Volume 5 Number 77 July Volume 5 Number July 1978 1978

Nucleic Acids Acids Research Research Nucleic

Histone HI1- DNA interaction. On the mechanism of DNA strands crosslinking by histone HI

B.O.Glotov, L.G.Nikolaev and E.S.Severin Institute of Molecular Biology, Academy of Sciences of the USSR, Moscow, 117312, USSR

Received 3 April 1978

ABSTRACT

Crosslinking of DNA fibers by histone Hi or phosphorylated on Ser-37 histone Hi, and by the individual fragments of the Hi polypeptide chain was studied by the method of turbidimetry. The dependence of the turbidity of DNA-protein complexes on the ionic strength in solution suggests that the condensation of Hl.DNA complexes in vitro is apparently due to both specific histone-DDA interactIngstlth the contribution of hydrogen and/or hydrophobic bonds and the formation of polycationic "bridges" fastening the DNA fibers. The effectiveness of the condensation is postulated to be a function of a proportion between the two mechanisms which in turn can be controlled by slight changes in ionic surroundings. The sharp dependence of. shrinkage of Hl-DNA complexes on ionic strength at "physiological" salt concentrations could provide a mechanism to regulate density and consequently the total activity of chromatin in the cell nuclei. The phosphorylation of histone Hi on Ser-37 by a specific histone kinase does not noticeably affect the pattern of DNA crosslinking by the Hi. INTRODUCTION Recently Olins and Olins have suggested that the structure of solubilized chromatin consists of repeating "globular" DNP-particles ("1 -bodies") separated by short DNA-like threads. This conception has been substantiated mainly by the approach of nuclease treatment of chromatin or whole nuclei2'3, and the term "nucleosome" for the chromatin subunit was often used equally with " J -body". Kornberg and Thomas have shown that "inner" histones H2A, H2B, H3 and H4 play an important structural role forming an octamer protein core4'5 which is presumably in some or other way wound by DNA double strand to produce a nucleosome. The fifth major histone Hi varies from the other histones in It has long been thought that histone Hi many respects. C Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England

2587

Nucleic Acids Research weakly, if any, interacts with the "inner" histones, but readily forms almost pure homopolymers, as judged from histone crosslinking patterns obtained for chromatin with bifunctional reagents7'8. However, with one of such reagents, methyl-4-mercaptobutyrimidate hydrochloride, we have recently found that histone Hi can be almost quantitatively covalently crosslinked to histone H3 in intact nuclei thus forming not exactly homo- but rather heteropolymers with the H3 and also with the histone H4 molecules apparently attached to H3. One of the conclusions is that histone Hi probably occupies the "core particle" regions of DNA and somehow interacts with the inner histones (this laboratory, manuscript in preparation). Nevertheless, neutron diffraction studies show that both DNA and histone HI are on the outside of the nucleosomal protein core9' More or less independent interaction between Hi and DNA within a nucleosome is supported by the almost identical appearance of nuclear magnetic resonance (NER) spectra for histone Hi in chromatin and in artificial complexes with DNA11. External location of histone Hi and the character of its interaction with DNA made it reasonable to assume that this histone contributes the chromatin structure at the level of spatial arrangement of polynucleosomal chains. Electron microscopic investigations carried out by Littau et al.12 with the cell nuclei in situ, have demonstrated that the removal of lysine-rich histones (mostly H1) loosened the structure of condensed nuclear chromatin. The dense chromatin reappeared on reassociation with the lysine-rich histones. These, obtained more than 10 years ago, data appear to be the first evidence supporting a possible role of histone Hi in chromatin condensation. The uneven and changing in the course of a cell cycle density of different regions of chromatin13 is one of the most demonstrative features, reflecting a hierarchy of genome regions and the dynamics of intranuclear processes. Thus, transcription proceeds less actively in dense chromatin regions than in those of diffuse euchromatin14. The gene activation in polytene chromosomes is accompanied by the appearance of puffs representing the swollen segments of these giant multistranded structures15. The gene deficiency for the .

