them to hydroxylapatite. Excess dimethyl sulfate was removed by washing with 10 mM phosphate (pH 6.9), treated histones were released with 2 M NaCI/5 M urea in 10 mM phosphate (11), and finally DNA was released by 2 M NaCI/5 M urea/0.5 M phosphate, pH 6.9. Histones were examined by fluorography after sodium dodecyl sulfate gel electrophoresis.
RESULTS The reaction of dimethyl sulfate with guanine in the major groove of nucleosome DNA The basic experiment in this study involved the reaction of 5' terminally labeled nucleosome core particles with dimethyl sulfate. This results in the methylation of guanines at the N7 position in the major groove and adenines at the N3 position in the minor groove (see refs. 12 and 13 for more details of the reaction). The dimethyl sulfate-treated core particles were reisolated as 11S particles on a sucrose gradient, proteins were removed, and the backbone was cleaved exclusively at the position of the original 7-methylguanine derivatives. When the single-stranded cleavage products were electrophoresed on a denaturing polyacrylamide gel and the gel was autoradiographed, the length of each observed fragment measured the
Proc. Natl. Acad. Sci. USA 76 (1979)
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distance between the 5' end of the nucleosome DNA and the original site of methylation in the nucleosome core particle. It is our expectation that if the N7 site of guanine were involved in a bond with histones, it would not be available for reaction, the DNA backbone would then not be cleaved at this point, and hence a blank area would appear on the gel at a position corresponding to the distance of the protected area from the 5' end of the DNA. Fig. 1 shows the gel autoradiograph from such an experiment. Lanes a, b, and c represent core particles treated with 50 niM dimethyl sulfate for 0, 10, or 30 min at 200C, respectively. Lianes d, e, and f represent the identical experiment but with protein-free DNA-i.e., DNA isolated from labeled nucleosWmes prior to the dimethyl sulfate reaction. In both cases, inc&easing reaction times led to increasing degradation of the DNA. (We estimate 1 or 2 and 3 or 4 methyl groups per DNA stand in lanes b and c, respectively.) However, the most striking observation is that little difference can be detected between the iraction pattern of DNA in nucleosomes and that of DNA free in solution. The gel resolved individual DNA fragment sizes up td about 50 nucleotides long; in the treated nucleosome, every oke of these bands appeared on the gel. The upper region of the gel (where sequence-dependent migration lowers resolution) cfih be expanded by using gels of lower aprylamide concentthtion or a longer running time, but in neither case was a diffetence between the nucleosome and free DNA reaction pattetns detected. There are, however, two minor features of the gels in Fig. 1 that reproducibly differentiate the dimethyl sulfate reaction with the nucleosome from that with free DNA: (i) judging from the relative concentration of intact unreacted DNA remaining, the guanines in the nucleosome reacted about 20-30% faster than in the isolated DNA; (ii) a reasonably intense band about 62 nucleotides from the 5' end appeared in the reaction with nucleosomes but not in the reaction with free DNA. a
b
c.
d
C.
When the above experiment was repeated with nucleosomes labeled with the enzyme terminal deoxynucleotidyl transferase at the 3' end of the DNA, gel patterns were obtained that were essentially identical to those of Fig. 1, except that the region of enhanced reactivity occurred at 85-90 nucleotides from the labeled end, as expected (data not shown). To analyze the reaction patterns in a more detailed and quantitative fashion, film intensities of the gel autoradiograms were digitized, and corrections were made for film background, for different amounts of total radioactivity loaded, and for small differences in migration distances among the individual gel lanes. Examples of such corrected traces are shown in Fig. 2a (corresponding to lanes c and f of Fig. 1). Overall, the two traces were extremely similar. The lower concentration of intact unreacted DNA as well as the region of heightened reactivity around 62 nucleotides was apparent in the nucleosome trace. In the gel region below 60 nucleotides, there was some indication of a slight modulation of the film intensity; however, this same modulation also appeared in the naked DNA control and can undoubtedly be traced to the presence of a low level of micrococcal nuclease nicks in the starting material (see Fig. 1, lanes a and d). Although the gels lost resolution above 60 nucleotides, the nucleosome trace definitely had less intensity in this size region than did the DNA control. Since, as mentioned above, 3'-endlabeled nucleosomes also exhibited little groove protection, this intensity difference was probably due to depletion of the larger
g
f
1.5 -
b
In W
~ .0
4Q)0
FIG. 1. Nucleosome
core
particles (5' end labeled) were treated at 20'C with 50 mM dimethyl sulfate for 0 min (lane a),
.cne
10 min (lane b), and 30 min (lane c). The control (protein-free DNA) was run in parallel for 0 min (lane d), 10 min (lane e), and 30 min (lane f0. After the reaction was quenched with excess 2mercaptoethanol, nucleosomes were reisolated on isokinetic sucrose
AM
4 *| ."j
4l-*
41,
IP
1.
gradients, deproteinized,
and cleaved at the position of 7-methylguanine residues. The cleavage products were then electrophoresed on 10% polyacrylamide/7 M urea denaturing gels. Lane g is a set of sequenced restriction fragments used as size standards.
