1 January 1979 Nucleic Acids Research Volume 66 Number ubr1Jnay17NcecAisRsac
Vlum
Nucleosome packing in chromatin as revealed by nuclease digestion
V.A.Pospelov, S.B.Svetlikova and V.I.Vorob'ev
Institute of Cytology of the Academy of Sciences of the USSR, Leningrad, USSR Received 30 May 1978
ABSTRACT Chromatin DNA of rat thymus nuclei was cleaved by Serratia marcescens endonuclease. The fragments have been examinedby7 polyacrylemide gel electrophoresis under denaturing conditions. The results obtained are interpreted to mean that the internucleosomal DNA is cleaved by the endonuclease into fragments which are multiples of 10 nucleotides. The 10 nucleotide periodicity in fragmentation of internucleosomal DNA is independent of the presence of histone HI and is lilcely to be determined by the interaction of this DNA stretch with the nistone core of nucleosomes. Such interaction implies a close association between the nucleosomes in the chromatin thread. Quasi-limit chromatin digest (50-55% of DNA hydrolysis) contains undegraded DNA fragments with length of up to 1000 nucleotides or more. A part of this resistant DNA consists of single-stranded fragments or contains single-stranded regions. These data may be accounted for by a very compact nucleosome packing in the resistant chromatin in which one of the DNA strands is more accessible to the endonuclease action.
INTROD) TION Chromatin fiber is a thread of subunits (nucleosomes) each of which contains a DNA stretch of about 200 nucleotide pairs two molecules each of H2A, H2B, H3 and H4 histones and one molecule of histone HI -6. Enzymatic removal from the particle of the DNA terminal fragment with the length 40-60 nucleotide pairs, probably associated with histone HI, yields an incomplete nucleosome-containing DNA stretch of 140 nucleotide pairs and a histone octamer 2-7. The DINA fragment, which is rather easy to remove, proved to be the internucleosomal DNA sometimes visible under the electron microscope 8 9. The DNA length in complete nucleosomes of different cells and different organisms can vary between 155 and 240 nucleotide pairs depenC Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
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Nucleic Acids Research dent upon the variation in size of internucleosomal DNA 10-12 It was suggested that there might be a relationship between the type of histone HI and the size of internucleosomal DNA 10-12 It has been shown earlier that the endonuclease from Serratia marcescens splits simultaneously internucleosomal DNA and nucleosomal DNA at early stages of chromatin digestion 13. Moreover, the electrophoretic analysis revealed that internucleosomal DNA as well as DNA of nucleosomes is split by Serratia endonuclease into fragments which are multiples of 10 nucleotides. It has been assumed that the structure of a chromatin thread is supported by the histone interactions between neighbouring nucleosomes and that the internucleosomal DNA is twisted in such a manner that it is able to interact not only with HI histone but also with histone oomplexes of iucleosomes. This investigation presents some additional data which support this assumption. It has been demonstrated that the removal of histone HI from chromatin does not change the pattern of internucleosomal DNA fragmentation. Consequently, the 10 nucleotide periodicity observed upon the fragmentation of internucleosomal DNA is independent of the presence of HI histone and is likely to be determined by the interaction of this DNA stretch with histones of nucleosome core. Such interaction implies a close association between the nucleosomes in the chromatin thread. de have also investigated the nature of DNA which is resistant to prolonged digestion of nuclei with Serratia endonuclease. This DNA attains a relatively large size (up to 1000 nucleotides) and contains single-stranded fragments or singlestranded regions. TL'hese peculiarities of the resistant DNA may shed light on the nature of nucleosome packing in chromatin. Z&ATERIAIS ANID IfTHODS Isolation and digestion of nuclei. Nuclei were isolated from thymus of outbred rats as described previously 13, 14 The nuclei were suspended in a digestion medium comprising 0.3 M sucrose, 0.01 M Tris-HCl, pH 7.5, 0.001 M 9lgCl2; 0.1 mM 400
Nucleic Acids Research phenylmethylsulphonyl fluoride was added to inactivate protease activity 15. Incubation with endonuclease from Serratia marcescens was carried out at 370 C. The reaction was stopped by adding EDTA up to 4 mM. The endonuclease from Serratia marcescens (E.C.3.1.4.9) was isolated by the method described in ref. 16. The enzyme characteristics were similar to those described by these authors - 'i.e. the endonuclease proved to be equally capable of hydrolyzing both the denatured and native DNA and RNA. The enzyme did not exhibit marked preference towards purine or pyrimidine bases. Exonuclease activity was negligible. Nucleosomes were obtained from Serratia endonuclease digest of nuclei by chromatography on Sepharose 4B (Pharmacia, Sweden) as described in ref. 13, 14. Isolation of DNA. The endonuclease reaction was stopped by addition of EDTA. The chromatin digest was then incubated with 100 Pg/ml pronase (Serva, BRD) and 1% SDS (Serva, BR)) for one hour at 370. Deproteinization was carried out by addition of NaCl up to 1 M and by shaking the mixture with an equal volume of chloroform-isoamyl alcohol (24:1) at room temperature. The mixture was then centrifuged at 4000 g and the aqueous phase was repeatedly deproteinized. DNA from the aqueous phase was precipitated by the addition of two volumes of ethanol and after reprecipitation was dissolved in the elecvrophoretic buffer. DNA electrophoresis. Electrophoresis of the denatured DNA was performed in 7.5% polyacrylamide slab gels in the presence of 6 M deionized urea 13. The DNA samples were dissolved in the electrophoretic buffer containing 10% sucrose and 6 M urea and denatured by heating on a boiling water bath for 5 min. Trisborate buffer 0.02 M, pH 8.3 with 0.002 M EDTA was used as the electrophoretic buffer. Two-dimensional electrophoresis of nucleosomal DNA was performed as follows: after the low ionic strength electrophoresis of nucleosomes 139 14 (direction I) a longitudinal strip was cut from 5% polyacrylamide gel and incubated for 30 min in the electrophoretic buffer containing 1% SDS and 6M urea. DNA was denatured by heating the strip of gel on a boiling water 401
Nucleic Acids Research bath for 10 min. Then the strip of gel was placed on the top of a 10% polyacrylamide gel block. Electrophoresis was then performed in the cross direction II for 2-3 h at 200 V per slab. The detergent was washed from the gel with 50% ethanol and the gel was stained with ethidium bromide (Serva, BRD, 1 pg/ml) in 1 mM EDTA, pH 7.4. DNA bands were visualized under ultraviolet light and photographed through a red filter on Micrat-300 film. Chromatography on Hy4droxyapatite. DNA was dissolved in 0.03 M sodium phosphate buffer (PB), pH 7.0. About 1.8 mg DNA was put onto a 5 cm3 column of Hydroxyapatite (Bio Gel lHT, BioRad, ULA); the colujmn was washed with 0.03 M PB at 600 C. The DNA was eluted successively with 0.12 M PB (fraction of single-stranded molecules) and 0.5 M PB (fraction of doublestranded molecules). All elution procedures were performed in a water-jacket column at 600. -NA digestion b- 51-nuclease. 51-nuclease preparation (specific activity 93000 units per mg of protein) produced by Special Bureau of Biologically Active Substances (Novosibirsic) was used for the experiment. The reaction sample (0.5 ml) contained 200 pg of DNA, 0.02 M acetate buffer, pH 4.6, 0.1 mM ZnSO4, 0.15 M NaCl. The enzyme was added up to the concentration of 3 units per pg DNA. Incubation was carried out at 370 for 45 min. Then DNA was deproteinized by pronase treatment in the presence of 1% SDS and shaked with the chloroform-isoamyl alcohol mixture. Ethanol precipitated DNA was dissolved in 0.02 M Tris-borate buffer, pH 8.3, containing 6 M urea and 10% sucrose, and subjected to electrophoresis under the denaturing conditions.