2588

Nucleic Acids Research lack of one X-chromosome in the cells of male Drosophila is apparently compensated by virtue of decreased density of the X-chromosome material in the interphase cell nuclei of males as compared to those of females.6 Finally, the formation of chromosomes during the mitosis is also a striking example of the functional change in chromatin density. It implies that changing chromatin density (potentially determined by the chromatin "superstructure") could appreciably affect the chromatin function at all the stages of cell development 16. If the chromatin "superstructure" did in effect depend on the histone Hi then it could be controlled by the directed chemical modification of this histone, with the Hi playing an important role of a mediator for regulating chromatin properties. It follows that investigations of histone Hi binding to DNA and concomitant phenomena, including effects of chemical modifications, seem to be an important step for understanding the dynamic aspects of the chromatin structure at the molecular level. In our previous works we investigated the electrostatic binding of histone Hi and its individual polypeptide fragments to DNA and the influence on this process of the phosphorylation ofjhistone Hi on Ser-376s17. Unlike these studies where attention has been drawn to immediate protein-DEA interaction, crosslinking of the complexes being a side event, the present investigation is devoted to the mechanism of DNA strands crosslinking by histone Hi and the influence of specific phosphorylation on this process. To assess the crosslinking in solutions of artificially prepared complexes of histone Hi and its fragments with DNA we used light-scattering properties of the solutions at a variety of the ionic strengths. The validity of the Hl.DNA complexes as a model of chromatin was discussed elsewhere6. The questions analysed in the present paper were as follows: 1) What is the molecular mechanism of DNA condensation by histone Hi? 2) What features of the HI-DiA complexes could be a basis for the regulation of chromatin condensation? 2589

Nucleic Acids Research 3) What is the effect of the specific Hi phosphorylation on the condensation of H1*DNA complexes? Some of the authors believe that the mechanism of chromatin precipitation is primarily an electrostatic phenomenon involving the neutralization of net negative charges on the nucleoprotein complex18'119. From this point of view the participation of histones in condensation comes essentially to the trivial effect of counterions. Other reports20-23 claim the occurence of protein crosslinks and their significance in the condensation of native and model DNP§, histones Hi (and sometimes H5) being most likely involved in this process. MATERIALS AND METHODS Histone Hi, phosphorylated on Ser-37 Hi, and linear fragmented DNA from calf thymus were prepared as described previously6l Highly purified DNA from E. coli was a gift from Prof. G.P. Georgiev. This DNA was fragmented similar to that from calf thymus. The individual fragments of the histone Hi polypeptide chain, comprising amino acid residues 1-72 (fragment N1), 73-213 (C1), 1-106 (N2), and 107-213 (C2) were generated by the treatment of the Hi molecules with N-bromosuccinimide or chymoThe fragments E2 and C2 were isolated by a modified procedure as follows. 50 mg of Hi was dissolved in 5 ml of 50 mM Tris-HCl buffer, pH 8.0, and 50,l of 1 mg/ml oL-chymotrypsin (Calbiochem) in the same buffer was added. After 90-sec digestion at 300 the reaction was stopped by the addition of 1 ml of 1 mg/ml PMSF solution in alcohol. The resulting mixture was allowed to stay for 30 min at room temperature and then directly applied to a 0.9x30 cm column of Bio-Rex 70 cation exchange resin, equilibrated with 50 mM Na-phosphate buffer, pH 7.0. The column was eluted with 200 ml of the linear gradient of 0-2 M NaCl in the same buffer, at a pumping rate of 10 ml/h. A typical elution profile is depicted in Fig. 1. The fractions corresponding to the N2 and C2 fragments were collected, desalted on a 3x40 cm Sephadex G-25 column, equilibrated with 10 ml HCl, lyophilized, redissolved in distilled water, and finally lyophilized once more. The procedure

trypsin17.