0
cr
0.5
_0
lU
50 N ucleotides
FIG. 2. (a) Densitometer scans of lanes c and f of Fig. 1. -, Nucleosome; ---, protein-free DNA. Film backgrounds were subtracted and each tracing was normalized to the total intensity in the gel lane to correct for slightly different amounts of radioactivity applied. The traces were aligned at the peak of intact DNA and then one trace was numerically contracted so that a particular low molecular weight band (usually 10-12 nucleotides long) matched in the two traces (this corrects for slight differences in migration in different wells, a correction of the order of 0.1-0.2 %). (b) Ratio of corrected film intensity in nucleosome reaction to that in DNA reaction, plotted against fragment size in nucleotides.
Biochemistry: McGhee and Felsenfeld DNA fragments as a result of multiple reactions (see curve b of Fig. 3). To compare densitometer traces more directly and to display them in a manner better designed to reveal reaction periodicity, we calculated the ratio of the film intensity for the nucleosome to that of the naked DNA at each point in the scans of Fig. 2a and then plotted this ratio vs. the length of the corresponding DNA fragment (using a set of sequenced restriction fragments and equation 4 of ref. 14 to transform gel migration distances into DNA fragment length). Such a plot is shown in Fig. 2b. In the region from 0 to 60 nucleotides from the 5' end, where the gel resolved individual bands, this intensity ratio was slightly greater than one. More importantly, there is no evidence for any significant periodicity in the reaction. The region of heightened reactivity around 62 nucleotides is apparent and supports our contention (see Fig. 3) that any other region of significant protection or enhancement would have been detected. In a later section we will calculate the expected reaction patterns for various hypothetical degrees of groove protection, and compare them to the results shown in Fig. 2b. Similar reaction patterns were obtained with four different batches of nucleosomes. Two sets of reaction conditions were used: (i) 0-50 mM dimethyl sulfate at 20'C for 0-60 min in 100 mM sodium cacodylate/I mM EDTA at pH 8.0; or (ii) 0-10 mM dimethyl sulfate for 20 hr at 00C in 25 mM sodium cacodylate/0.25 mM EDTA at pH 8.0. (Under both sets of conditions, the final pH never dropped below 6.7.) The same apparent lack of large groove protection was observed under both sets of buffer conditions, with or without the addition of 5 mM MgCl2, whether or not the reaction was quenched with excess sulfhydryl reagent, whether or not the nucleosomes were reisolated on sucrose gradients after reaction, and at extents of reaction varying from 10 methylguanines formed per nucleosome. We also did not detect regions of preferential protection when unlabeled nucleosomes were compared with unlabeled DNA, using either ethidium bromide fluorescence or "Stains-All" to detect the DNA cleavage products. In preliminary experiments, no significant degree of protection was detected by using the larger probe, diethyl sulfate. The above results suggest an almost complete lack of protection of the major groove of the nucleosome DNA, but there are two obvious ways in which this interpretation could be in error. The first possibility is that during the reaction the nucleosome structure was disrupted in such a way that the DNA became exposed to solvent. The second possibility is that, for reasons such as length heterogeneity of the original nucleosome DNA, the gel patterns are deceptive and are an insensitive measure of protection. In the following two sections, we eliminate these two possible sources of error.