RESULTS Electrophoretio analysis of denatured DNA from the Serratia endonuclease digest of nuclei revealed a series of distinct bands of single-stranded DNA fragments up to 1000 nucleotides and more (Fig.1). The size of small DNA fragments (up to 200 nucleotides) was determined by comparing their migration mobility with that of the DNA fragments produced by treatment of 402
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300-
Figure 1. Electrophoresis of chLromat'in DNA fragments produced by nucleases. DNA samples were dissolved in electrophoretic buffer containing 6 ML urea and were denatured by heating on a boiling water bath for 10 mm. a - DNAase I (100 units,, 2 mmn .b - Serratia endonuclease (100 units,, 2 min), c - Serratia endonuclease (1000 units, 2 mi) Numeral.s denote the length of DNA fragments (in nucleotides). nuclei with pancreatic DNAase I. It is known that these latter fragments are separated electrophoretically under denaturing conditions into 16-20 distinct bands of single st-randed f rag-. ments which are multiples of 10 nucleotides 99 17 The size of 403
Nucleic Acids Research long DNA fragments in Serratia endonuclease digest was determined from the dependence of electrophoretic mobilities on DNA fragment size in a DNAase I digest extrapolated to the region of long fragments (up to 300 nucleotides). It was calculated that for long DNA fragments in Serratia endonuclease digest (200-300 nucleotides) the difference in the size of neighbouring fragments was on the average also about 10 nucleotides. The data presented in Fig.1 show that Serratia endonuclease reveals a specific 10 nucleotide periodicity of DNA fragmentation at least within two nucleosomes. We suppose that internucleosomal DNA as well as DNA of nucleosomes may be cleaved by Serratia endonuclease into fragments which are multiples of 10 nucleotides. It appears that this pattern of fragmentation is due to the presence of histones which are able to protect DNA in chromatin leaving sites accessible to endonuclease at regular intervals of 10 nucleotides. This conclusion is also supported by the results of experiments with nucleosomes isolated chromatographically from chromatin digest. It is known that mononucleosomes in a micrococcal nuclease digest of chromatin can be separated by low ionic strength electrophoresis into two main fractions: a slowly migrating fraction of complete nucleosomes which contain 180-200 base pairs of DNA and one molecule of HI histone and a rapidly migrating one in which nucleosomes contain 140 base pairs of DNA and no HI histone 3, 13, 18 Nucleosomes of the both types can be ootained also from Serratia endonuclease chromatin digest. As has been shown earlier, these nucleosomes contain internally degraded DNA 13* Such nucleosome particles were isolated from a Berratia endonuclease digest of chromatin by chromatography on Sepharose 4B and then were subjected to two-dimensional polyacrylamide gel electrophoresis (Fig. 2). NIucleosomaiL particles were separated by low ionic strength electrophoresis (direction I). Electrophoresis in the presence of 0.1% SDS and 6 M urea (direction II) was used to separate DNA fragments after its denaturation directly in the gel (Fig. 2).
404
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_ _ 140 t2o-
100800
4020-
Figure 2. Two-dimensional electrophoresis of nucleosomal DNA. The nucleosomes were obtained by chromatography on Sepharose 4B after treatment of the nuclei by Serratia endonuclease (30 units, 15 min). I .- first electrophoresis run of nucleosomes under low ionic strength conditions in 5% polyacrylamide gel. II second electrophoresis run after incubation of She longitudinal strip of gel in 0.1% SLDS and 6 M urea at 95 for 10 min. After cooling, the DNA fragments were electrophoretically separated in the cross direction through a 10% gel. The numerals denote the DNA fragments which are multiples of 10 bases. A - nucleosomes lacking HI histone, B - nucleosomes containing HI histone. A number of DNA fragments which are multiples of 10 nucleotides can be identified electrophoretically in both mononucleosomal fraction (Fig. 2). The maximum size of DNA fragments corresponds to the length of DNA in both nucleosome fractions: in complete nucleosomes - 180 nucleotides and in nucleosomes lacking histone HI -- 140 nucleotides 29 4. In Fig. 2 the bands of different intensity are seen within the spectrum of DNA fragments from 10 to 140 or 180 nucleotides. The fragments of 20, 40 and 100 nucleotides long are present in maximuim quantities. Fragments of 50 and 80 405
Nucleic Acids Research nueleotides in length have, however, low intensities (Fig. 2) in both mononucleosomal fractions. The latter fact is in good agreement with Simpson's results of electrophoretic analysis of DNA fragments produced by DNAase I digestion of nucleosomes . Consequently, it must be concluwith 5'-end labelled DNA ded that the pattern of DNA fragmentation at an interval of 10 nucleotides is similar in both types of mononucleosomes despite the presence cf the internucleosomal DNA stretch in the complete nucleosome. Moreover, this short DNA stretch bound with the histone HI is protected from random endonuclease degradation: it is fragmented regularly at intervals of 10 nucleotides, which is characteristic of intranucleosomal DNA. There is an alternative interpretation of these data: the materials in the electrophoretic 10 nucleotide ladder may be derived only from cutting within nucleosomes. In such case, however, internucleosomal DNA must be resistant to nuclease attack. The last assumption seems to be scarcely probable. To check whether HI histone, which has been suggested to be associated with internucleosomal DNA 24, participates in the protection of this DNA, endonuclease digestion of chromatin, devoid of histone HI, was performed. Nuclei were mildly digested with micrococcal nuclease (Worthington, USA) (30 units, 10 min), then were immediately precipitated by centrifugation and separated into two portions. One of them was suspended in a solution of low ionic strength (0.005 M TrisHCl, pH 7.4, 0.002 M EDTA) while the other was treated with 0.6 hI NaCl in the same buffer in order to dissociate histone HI. Both suspensions were then clarified by centrifugation and supernatants containing soluble chromatin fragments were put onto Sepharose 4B columns equilibrated with appropriate solutions. The same solutions were used for the elution procedure. Fractions excluded from the columns contained large chromatin fragments (the average sedimentation coefficient 32-33 S in 0.005 M Tris-HOl, pH V.4). It was found electrophoretically that histone HI was wholly absent from the excluded fraction of chromatin eluted with 0.6 M NaCl. NaCl was removed by dialysis and the chromatins were transferred to the appropriate solutions for digestion wita Serratia endonuclease or micro406
Nucleic Acids Research coccai nuc.Lease. After iigestion, DNA was isolated from the chromatin digests, denatured and analyzed by polyacrylamide gel electrophoresis. The results of control experiments indicate that the DNA repeat length (191 nucleotides) does not change after HI histone removal. Therefore the procedure used is not likely to cause major nucleosome rearrangement (data not shown). The comparison of the DNA fragments isolated from digests of complete chromatin (Fig. 3a) and of chromatin without HI histone (Fig. 3b) shows that in both cases DNA fragments, which are multiples of 10 nucleotides, can be traced up to the size of 250 nucleotides, i.e. to a size larger than that of DNA in a complete nucleosome. Consequently, the removal of histone HI does not change the pattern of specific splitting of internucleosomal DNA. Had DNA sites between nucleosomes liable to
U'Figure 3. Comparison of DNA fragments produced by Serratia endonuclease and micrococcal nuclease 'in complete chromatin and HI-depleted chromatin. Serratia endonuclease: a) complete chromatin: b) HI-depleted chromatin: micrococcal nuclease; c) complete chromatin; d) HI-depleted chromatin. Nuclease concentration - 20 units,, time of hydrolysis - 30 mi. 407
Nucleic Acids Research degradation with Serratia endonuclease been revealed upon removal of histone HI, the size of the remaining DNA fragments would not have exceeded 140 nucleotides (size of DNA fragment in core nucleosome lacking of histone HI). Such breakdown, tirpical for micrococcal nuclease digestion of chromatin DNA between nucleosomes, can be seen in Fig. 3c, d. In this case the largest size of DNA fragments in the digest of the complete chromatin and chromatin without histone HI is 140 nucleotides. Subnucleosomal DNA fragments, the products of nucleosomal DNA degradation, are also seen owing to extensive chromatin digestion in our experimental conditions. The results of these experiments permit to suppose that the specific 10 nucleotide periodicity of degradation of internucleosomal DINA in chromatin does not depend on the protection of this DNA stretch by histone HI. Thus, the above findings indicate a close association of nucleosomes in chromatin thread with internucleosomal DNA. ;vhile studying the DNA digestion of nuclei with Serratia endonuclease we have received evidence demonstrating that an essential part of chromatin in the nuclei is in a very compact state and its DNA is resistant to endonuclease attack. Fig. I shows a set of fragments produced by mild treatment of nuclei with Serratia endonuclease. An increase in the endonuclease concentration and incubation time resulting in a further growth of acid-soluble DNA products, does not lead to the disappearance of high molecular weight DNA fragments. Fig. 4 presents the results of an electrophoretic analysis of DNA fragments produced by digestion of nuclei with endonuclease in high concentration (1000 units/mg DNA) at different intervals: 10 min, 30 min and 60 min (b, c, d). The quantity of acid soluble products was 22%, 31% and 48%, respectively. Under such conditions, free DLNA was completely hydrolyzed in less than 5 min. Incubation OIf control nuclei in the absence of added endonuclease for 1 h yielded less than 1% of the acid-soluble products. iFig. 4 shows that the electrophoretic mobilities of nearly all DNA fragments produced after treatment of the nuclei at different intervals practically coincided. No marked enrichment in the low molecular weight fragments at the expense 408
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Figure 4. Electrophoresis of reesistant DNA fragments. a - denatured DNA from micrococcal nuclease-treated chromatin (5% acid soluble products), b, c, d - denatured D)NA from Serratia endonuclease-treated chromatin. Enzyme/DNA ratio 1000 units/mg; time of digestion - 10 min (b), 30 min (c), 60 min (d), giving 22A, 31%, 48% of acid soluble produacts, respectively, e - the same as (b), but the DNA was not denatured. 6 M4 urea was in the 7.5% gel and all DNA samples.