Control experiments By all available criteria, the nucleosome core particles remain intact and the DNA conformation remains unchanged both during and after the reaction with dimethyl sulfate. In all experiments, the core particles after reaction still sedimented as 11S particles on sucrose gradients, even when there were as many as 20 DNA methyl groups per nucleosome. Moreover, these reacted core particles still gave a narrow discrete band when subjected to electrophoresis (data not shown). Even in the presence of 50 mM dimethyl sulfate, the circular dichroic spectrum of reacted nucleosomes still retained its characteristic low ellipticity relative to free DNA at wavelengths greater than 260 nm. This rules out the possibility that the nucleosome was unfolded during the reaction but refolded during the subsequent sucrose gradient sedimentation. When end-labeled
Proc. Natl. Acad. Sci. USA 76 (1979)
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nucleosomes were digested with pancreatic DNase under the same solvent conditions used for the dimethyl sulfate reaction and even in the presence of dimethyl sulfate, the usual distinctive DNase I cleavage pattern (8) was obtained at all extents of digestion. This rules out the possibility that the nucleosome DNA is able to slide back and forth on the nucleosome surface during the reaction, thus exposing each base to solvent a certain fraction of the time and thereby giving the appearance of a uniform lack of protection. We can also rule out the possibility that dimethyl sulfate reacts only with a small fraction of the DNA that is rapidly exchanging between the nucleosome surface and free solution because, when a trace amount of labeled DNA was added to the reaction of unlabeled nucleosomes with dimethyl sulfate, no significant incorporation of label into an IIS particle was observed. Under the most extreme reaction conditions used, the core particle histones remain intact as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The histones do however react with dimethyl sulfate and the degree of methylation can be estimated. Under several reaction conditions, for each methyl group bound to DNA, 0.7-1 methyl group was bound per histone octamer, distributed about equally among the four histone types. It seems unlikely that such a low level of protein modification would grossly perturb the DNA behaviour. Since the apparent lack of groove protection was observed even at very low levels of reaction (less than 0.1 methyl group per DNA) most of the observed DNA reaction must have come from nucleosomes carrying unreacted histones, provided that the protein and DNA reactions occurred independently. The base specificity both of the initial dimethyl sulfate reaction and of the piperidine cleavage reaction remained unchanged in the presence of histones, because, when the entire reaction sequence was conducted with a sequenced, end-labeled, 140-base pair-long restriction fragment, either added to the reaction with unlabeled nucleosomes or reconstituted into an 1IS particle (unpublished results), cleavage was observed only at the known positions of guanine residues. Finally, it seems unlikely that there is any significant intermolecular transfer of methyl groups during the deproteinization steps when the DNA becomes exposed to solvent, because we could observe no significant degradation of a labeled restriction fragment that was added to the pooled sucrose gradient fractions of unlabeled treated nucleosomes and then processed as usual. Simulation of the gel patterns It is important to analyze further the gel patterns shown in Figs. 1 and 2 in order to estimate the degree of groove protection that, if it existed, could have been detected. Probably the most obvious limitation in the overall approach (at least for regions in the nucleosome DNA where individual gel bands can be resolved) arises from heterogeneity in the length of the initial nucleosome DNA, because this would have the effect of smearing any protection pattern. To examine the effect of heterogeneity and of varying extents of protection, we simulated plots such as Fig. 2b by using a simple model for the reaction in which a specified number of contiguous bases out of every 10 bases were assumed to be blocked to a specified extent. This is described in more detail in the legend to Fig. 3. The length heterogeneity of the starting DNA then is superimposed on these calculations. The actual distribution of DNA lengths was obtained experimentally from a DNase I digest of the same batch of nucleosomes by analyzing the intensity of the individually resolved bands in the gel region around 20 nucleotides. If the DNase I cutting site is unique, this distribution should be an accurate measure of the heterogeneity at the 5' end of the
Proc. Nati. Acad. Sci. USA 76 (1979)
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2136
starting DNA (i.e., the heterogeneity needed for the calculation). If the DNase I cutting site is itself nonunique, then the length distribution estimated in this fashion is an overestimate of the true breadth of the distribution. Fig. 3 shows the calculated ratio of film intensities of nucleosome to DNA as a function of DNA size. The different sections of Fig. 3 represent models in which (i) a block of 5 bases out of every 10 is 30% protected; (ii) 1 base out of every 10 is completely protected; and (iii) 1 base out of every 10 is 50% protected. The actual degrees of methylation are chosen to correspond to those of Fig. 2. In curve b of Fig. 3, the simulated pattern is superimposed on the experimental data taken from Fig. 2b and shows that a large part of the overall decline in intensity towards the 3' end can be ascribed to multiple methylation events on each DNA strand. From a number of these simulations in which reaction extent and degree of protection were varied, we believe that the protection of as few as 1 nucleotide in 10 could have been detected, at least in the gel region below 60 nucleotides. If several bases in every 10 were appreciably protected, we feel certain that it would have been detected.