of cleaving of high molecular weight fragments was observed. DNA hydrolysis with the formation of about 50%o of the acid soluble products is the quasi-limit in the course of chromatin digestion with Serratia endonuclease in our conditions. The 409
Nucleic Acids Research same quasi-limit of hydrolysis was also characteristic for the action of micrococcal nuclease 20, 21 DNA resistant to Serratia endonuclease, however, includes comparatively large fragments (up to 1000 nucleotides or more) while DNA resistant to micrococcal nuclease contained only subnucleosomal fragnents of no more than 140 nucleotides in length. There is a great similarity between the electrophoretic mobility of denatured DNA fragnents (Fig. 4b, c, d) and the fragments which were not denatured but were dissolved in 6 M urea (Fig. 4e). The effect of heat denaturation is primarily manifested in the formation of low molecular weight fragments which are multiples of 10 nucleotides (20-140 nucleotides). Thus it may be supposed.that short fragents were formed as a result of heat denaturation of longer DNA fragments containing a great number of single-stranded breaks. As is seen from Fig. 4e, the quantity of such short fragments in DNA samples dissolved in 6 M urea is negligible as compared to heat denatured DNA (the dame results have been also obtained for DNA after 30 and 60 min endonuclease digestion of nuclei). Consequently, under our experimental conditions, urea did not induce DNA denaturation. Thus, the similarity of electrophoretic mobilities of high molecular weight DNA fragments after heat denaturation and those of undenatured fragments dissolved in urea cannot be accounted for by the denaturing effect of the latter. At the same time, our earlier findings show that in the absence of urea, distinct bands of high molecular weight fragments were not detected during electrophoresis of native DNKA fmm chromatin digest 13. It is known that urea improves the electrophoretic resolution of single stranded molecules since it is able to break their intermolecular and intramolecular interactions 22. It may be concluded that chromatin DNA which is resistant to Serratia endonuclease contains, along with native double-stranded molecules, a great number of single-stranded fragments or double-stranded ones with single-stranded regions. In such a case, however, the discrete pattern of electrophoretic bands of undenatured DNA fragments in the presence of urea similar to those of denatured ones may arise only when electrophoretic 410
Nucleic Acids Research mobilities of corresponding native and denatured fragnents of DNA are coincident. In this connection, it is desirable to compare the mobilities of native and denatured DNA fragments under our electrophoretic conditions. The electrophoretic mobility of nucleic acids, which in the first approximation was found to be inversely proportional to the molecular weight, depends also on the partial specific volume and the shape of the molecule. Although the mobility of single-stranded and double-stranded molecules may differ, the possibilities of their separation are determined by the size of the molecule and electrophoretic conditions. We compared the electrophoretic mobilities of native and denatured fragments of phage 1929 DNA produced by restriction endonuclease EcoRI (Fig. 5). It is seen that the two fragments of higher molecular weight (5.106 and 3-106) show different electrophoretic mobilities in the native and denatured state. However the mobilities of the three fragments with molecular weights of 3o105, 5 105 and 1.2a106, both in the native and denatured state practically coincide. Returning to the data presented in the Fig. 4 it may be supposed that in the course of electrophoresis under completely native conditions the single-stranded fragments yield overlapping diffuse bands which conceal the bands of native
Figure 5. Comparison of electrophoretic mobilities of native and denatured DNA fragments of P29 phage.
't ¢*l
3 tQS
a
-
native,
b
-
denatured.
I 1.2106 -0.5310'
a
b 411
Nucleic Acids Research fragments. The bands of single-stranded fragments are narrowing in the presence of urea. Since the mobilities of these fragments coincide with that of native fragments, distinct bands come into view. Thie presence of single-stranded fragments in chromatin DNA resistant to Serratia endonuclease may be checked by treatment with S1-nuclease specific to single-stranded DNA. After treatment of resistant DNA with S1-nuclease, all discrete and distinct bands disappear from electrophoretographs leaving a rather diffusely stained backgroung (Fig. 6). This indicates the presence of single-stranded DNA fragments or single-stranded regions in resistant DNA. Chromatography on hydroxyapatite is another method employed to reveal the presence of single-stranded fragments in resistant DNA. The results of one of these experiments are given in Fig. 7. The DNA single-stranded fragments are eluted with 0.12 M PB in the first fraction. Their quantity varied from 20 to 35% of the total resistant DNA depending on the
Figure 6. Influence of S -nuclease on electrophoratic mobility of resistant DNA. a - control DNA, b - resistant chromatin DNA after S -nuclease treatment (3 units/pg 9NA, 45 min, 370). Resistant DNA was isolated from nuclei treated by Serratia endonuclease (1000 _ DNA, 48% acid soluble s ........units/mg
products).
a 412
b
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E
cB eat
co 'A
2
2
4
012MPB
6
8 10 12 14 16 18 20 FRACTION NUMBER .5M PB
Figure 7. Chromatography of resistant DNA on hydroxyapatite. Single-stranded fragments were eluted with 0.12 M PB, double-stranded fragments - with 0.5 M PB. Resistant DNA was isolated from nuclei treated by Serratia endonuclease up to 55% acid soluble products of DNA. conditions of chromatin digestion. It must be noted that incompletely-paired DNA fragments including single-stranded regions are not eluted with 0.12 M PB and leave the coluimn in the fraction of double-stranded molecules. That is why our estimation of the quantity of single-stranded fragments in resistant DNA may be lowered. A similar quantity of singlestranded fragments in DlJA of chromatin resistant to DNAase I was found earlier by chromatographic analysis on hydroxyapatite 23, 24 Electrophoresis of the chromatographic fraction of single-stranded DNA fragments shows that this fraction consists of comparatively short fragments (no more than 120-140 nucleotides) which are multiples of 10 nucleotides (Fig. 8). In the fraction of double-stranded molecules, along with high molecular weight fragments characteristic of resistant DNA, there occur short DNA fragments of exactly the same size as in the first fraction. It has been already mentioned that partially paired DINA molecules are retarded on hydroxyapatite as 413
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Figure 8. Electrophoresis
of
fragments isolated during chromatography of resistant DNA on hydroxyapatite. a - 0.12 M PB fraction; b - 0.5 M PB fraction. Both DNA
fractions
were
dialysed
against ele ctrophoretic buffer and denatured in the presence of 6 M urea on a boiling water bath. Numerals denote the length of DNA fragments (in nucleotides).
so~~~~~.s x
completely resistant stranded these
native
DNA
molecules.
consists
molecules.
molecules
composed of resistant
is
short
DNA may
Therefore
mainly
It
is
long
not
while
fragments. contain
the
in
with
S1
nuclease
and
inconceivable the
Along
that
complementary with
such
perfect duplexes Thus
the
chromatography
presence
of
single-stranded fragments
DNA which
is
resistant
to
be
may
assumed
incompletely paired
of
tely single-stranded molecules. ments
it
Serratia
and
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our
HAP-columns
unpaired
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areas
endonuclease.