1.5
b
.0
.o .0-
-
Ei 0
a:io
1.5F 1.0
F
0.5F 50
100 N ucleotides
FIG. 3. Simulation of gel patterns, such as those in Fig. 2b, using the following simple model for the reaction. (i) There is a probability of 0.215 that any position in the DNA is occupied by a guanine residue (i.e., chicken DNA is 43% GC). (ii) Guanines react independently of each other. (iii) The probability of each guanine reacting can be calculated from the amount of intact, unreacted DNA remaining under each set of reaction conditions. Thus, for free DNA, the concentration of a particular length of DNA fragment, i nucleotides long, appearing on the gel, can be calculated as 0.215a (1 - 0.215a)( -1), where a is the fraction of guanine residues that reacted. The nucleosome reactivity is computed in a similar fashion but allowing for a region of n bases out of every 10 bases to be blocked by a specified factor. For the calculations shown, a = 0.12 for the nucleosome trace and 0.10 for the free DNA control, corresponding to the conditions of lanes c and f of Fig. 1. As described in the text, the length heterogeneity of the starting DNA is superimposed on these calculations. The figure shows the calculated ratio of film intensities for the nucleosome to that of DNA plotted as a function of DNA size for the cases in which (curve a) a block of 5 bases out of every 10 was 30% protected; (curve b) 1 base out of every 10 was completely protected; and (curve c) 1 base out of every 10 was 50% protected. On graph b is superimposed the experimental data from Fig. 2b.
,
2)
1.5
0.
.Y..-. %.
,C 1.0 E
..
._
0
.2
0.5
4r
100
50 Nucleotides
FIG. 4. Nucleosomes (5' terminally labeled) and protein-free DNA were treated with 10 mM dimethyl sulfate at 0C for 20 hr. After reisolation on a sucrose gradient and deproteinization, the reacted DNAs were treated under "adenine-cleavage conditions" (9). Gel autoradiograms were scanned and processed as described in the legend to Fig. 2. The ratio of film intensity at each point in the nucleosome trace to that of the free DNA control is plotted vs. the corresponding DNA fragment length.
Reaction of N3 of adenine in the minor groove The same kind of experiment can be used to examine protection in the DNA minor groove, utilizing the reaction of dimethyl sulfate with the N3 of adenine. Such an experiment is necessarily less definitive than that with guanine, because conditions have not yet been found that cause the DNA backbone to be cleaved exclusively at 3-methyladenine. When the cold acid depurination procedure of Maxam and Gilbert (9) was used, about half of the intensity at any position in the gel came from the cleavage at 3-methyladenine and the remaining half, from cleavage at guanine. However, there is the possibility that a striking pattern of adenine protection, if it existed, might be discerned above the background of guanine cleavage, which was shown above to be quite featureless. Nucleosomes were treated with dimethyl sulfate, reisolated on sucrose gradients, deproteinized, and treated under conditions most favorable for cleavage at 3-methyladenine. The cleavage products were subjected to electrophoresis, and autoradiograms were processed as described above. Fig. 4 shows the point-to-point ratio of the film intensity in the densitometer scan of the nucleosome autoradiogram to that of the free DNA control, plotted vs. DNA fragment size. Throughout the best resolving region of the gel, this ratio is close to unity, just as in the analysis of the major groove reaction discussed above. There is, however an indication of a region of enhanced reactivity at about 45 nucleotides from the 5' end as well as a region of strongly enhanced reactivity at about 65 nucleotides. DISCUSSION The reaction of dimethyl sulfate with chromatin has been investigated by Mirzabekov et al. (13) by comparing the rate of reaction of chromatin with that of free DNA. They concluded that the major groove was 10-20% blocked, but they had no means of determining whether this small kinetic difference was due to reaction within the core particle or within the linker region. Furthermore, it is difficult to interpret overall kinetic effects when there are local regions of enhanced reactivity as described above. The method used in the present paper allowed us to examine directly the protection of individual groups in the core particle DNA; our conclusion is that in most of the nucleosome DNA no protection can be detected, certainly none with a pattern repeating at 10-base intervals. Our results, combined with re-