DISCUSSION
IEndonucleolytic 414
splitting of
nuclear DNA by
an
of
enzyme
Nucleic Acids Research from Serratia marcescens is different from the action of micrococcal nuclease. The latter breaks DNA primarily between chromatin subunits producing nucleosomes and their oligomers. After splitting of a short stretch of internucleosomal DNA associated with histone HI, this endonuclease digests DNA inside nucleosomes 2, 3, 20 21. Serratia endonuclease cleaves simultaneously internucleosomal and nucleosomal DNA at the early stages of chromatin digestion. IMoreover,this endonuclease produces DNA fragments which form during electrophoresis a long 10 nucleotide interval ladder (up to 300 nucleotides) 13. A similar 10 nucleotide periodicity of DNA fragmentation in c4romatin was recently demonstrated in experiments with DNAase 25 I and Exonuclease III from E.coli 26 The results obtained with Serratia endonuclease allow to propose that internucleosomal DNA as well as DNA in nucleosomes interact with histones in such a manner that DNA sites every 10 nucleotides in length are sensitive to the enzyme. Since this peculiar chromatin structure is retained after the removal of histone HI, the latter does not apparently determine the arrangement of endonuclease-sensitive sites on the internucleosomal DNA. Consequently, it may be suggested that internucleosomal DNA interacts with the octamer of nucleosomal histones, which provides it with an ordered configurzation and protection from endonuclease. Such interaction suggests a tight association between nucleosomes along the chromatin fiber, i.e. complete absence of free DNA connecting nucleosomes. Mandel and Fasman 27 came to a similar conclusion when comparing the spectra of circular dichroism and denaturation curves of mononucleosomes and their oligomers. The authors assumed that nucleosomes in solution make up a compact structure where each particle has many contacts forming a kind of superhelix. Internucleosomal DNA was examined in an electron microscope by a great number of authors 79 8, who found that its 0 28 . However, electron length varied greatly (up to 240 A) microscopic studies of internucleosomal DNA in complete chromatin and in chromatin devoid of histone HI made by Finch et al. 8 showed that in most of the cases no connecting DNA is seen between nucleosomes. These investigators regard inter415
Nucleic Acids Research nucleosomal DNA which is visible in the electron microscope as an artifact arising in the process of sample preparation. Noteworthy is the evidence of a tight interaction between nucleosomes obtained from cross-linking of histones in chromatin by bifunctional reagents. Thomas and Kornberg showed that dimethyl suberimidate treatment of chromatin induces formation of histone oligomers containing not only 8 but also 16 and 24 histone molecules 29. Consequently, the octamers of histones of neighbouring nucleosomes exist in such proximity that they can be crosslinked by a bifunctional reagent in which the distance between reactive groups in no more than 13.5 A 3°. This length corresponds to that of 4 nucleotide pairs in B-form DNA. It must also be pointed out that isolated nucleosomes show a great potential ability to interact. In the presence of Mg++ their interaction results in the formation of fibrillar structures 31In the process of prolonged digestion Serratia endonuclease, similarly to other nucleases, degrades about 50% of the nuclear DNA. However, DNA resistant to this endonuclease differs qualitatively from DNA resistant to micrococcal nuclease as it consists of rather large fragments (up to 1000 nucleotides and more). The presence of the long fragments of resistant DNA is accounted for by the resistance of nucleosomal and internucleosomal DNA to endonuclease attack. This provides additional evidence in favour of a compact arrangement of nucleosomes in chromatin. The degree of DNA resistance to endonucleases is likely to be determined by the state of chromatin during nuclease treatment. Burgoyne et al. 32 have demonstrated that nuclear DNA in rat liver is resistant to pancreatic D.NAase I, but it becomes more sensitive to enzyme in the presence of Ca++ ions. This is not the case in our experiments, since addition of calcium ions (1 ralvI) to the digestion medium had no detectable effect on the results. An electrophoretic analysis revealed another interesting peculiarity in the length distribution of DNA fragments in endonuclease-resistant chromatin. Besides the fragments which are multiples of 10 bases, one can trace the second order periodicity. The most intense bands are concentrated in the 416
Nucleic Acids Research regions of electrophoretographs which correspond to the lengths of mononucleosomal, dinucleosomal and trinucleosomal DNA of micrococcal digest (Fig. 4). Each of them is represented by at least two bands of similar intensity. It may be supposed that occurence of doublets in the second order periodicity reflects the presence of the different nucleosomal DNA repeats. Since discrete bands of DNA fragments of the resistant chromatin show up only (luring electrophoresis after denaturation, nucleolytic fragmentation of both DNA strands seems to proceed independentlyu Hence, accessible sites do not necessarily localize in both DNA strands opposite each other. Moreover, the presence of single-stranded regions and DNA fragments may indicate that Serratia endonuclease is able to split preferenticaly one of the DNA strands in the resistant chromatin due to the compact association between the nucleosomes along the chromatin thread. A ,part of the present work was reported at the 3rd SMEA Symposium on Biophysics of Nucleic Acids, Brno, Czechoslovakya, September 1977 33.
ACKNOWILEDGMENT The authors thank Dr. R.I.Salganik for his gift of the Serratia endonuclease, Dr. Bryan for providing us with the EcoRI digest of the P29 DNA and Dr. T.N.Osipova for performing the ultracentrifugation for us.
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Oudet, P., Gross-Bellard, MI. and Chambon, P. (1975) Cell 4, 281-300 Finch, J.T., Noll, M. and Kornberg, R. (1975) Proc. Nat, Acad. Sci. USA 72, 3320-3322 Compton, J.I., Bellard, M. and Chambon, P. (1976) Proc. Nat. Acad. Sci. L;A 73, 4382-4386 Spadafora, C., Bellard, M., Compton, J.L. and Chambon, P. (1976) FEBS letters 69, 281-285 Lohr, D., Corden J., Tatchell, K., Kovacic, R.T. and Van Holde, K.E. Proc. Nat. Acad. Sci. USA 74, 7983 Pospelov, V.A., Svetlikova, S.B. and Vorob'ev, V.I. (1977) Nucl. Acids Res. 4, 3267-3279 Pospelov, V.A., Svetlikova, S.B. and Vorob'ev,V.I. (1977) Molec. Biol. (USS) 11, 781-789 Ballal, N.R., Goldberg, D.A. and Busch, H. (1975) Biochem. Biophys. Res. Commun. 62, 972-984 Nestle, l. and Roberts, W.K. (1969) J. Biol. Chem. 244, 5213-5218 ibid. 5219-5225 Noll, M. (1974) Nuci. Acids Res. 1, 1573-1578 Pospelov, V.A., Svetlikova, S.B. and Voroblev V.I. (1977) FEBS letters 74, 224-233 Simpson, R.T. and Whitlock, J.P. (1976) Cell 9, 347-353 Sollner-Webb, B. and Felsenfeld, G. (1975) Biochemistry 14, 2915-2920 Lacy, E. and Axel, R. (1975) Proc. Nat. Acad. Sci. USA 72, 3978-3982 Tennov, A.V. (1975) In: "Molecular Biology" 4, Kisselev, L.L., ed., £Aoscow, pp. 130-175 Pospelov, V.A., Sokolenko, A.A. and Dianov, G.L. (1975) Molec. Biol. 9, 691-697 Oliver, 1). and Chalkley, Ro (1974) Biochemistry 13, 50935098 Lohr, D., Tatchell, K. and Van Holde, K.E. (1977) Cell
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12, 829-836 Riley, D. and 8Weintraub, H. (1978) Cell 13, 281-293 ldandel, R. and Fasman, G. (1976) NuCl. Acids Res. 3, 1839-1855 Van Holde, K.E., Sahasrabuddhe, C.G., Shaw, B.R., Van Bruggen, E.F.J. and Arnberg, A.C. (1974) Biochem. Biophys. Res. Commun. 60, 1365-1370 Thomas, J.0. and Kornberg, R.D. (1975) Proc. Nat. Acad. Sci. USA 72, 2626-2630 Chalkley, R. (1975) Biochem. Biophys. Res. Commun. 64,
587-594
Finch, J.T. and Klug, A. (1976) Proc. Nat. Acad. Sci. LEA 73, 1897-1901 Burgoyne, L,A., M4obbs J.0. and Marshall, A.J. (1976) Nucl. Acids Res, 3, 3f93-3304 Vorob'ev, V.o., Pospelov, V.A. and Svetlikova, S.B. (1978) Studia biophysica, Berlin, 67, 3-4
Vlum
Nucleosome packing in chromatin as revealed by nuclease digestion
V.A.Pospelov, S.B.Svetlikova and V.I.Vorob'ev
Institute of Cytology of the Academy of Sciences of the USSR, Leningrad, USSR Received 30 May 1978
ABSTRACT Chromatin DNA of rat thymus nuclei was cleaved by Serratia marcescens endonuclease. The fragments have been examinedby7 polyacrylemide gel electrophoresis under denaturing conditions. The results obtained are interpreted to mean that the internucleosomal DNA is cleaved by the endonuclease into fragments which are multiples of 10 nucleotides. The 10 nucleotide periodicity in fragmentation of internucleosomal DNA is independent of the presence of histone HI and is lilcely to be determined by the interaction of this DNA stretch with the nistone core of nucleosomes. Such interaction implies a close association between the nucleosomes in the chromatin thread. Quasi-limit chromatin digest (50-55% of DNA hydrolysis) contains undegraded DNA fragments with length of up to 1000 nucleotides or more. A part of this resistant DNA consists of single-stranded fragments or contains single-stranded regions. These data may be accounted for by a very compact nucleosome packing in the resistant chromatin in which one of the DNA strands is more accessible to the endonuclease action.
INTROD) TION Chromatin fiber is a thread of subunits (nucleosomes) each of which contains a DNA stretch of about 200 nucleotide pairs two molecules each of H2A, H2B, H3 and H4 histones and one molecule of histone HI -6. Enzymatic removal from the particle of the DNA terminal fragment with the length 40-60 nucleotide pairs, probably associated with histone HI, yields an incomplete nucleosome-containing DNA stretch of 140 nucleotide pairs and a histone octamer 2-7. The DINA fragment, which is rather easy to remove, proved to be the internucleosomal DNA sometimes visible under the electron microscope 8 9. The DNA length in complete nucleosomes of different cells and different organisms can vary between 155 and 240 nucleotide pairs depenC Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
399
Nucleic Acids Research dent upon the variation in size of internucleosomal DNA 10-12 It was suggested that there might be a relationship between the type of histone HI and the size of internucleosomal DNA 10-12 It has been shown earlier that the endonuclease from Serratia marcescens splits simultaneously internucleosomal DNA and nucleosomal DNA at early stages of chromatin digestion 13. Moreover, the electrophoretic analysis revealed that internucleosomal DNA as well as DNA of nucleosomes is split by Serratia endonuclease into fragments which are multiples of 10 nucleotides. It has been assumed that the structure of a chromatin thread is supported by the histone interactions between neighbouring nucleosomes and that the internucleosomal DNA is twisted in such a manner that it is able to interact not only with HI histone but also with histone oomplexes of iucleosomes. This investigation presents some additional data which support this assumption. It has been demonstrated that the removal of histone HI from chromatin does not change the pattern of internucleosomal DNA fragmentation. Consequently, the 10 nucleotide periodicity observed upon the fragmentation of internucleosomal DNA is independent of the presence of HI histone and is likely to be determined by the interaction of this DNA stretch with histones of nucleosome core. Such interaction implies a close association between the nucleosomes in the chromatin thread. de have also investigated the nature of DNA which is resistant to prolonged digestion of nuclei with Serratia endonuclease. This DNA attains a relatively large size (up to 1000 nucleotides) and contains single-stranded fragments or singlestranded regions. TL'hese peculiarities of the resistant DNA may shed light on the nature of nucleosome packing in chromatin. Z&ATERIAIS ANID IfTHODS Isolation and digestion of nuclei. Nuclei were isolated from thymus of outbred rats as described previously 13, 14 The nuclei were suspended in a digestion medium comprising 0.3 M sucrose, 0.01 M Tris-HCl, pH 7.5, 0.001 M 9lgCl2; 0.1 mM 400
Nucleic Acids Research phenylmethylsulphonyl fluoride was added to inactivate protease activity 15. Incubation with endonuclease from Serratia marcescens was carried out at 370 C. The reaction was stopped by adding EDTA up to 4 mM. The endonuclease from Serratia marcescens (E.C.3.1.4.9) was isolated by the method described in ref. 16. The enzyme characteristics were similar to those described by these authors - 'i.e. the endonuclease proved to be equally capable of hydrolyzing both the denatured and native DNA and RNA. The enzyme did not exhibit marked preference towards purine or pyrimidine bases. Exonuclease activity was negligible. Nucleosomes were obtained from Serratia endonuclease digest of nuclei by chromatography on Sepharose 4B (Pharmacia, Sweden) as described in ref. 13, 14. Isolation of DNA. The endonuclease reaction was stopped by addition of EDTA. The chromatin digest was then incubated with 100 Pg/ml pronase (Serva, BRD) and 1% SDS (Serva, BR)) for one hour at 370. Deproteinization was carried out by addition of NaCl up to 1 M and by shaking the mixture with an equal volume of chloroform-isoamyl alcohol (24:1) at room temperature. The mixture was then centrifuged at 4000 g and the aqueous phase was repeatedly deproteinized. DNA from the aqueous phase was precipitated by the addition of two volumes of ethanol and after reprecipitation was dissolved in the elecvrophoretic buffer. DNA electrophoresis. Electrophoresis of the denatured DNA was performed in 7.5% polyacrylamide slab gels in the presence of 6 M deionized urea 13. The DNA samples were dissolved in the electrophoretic buffer containing 10% sucrose and 6 M urea and denatured by heating on a boiling water bath for 5 min. Trisborate buffer 0.02 M, pH 8.3 with 0.002 M EDTA was used as the electrophoretic buffer. Two-dimensional electrophoresis of nucleosomal DNA was performed as follows: after the low ionic strength electrophoresis of nucleosomes 139 14 (direction I) a longitudinal strip was cut from 5% polyacrylamide gel and incubated for 30 min in the electrophoretic buffer containing 1% SDS and 6M urea. DNA was denatured by heating the strip of gel on a boiling water 401
Nucleic Acids Research bath for 10 min. Then the strip of gel was placed on the top of a 10% polyacrylamide gel block. Electrophoresis was then performed in the cross direction II for 2-3 h at 200 V per slab. The detergent was washed from the gel with 50% ethanol and the gel was stained with ethidium bromide (Serva, BRD, 1 pg/ml) in 1 mM EDTA, pH 7.4. DNA bands were visualized under ultraviolet light and photographed through a red filter on Micrat-300 film. Chromatography on Hy4droxyapatite. DNA was dissolved in 0.03 M sodium phosphate buffer (PB), pH 7.0. About 1.8 mg DNA was put onto a 5 cm3 column of Hydroxyapatite (Bio Gel lHT, BioRad, ULA); the colujmn was washed with 0.03 M PB at 600 C. The DNA was eluted successively with 0.12 M PB (fraction of single-stranded molecules) and 0.5 M PB (fraction of doublestranded molecules). All elution procedures were performed in a water-jacket column at 600. -NA digestion b- 51-nuclease. 51-nuclease preparation (specific activity 93000 units per mg of protein) produced by Special Bureau of Biologically Active Substances (Novosibirsic) was used for the experiment. The reaction sample (0.5 ml) contained 200 pg of DNA, 0.02 M acetate buffer, pH 4.6, 0.1 mM ZnSO4, 0.15 M NaCl. The enzyme was added up to the concentration of 3 units per pg DNA. Incubation was carried out at 370 for 45 min. Then DNA was deproteinized by pronase treatment in the presence of 1% SDS and shaked with the chloroform-isoamyl alcohol mixture. Ethanol precipitated DNA was dissolved in 0.02 M Tris-borate buffer, pH 8.3, containing 6 M urea and 10% sucrose, and subjected to electrophoresis under the denaturing conditions.