Biochemistry: McGhee and Felsenfeld cent nuclease digestion studies (4), suggest that the DNA in the core particle is remarkably accessible to solvent in both the major and the minor groove. Goodwin and Brahms (15) have also concluded, from Raman spectroscopy, that the N7 of guanine is unbonded in the core particle. At some point in each 10-base pair interval, each DNA groove must face inward towards the central histone core. Our results thus imply that, at these points, there must be some groove or cleft in the histone core that still allows free solvent access to the DNA. Such a cleft need not be very large as illustrated by the example of lac or X repressor's protection of their respective operators from the dimethyl sulfate reaction (5, 6). In these cases, it is clear that protected and unprotected guanines can be adjacent to one another, and thus protection against dimethyl sulfate can be a very local phenomenon. The lac or X repressor systems also give a precedent for the enhanced reactivity of certain bases. It is a present limitation in the use of dimethyl sulfate as a probe that there is no detailed chemical knowledge of the causes of reaction inhibition or enhancement. The obvious explanations of the enhanced reactivities of certain nucleosome regions are that, at about 62 nucleotides from the 5' end in the major groove and at about 65 nucleotides from the 5' end in the minor groove, there is some altered DNA conformation or some protein constellation that enhances reaction. It is of interest that the DNase I cutting site falls exactly between these two reaction sites (data not shown) and that this is a low-frequency cutting site (8). Mirzabekov et al. (16) have crosslinked histones to DNA by first treating nucleosome core particles with dimethyl sulfate, depurinating in situ, and then reducing any Schiff base crosslinks formed. The ability to crosslink with dimethyl sulfate is not necessarily in conflict with the present results, because crosslinking (and hence backbone cleavage) may occur at only a small fraction of all methylpurine residues or may trap conformations too infrequent to be recorded as reaction protection. Alternatively, the sugar aldehyde created by depurination may be close to histone reactive groups or be capable of migrating some (not necessarily large) distance prior to its reaction. We mention two further corroborations of the suggestion that nucleosome DNA is quite accessible to solvent (unpublished results). (i) We have repeated the dimethyl sulfate reaction with nucleosomes reconstituted onto a unique restriction fragment 140 base pairs long and have seen no significant degree of protection. This experiment avoids all the problems of DNA sequence and length heterogeneity but depends upon establishing that reconstitution is correct. (ii) We have been able to reconstitute histones onto T4 DNA and obtain complexes showing properties of nucleosomes. This DNA has every cytosine replaced by glucosylated hydroxymethylcytosine, with
Proc. Natl. Acad. Sci. USA 76 (1979)
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the glucose residue protruding into the large groove. Our results point to a model for the nucleosome core in which both grooves of DNA are almost completely accessible to solvent, and suggest that contacts with the histone core must be predominantly localized to the DNA phosphodiester backbone. The volume occupied by the histone core appears to be considerably less than the total volume available in the region circumscribed by the coiled DNA (see for example the calculations in ref. 17). This suggests that DNA might be held suspended on the surface of the nucleosome in a manner designed to maximize exposure to the surroundings. The accessibility revealed by these experiments suggests that the histones of the nucleosome may be so designed that they can render the DNA more compact while interfering as little as possible with its biological and chemical activity. We thank Dr. Manuel Navia who very kindly provided the digitized densitometer scans of the gel autoradiographs and Drs. D. CameriniOtero, M. Gellert, and M. Zasloff for reading the manuscript. 1. Kornberg, R. D. (1977) Annu. Rev. Biochem. 46,931-954. 2. Felsenfeld, G. (1978) Nature (London) 271, 115-122. 3. Cold Spring Harbor Laboratory (1978) Chromatin, Cold Spring Harbor Symposia on Quantitative Biology (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), Vol. 42. 4. Sollner-Webb, B., Melchior, W., Jr. & Felsenfeld, G. (1978) Cell 14,611-627. 5. Gilbert, W., Maxam, A. & Mirzabekov, A. D. (1976) in Control of Ribosome Synthesis, The Alfred Benzon Symposium IX, eds. Kjeldgaard, N. C. & Maalle, 0. (Munksgaard, Copenhagen), pp. 139-148. 6. Humayun, Z., Kleid, D. & Ptashne, M. (1977) Nucleic Acids Res. 4, 1595-1607. 7. Olins, A. L., Carlson, R. D., Wright, E. B. & Olins, D. E. (1976) Nuicleic Acids Res. 3, 3271-3291. 8. Simpson, R. & Whitlock, J. P., Jr. (1976) Cell 9, 347-353. 9. Maxam, A. & Gilbert, W. (1977) Proc. Natl. Acad. Sci. USA 74, 560-564. 10. Maniatis, T., Jeffrey, A. & van deSande, H. (1976) Biochemistry 14,3787-3794. 11. Bloom, K. S. & Anderson, J. N. (1978) J. Biol. Chem. 253, 4446-4450. 12. Singer, B. (1975) Prog. Nucleic Res. Mol. Biol. 15, 219-284. 13. Mirzabekov, A. D., San'ko, D. F., Kolchinsky, A. M. & Melnikova, A. F. (1977) Eur. J. Biochem. 75, 379-389. 14. Kovacic, R. T. & van Holde, K. E. (1977) Biochemistry 16,
1490-1498. 15. Goodwin, D. C. & Brahms, J. (1978) Nucleic Acids Res. 5, 835-850. 16. Mirzabekov, A. D., Shick, V. V., Belyavsky, A. V. & Bavykin, S. G. (1978) Proc. Natl. Acad. Sci. USA 75,4184-4188. 17. Voordouw, G. & Eisenberg, H. (1978) Nature (London) 273, 446-448.