RESULTS Electrophoretio analysis of denatured DNA from the Serratia endonuclease digest of nuclei revealed a series of distinct bands of single-stranded DNA fragments up to 1000 nucleotides and more (Fig.1). The size of small DNA fragments (up to 200 nucleotides) was determined by comparing their migration mobility with that of the DNA fragments produced by treatment of 402
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300-
Figure 1. Electrophoresis of chLromat'in DNA fragments produced by nucleases. DNA samples were dissolved in electrophoretic buffer containing 6 ML urea and were denatured by heating on a boiling water bath for 10 mm. a - DNAase I (100 units,, 2 mmn .b - Serratia endonuclease (100 units,, 2 min), c - Serratia endonuclease (1000 units, 2 mi) Numeral.s denote the length of DNA fragments (in nucleotides). nuclei with pancreatic DNAase I. It is known that these latter fragments are separated electrophoretically under denaturing conditions into 16-20 distinct bands of single st-randed f rag-. ments which are multiples of 10 nucleotides 99 17 The size of 403
Nucleic Acids Research long DNA fragments in Serratia endonuclease digest was determined from the dependence of electrophoretic mobilities on DNA fragment size in a DNAase I digest extrapolated to the region of long fragments (up to 300 nucleotides). It was calculated that for long DNA fragments in Serratia endonuclease digest (200-300 nucleotides) the difference in the size of neighbouring fragments was on the average also about 10 nucleotides. The data presented in Fig.1 show that Serratia endonuclease reveals a specific 10 nucleotide periodicity of DNA fragmentation at least within two nucleosomes. We suppose that internucleosomal DNA as well as DNA of nucleosomes may be cleaved by Serratia endonuclease into fragments which are multiples of 10 nucleotides. It appears that this pattern of fragmentation is due to the presence of histones which are able to protect DNA in chromatin leaving sites accessible to endonuclease at regular intervals of 10 nucleotides. This conclusion is also supported by the results of experiments with nucleosomes isolated chromatographically from chromatin digest. It is known that mononucleosomes in a micrococcal nuclease digest of chromatin can be separated by low ionic strength electrophoresis into two main fractions: a slowly migrating fraction of complete nucleosomes which contain 180-200 base pairs of DNA and one molecule of HI histone and a rapidly migrating one in which nucleosomes contain 140 base pairs of DNA and no HI histone 3, 13, 18 Nucleosomes of the both types can be ootained also from Serratia endonuclease chromatin digest. As has been shown earlier, these nucleosomes contain internally degraded DNA 13* Such nucleosome particles were isolated from a Berratia endonuclease digest of chromatin by chromatography on Sepharose 4B and then were subjected to two-dimensional polyacrylamide gel electrophoresis (Fig. 2). NIucleosomaiL particles were separated by low ionic strength electrophoresis (direction I). Electrophoresis in the presence of 0.1% SDS and 6 M urea (direction II) was used to separate DNA fragments after its denaturation directly in the gel (Fig. 2).
404
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_ _ 140 t2o-
100800
4020-
Figure 2. Two-dimensional electrophoresis of nucleosomal DNA. The nucleosomes were obtained by chromatography on Sepharose 4B after treatment of the nuclei by Serratia endonuclease (30 units, 15 min). I .- first electrophoresis run of nucleosomes under low ionic strength conditions in 5% polyacrylamide gel. II second electrophoresis run after incubation of She longitudinal strip of gel in 0.1% SLDS and 6 M urea at 95 for 10 min. After cooling, the DNA fragments were electrophoretically separated in the cross direction through a 10% gel. The numerals denote the DNA fragments which are multiples of 10 bases. A - nucleosomes lacking HI histone, B - nucleosomes containing HI histone. A number of DNA fragments which are multiples of 10 nucleotides can be identified electrophoretically in both mononucleosomal fraction (Fig. 2). The maximum size of DNA fragments corresponds to the length of DNA in both nucleosome fractions: in complete nucleosomes - 180 nucleotides and in nucleosomes lacking histone HI -- 140 nucleotides 29 4. In Fig. 2 the bands of different intensity are seen within the spectrum of DNA fragments from 10 to 140 or 180 nucleotides. The fragments of 20, 40 and 100 nucleotides long are present in maximuim quantities. Fragments of 50 and 80 405
Nucleic Acids Research nueleotides in length have, however, low intensities (Fig. 2) in both mononucleosomal fractions. The latter fact is in good agreement with Simpson's results of electrophoretic analysis of DNA fragments produced by DNAase I digestion of nucleosomes . Consequently, it must be concluwith 5'-end labelled DNA ded that the pattern of DNA fragmentation at an interval of 10 nucleotides is similar in both types of mononucleosomes despite the presence cf the internucleosomal DNA stretch in the complete nucleosome. Moreover, this short DNA stretch bound with the histone HI is protected from random endonuclease degradation: it is fragmented regularly at intervals of 10 nucleotides, which is characteristic of intranucleosomal DNA. There is an alternative interpretation of these data: the materials in the electrophoretic 10 nucleotide ladder may be derived only from cutting within nucleosomes. In such case, however, internucleosomal DNA must be resistant to nuclease attack. The last assumption seems to be scarcely probable. To check whether HI histone, which has been suggested to be associated with internucleosomal DNA 24, participates in the protection of this DNA, endonuclease digestion of chromatin, devoid of histone HI, was performed. Nuclei were mildly digested with micrococcal nuclease (Worthington, USA) (30 units, 10 min), then were immediately precipitated by centrifugation and separated into two portions. One of them was suspended in a solution of low ionic strength (0.005 M TrisHCl, pH 7.4, 0.002 M EDTA) while the other was treated with 0.6 hI NaCl in the same buffer in order to dissociate histone HI. Both suspensions were then clarified by centrifugation and supernatants containing soluble chromatin fragments were put onto Sepharose 4B columns equilibrated with appropriate solutions. The same solutions were used for the elution procedure. Fractions excluded from the columns contained large chromatin fragments (the average sedimentation coefficient 32-33 S in 0.005 M Tris-HOl, pH V.4). It was found electrophoretically that histone HI was wholly absent from the excluded fraction of chromatin eluted with 0.6 M NaCl. NaCl was removed by dialysis and the chromatins were transferred to the appropriate solutions for digestion wita Serratia endonuclease or micro406
Nucleic Acids Research coccai nuc.Lease. After iigestion, DNA was isolated from the chromatin digests, denatured and analyzed by polyacrylamide gel electrophoresis. The results of control experiments indicate that the DNA repeat length (191 nucleotides) does not change after HI histone removal. Therefore the procedure used is not likely to cause major nucleosome rearrangement (data not shown). The comparison of the DNA fragments isolated from digests of complete chromatin (Fig. 3a) and of chromatin without HI histone (Fig. 3b) shows that in both cases DNA fragments, which are multiples of 10 nucleotides, can be traced up to the size of 250 nucleotides, i.e. to a size larger than that of DNA in a complete nucleosome. Consequently, the removal of histone HI does not change the pattern of specific splitting of internucleosomal DNA. Had DNA sites between nucleosomes liable to
U'Figure 3. Comparison of DNA fragments produced by Serratia endonuclease and micrococcal nuclease 'in complete chromatin and HI-depleted chromatin. Serratia endonuclease: a) complete chromatin: b) HI-depleted chromatin: micrococcal nuclease; c) complete chromatin; d) HI-depleted chromatin. Nuclease concentration - 20 units,, time of hydrolysis - 30 mi. 407
Nucleic Acids Research degradation with Serratia endonuclease been revealed upon removal of histone HI, the size of the remaining DNA fragments would not have exceeded 140 nucleotides (size of DNA fragment in core nucleosome lacking of histone HI). Such breakdown, tirpical for micrococcal nuclease digestion of chromatin DNA between nucleosomes, can be seen in Fig. 3c, d. In this case the largest size of DNA fragments in the digest of the complete chromatin and chromatin without histone HI is 140 nucleotides. Subnucleosomal DNA fragments, the products of nucleosomal DNA degradation, are also seen owing to extensive chromatin digestion in our experimental conditions. The results of these experiments permit to suppose that the specific 10 nucleotide periodicity of degradation of internucleosomal DINA in chromatin does not depend on the protection of this DNA stretch by histone HI. Thus, the above findings indicate a close association of nucleosomes in chromatin thread with internucleosomal DNA. ;vhile studying the DNA digestion of nuclei with Serratia endonuclease we have received evidence demonstrating that an essential part of chromatin in the nuclei is in a very compact state and its DNA is resistant to endonuclease attack. Fig. I shows a set of fragments produced by mild treatment of nuclei with Serratia endonuclease. An increase in the endonuclease concentration and incubation time resulting in a further growth of acid-soluble DNA products, does not lead to the disappearance of high molecular weight DNA fragments. Fig. 4 presents the results of an electrophoretic analysis of DNA fragments produced by digestion of nuclei with endonuclease in high concentration (1000 units/mg DNA) at different intervals: 10 min, 30 min and 60 min (b, c, d). The quantity of acid soluble products was 22%, 31% and 48%, respectively. Under such conditions, free DLNA was completely hydrolyzed in less than 5 min. Incubation OIf control nuclei in the absence of added endonuclease for 1 h yielded less than 1% of the acid-soluble products. iFig. 4 shows that the electrophoretic mobilities of nearly all DNA fragments produced after treatment of the nuclei at different intervals practically coincided. No marked enrichment in the low molecular weight fragments at the expense 408
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Figure 4. Electrophoresis of reesistant DNA fragments. a - denatured DNA from micrococcal nuclease-treated chromatin (5% acid soluble products), b, c, d - denatured D)NA from Serratia endonuclease-treated chromatin. Enzyme/DNA ratio 1000 units/mg; time of digestion - 10 min (b), 30 min (c), 60 min (d), giving 22A, 31%, 48% of acid soluble produacts, respectively, e - the same as (b), but the DNA was not denatured. 6 M4 urea was in the 7.5% gel and all DNA samples.
of cleaving of high molecular weight fragments was observed. DNA hydrolysis with the formation of about 50%o of the acid soluble products is the quasi-limit in the course of chromatin digestion with Serratia endonuclease in our conditions. The 409
Nucleic Acids Research same quasi-limit of hydrolysis was also characteristic for the action of micrococcal nuclease 20, 21 DNA resistant to Serratia endonuclease, however, includes comparatively large fragments (up to 1000 nucleotides or more) while DNA resistant to micrococcal nuclease contained only subnucleosomal fragnents of no more than 140 nucleotides in length. There is a great similarity between the electrophoretic mobility of denatured DNA fragnents (Fig. 4b, c, d) and the fragments which were not denatured but were dissolved in 6 M urea (Fig. 4e). The effect of heat denaturation is primarily manifested in the formation of low molecular weight fragments which are multiples of 10 nucleotides (20-140 nucleotides). Thus it may be supposed.that short fragents were formed as a result of heat denaturation of longer DNA fragments containing a great number of single-stranded breaks. As is seen from Fig. 4e, the quantity of such short fragments in DNA samples dissolved in 6 M urea is negligible as compared to heat denatured DNA (the dame results have been also obtained for DNA after 30 and 60 min endonuclease digestion of nuclei). Consequently, under our experimental conditions, urea did not induce DNA denaturation. Thus, the similarity of electrophoretic mobilities of high molecular weight DNA fragments after heat denaturation and those of undenatured fragments dissolved in urea cannot be accounted for by the denaturing effect of the latter. At the same time, our earlier findings show that in the absence of urea, distinct bands of high molecular weight fragments were not detected during electrophoresis of native DNKA fmm chromatin digest 13. It is known that urea improves the electrophoretic resolution of single stranded molecules since it is able to break their intermolecular and intramolecular interactions 22. It may be concluded that chromatin DNA which is resistant to Serratia endonuclease contains, along with native double-stranded molecules, a great number of single-stranded fragments or double-stranded ones with single-stranded regions. In such a case, however, the discrete pattern of electrophoretic bands of undenatured DNA fragments in the presence of urea similar to those of denatured ones may arise only when electrophoretic 410
Nucleic Acids Research mobilities of corresponding native and denatured fragnents of DNA are coincident. In this connection, it is desirable to compare the mobilities of native and denatured DNA fragments under our electrophoretic conditions. The electrophoretic mobility of nucleic acids, which in the first approximation was found to be inversely proportional to the molecular weight, depends also on the partial specific volume and the shape of the molecule. Although the mobility of single-stranded and double-stranded molecules may differ, the possibilities of their separation are determined by the size of the molecule and electrophoretic conditions. We compared the electrophoretic mobilities of native and denatured fragments of phage 1929 DNA produced by restriction endonuclease EcoRI (Fig. 5). It is seen that the two fragments of higher molecular weight (5.106 and 3-106) show different electrophoretic mobilities in the native and denatured state. However the mobilities of the three fragments with molecular weights of 3o105, 5 105 and 1.2a106, both in the native and denatured state practically coincide. Returning to the data presented in the Fig. 4 it may be supposed that in the course of electrophoresis under completely native conditions the single-stranded fragments yield overlapping diffuse bands which conceal the bands of native
Figure 5. Comparison of electrophoretic mobilities of native and denatured DNA fragments of P29 phage.
't ¢*l
3 tQS
a
-
native,
b
-
denatured.
I 1.2106 -0.5310'
a
b 411
Nucleic Acids Research fragments. The bands of single-stranded fragments are narrowing in the presence of urea. Since the mobilities of these fragments coincide with that of native fragments, distinct bands come into view. Thie presence of single-stranded fragments in chromatin DNA resistant to Serratia endonuclease may be checked by treatment with S1-nuclease specific to single-stranded DNA. After treatment of resistant DNA with S1-nuclease, all discrete and distinct bands disappear from electrophoretographs leaving a rather diffusely stained backgroung (Fig. 6). This indicates the presence of single-stranded DNA fragments or single-stranded regions in resistant DNA. Chromatography on hydroxyapatite is another method employed to reveal the presence of single-stranded fragments in resistant DNA. The results of one of these experiments are given in Fig. 7. The DNA single-stranded fragments are eluted with 0.12 M PB in the first fraction. Their quantity varied from 20 to 35% of the total resistant DNA depending on the
Figure 6. Influence of S -nuclease on electrophoratic mobility of resistant DNA. a - control DNA, b - resistant chromatin DNA after S -nuclease treatment (3 units/pg 9NA, 45 min, 370). Resistant DNA was isolated from nuclei treated by Serratia endonuclease (1000 _ DNA, 48% acid soluble s ........units/mg
products).
a 412
b
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E
cB eat
co 'A
2
2
4
012MPB
6
8 10 12 14 16 18 20 FRACTION NUMBER .5M PB
Figure 7. Chromatography of resistant DNA on hydroxyapatite. Single-stranded fragments were eluted with 0.12 M PB, double-stranded fragments - with 0.5 M PB. Resistant DNA was isolated from nuclei treated by Serratia endonuclease up to 55% acid soluble products of DNA. conditions of chromatin digestion. It must be noted that incompletely-paired DNA fragments including single-stranded regions are not eluted with 0.12 M PB and leave the coluimn in the fraction of double-stranded molecules. That is why our estimation of the quantity of single-stranded fragments in resistant DNA may be lowered. A similar quantity of singlestranded fragments in DlJA of chromatin resistant to DNAase I was found earlier by chromatographic analysis on hydroxyapatite 23, 24 Electrophoresis of the chromatographic fraction of single-stranded DNA fragments shows that this fraction consists of comparatively short fragments (no more than 120-140 nucleotides) which are multiples of 10 nucleotides (Fig. 8). In the fraction of double-stranded molecules, along with high molecular weight fragments characteristic of resistant DNA, there occur short DNA fragments of exactly the same size as in the first fraction. It has been already mentioned that partially paired DINA molecules are retarded on hydroxyapatite as 413
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Figure 8. Electrophoresis
of
fragments isolated during chromatography of resistant DNA on hydroxyapatite. a - 0.12 M PB fraction; b - 0.5 M PB fraction. Both DNA
fractions
were
dialysed
against ele ctrophoretic buffer and denatured in the presence of 6 M urea on a boiling water bath. Numerals denote the length of DNA fragments (in nucleotides).
so~~~~~.s x
completely resistant stranded these
native
DNA
molecules.
consists
molecules.
molecules
composed of resistant
is
short
DNA may
Therefore
mainly
It
is
long
not
while
fragments. contain
the
in
with
S1
nuclease
and
inconceivable the
Along
that
complementary with
such
perfect duplexes Thus
the
chromatography
presence
of
single-stranded fragments
DNA which
is
resistant
to
be
may
assumed
incompletely paired
of
tely single-stranded molecules. ments
it
Serratia
and
and
doublestrand
strand
fragments short
results on
one
of
that
is the
comple-
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our
HAP-columns
unpaired
shLow
areas
endonuclease.
DISCUSSION
IEndonucleolytic 414
splitting of
nuclear DNA by
an
of
enzyme
Nucleic Acids Research from Serratia marcescens is different from the action of micrococcal nuclease. The latter breaks DNA primarily between chromatin subunits producing nucleosomes and their oligomers. After splitting of a short stretch of internucleosomal DNA associated with histone HI, this endonuclease digests DNA inside nucleosomes 2, 3, 20 21. Serratia endonuclease cleaves simultaneously internucleosomal and nucleosomal DNA at the early stages of chromatin digestion. IMoreover,this endonuclease produces DNA fragments which form during electrophoresis a long 10 nucleotide interval ladder (up to 300 nucleotides) 13. A similar 10 nucleotide periodicity of DNA fragmentation in c4romatin was recently demonstrated in experiments with DNAase 25 I and Exonuclease III from E.coli 26 The results obtained with Serratia endonuclease allow to propose that internucleosomal DNA as well as DNA in nucleosomes interact with histones in such a manner that DNA sites every 10 nucleotides in length are sensitive to the enzyme. Since this peculiar chromatin structure is retained after the removal of histone HI, the latter does not apparently determine the arrangement of endonuclease-sensitive sites on the internucleosomal DNA. Consequently, it may be suggested that internucleosomal DNA interacts with the octamer of nucleosomal histones, which provides it with an ordered configurzation and protection from endonuclease. Such interaction suggests a tight association between nucleosomes along the chromatin fiber, i.e. complete absence of free DNA connecting nucleosomes. Mandel and Fasman 27 came to a similar conclusion when comparing the spectra of circular dichroism and denaturation curves of mononucleosomes and their oligomers. The authors assumed that nucleosomes in solution make up a compact structure where each particle has many contacts forming a kind of superhelix. Internucleosomal DNA was examined in an electron microscope by a great number of authors 79 8, who found that its 0 28 . However, electron length varied greatly (up to 240 A) microscopic studies of internucleosomal DNA in complete chromatin and in chromatin devoid of histone HI made by Finch et al. 8 showed that in most of the cases no connecting DNA is seen between nucleosomes. These investigators regard inter415
Nucleic Acids Research nucleosomal DNA which is visible in the electron microscope as an artifact arising in the process of sample preparation. Noteworthy is the evidence of a tight interaction between nucleosomes obtained from cross-linking of histones in chromatin by bifunctional reagents. Thomas and Kornberg showed that dimethyl suberimidate treatment of chromatin induces formation of histone oligomers containing not only 8 but also 16 and 24 histone molecules 29. Consequently, the octamers of histones of neighbouring nucleosomes exist in such proximity that they can be crosslinked by a bifunctional reagent in which the distance between reactive groups in no more than 13.5 A 3°. This length corresponds to that of 4 nucleotide pairs in B-form DNA. It must also be pointed out that isolated nucleosomes show a great potential ability to interact. In the presence of Mg++ their interaction results in the formation of fibrillar structures 31In the process of prolonged digestion Serratia endonuclease, similarly to other nucleases, degrades about 50% of the nuclear DNA. However, DNA resistant to this endonuclease differs qualitatively from DNA resistant to micrococcal nuclease as it consists of rather large fragments (up to 1000 nucleotides and more). The presence of the long fragments of resistant DNA is accounted for by the resistance of nucleosomal and internucleosomal DNA to endonuclease attack. This provides additional evidence in favour of a compact arrangement of nucleosomes in chromatin. The degree of DNA resistance to endonucleases is likely to be determined by the state of chromatin during nuclease treatment. Burgoyne et al. 32 have demonstrated that nuclear DNA in rat liver is resistant to pancreatic D.NAase I, but it becomes more sensitive to enzyme in the presence of Ca++ ions. This is not the case in our experiments, since addition of calcium ions (1 ralvI) to the digestion medium had no detectable effect on the results. An electrophoretic analysis revealed another interesting peculiarity in the length distribution of DNA fragments in endonuclease-resistant chromatin. Besides the fragments which are multiples of 10 bases, one can trace the second order periodicity. The most intense bands are concentrated in the 416
Nucleic Acids Research regions of electrophoretographs which correspond to the lengths of mononucleosomal, dinucleosomal and trinucleosomal DNA of micrococcal digest (Fig. 4). Each of them is represented by at least two bands of similar intensity. It may be supposed that occurence of doublets in the second order periodicity reflects the presence of the different nucleosomal DNA repeats. Since discrete bands of DNA fragments of the resistant chromatin show up only (luring electrophoresis after denaturation, nucleolytic fragmentation of both DNA strands seems to proceed independentlyu Hence, accessible sites do not necessarily localize in both DNA strands opposite each other. Moreover, the presence of single-stranded regions and DNA fragments may indicate that Serratia endonuclease is able to split preferenticaly one of the DNA strands in the resistant chromatin due to the compact association between the nucleosomes along the chromatin thread. A ,part of the present work was reported at the 3rd SMEA Symposium on Biophysics of Nucleic Acids, Brno, Czechoslovakya, September 1977 33.
ACKNOWILEDGMENT The authors thank Dr. R.I.Salganik for his gift of the Serratia endonuclease, Dr. Bryan for providing us with the EcoRI digest of the P29 DNA and Dr. T.N.Osipova for performing the ultracentrifugation for us.
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