2590

Nucleic Acids Research

15

20 25 30 Fraction number

35

Fig. 1. Elution profile for histone Hi digested with d -chymotrypin and applied to a Bio-Rex 70 column. Optical density of the eluate at 230 and 280 nm as well as NaCl gradient concentration are plotted against the number of fraction. Total volume of each fraction is 4 ml. For further explanations see the text. allows to obtain e 10 mg of each fragment without any protein cross-contamination. If necessary, the input of Hi can be severalfold increased. Denatured DNA from calf thymus was prepared by heating a solution of fragmented DNA in low salt buffer for 10 min at 1000 and subsequent fast cooling the preparation in ice-cold water. As judged from the percentage of hyperchromicity, more than 60% of the bases turned to be unpaired after such treatment. Recombination of histone Hi and its fragments with DNA was achieved by a stepwise gradient dialysis of DNA-protein mixture in high salt6917. For histone Hi, its phosphoform and the C-terminal fragments the last step of dialysis was carried out against 5 mM Na-phosphate buffer, pH 7.0, 20 mM NaCl and 0.1 mE Na2EDTA. For the N-terminal fragments the final buffer mixture was the same except for sodium chloride was omitted. H1-DNA complexes to be studied at different pH were additionally dialyzed against deionized water, and then the required 2591

Nucleic Acids Research quantities of salt or concentrated (0.2 M) buffer were added dropwise while stirring. To remove occasional turbidity of the DNA-protein complexes the solutions were centrifuged for 10 min at 1500Oxg. Concentrations of DNA and proteins were determined from optical density at 258 nm, using E258 (mole nucleotides) = 6800, and by the method of Lowry et al./24/ respectively, as described previously 6 Electron microcopwy. H1-DNA complexes in solutions at a concentration of DNA between 5 and 10 pg/ml were fixed with 1% formaldehyde for 3 min at room temperature and then placed onto 1% Parlodion-coated grids. The excess liquid was removed from the grids with a filter paper. The samples were rotatory shadowed with Pt-Pd (3:1) at low angles. ElectrQn micrographs were obtained with a JEM 100B electron microscope (Jeol) at an initial magnification of 20,000 or 25,000. Measurements of light-scattering. To evaluate the light-scattering in the solutions and "gels" of DNA-protein complexes we measured optical density at 400 nm (A400) of the preparations placed in a 1-cm path length spectrophotometer cell at a temperature of 200 using a Model 25 spectrophotometer (Beckman). The value of A400 is proportional to the turbidity (at 400 nm) of a solution and may be regarded as a measure of light-scattering25. The dependence of A400 on ionic strength in solutions was measured by addition of small volumes of 5.0 M NaCl or 2.0 M MgCi2 to the initially transparent DNA-protein preparations. Salt concentrations were checked with a Zeiss refractometer. Solutions with the salt added were being gently stirred with a polyethylene stick for the time required to reach equilibrium (about 30-40 min in 0.1-0.3 M baCl). The equilibrium was assumed to be attained when after 5-min stirring a change in the value of A400 was less than 5x103 o.D. units.

RESULTS AND DISCUSSION The meaning2of changes in turbidity. It will be noted that at the concentrations used ( 100 pg/ml) the aqueous solutions of histone Hi, its fragments, or DNA separately do not practically scatter visible 2592

Nucleic Acids Research light over the wide range of ionic strengths (0-2 1 NaCl). All of the samples of DNA-protein complexes are also almost For an ensemble transparent in low salt (A400ev5x1033). of unordered and non-interacting fragments of DNA covered by the histone or smaller molecules the size of inhomogeneity (i.e. of a scattering center) should be within the range between 2-5 nm (approximate diameter of the linearized complexes) and 60 nm (taking into account the length and flexibility of DNA fragments). In any case this size is much smaller than the wavelength of probing light (400 nm) that meets the requirements of the Rayleigh's scattering law. The average distance between DNA fragments in solution is also about 50 nm. In low salt the fragments of DNA-protein complexes do not apparently interact with each other (see below) and hence the existing distribution of inhomogeneities in the medium fails to produce considerable light-scattering. The intensity of Raleigh's scattering is known to be proportional to the squared volume of the scattering particles or, in our case, to the sixth power of the size of inhomogeneity. The dependence of light-scattering on the other parameters of the system is incomparably less critical. Hence, a considerable increase in light-scattering of the systems under study should be first and foremost due to an increase in size of scattering centers. In the solutions or "gels" of DNA-protein complexes such an increase could be apparently produced by bringing heretofore distant DNA strands together. Although herewith the total number of scattering centers naturally diminishes, every new center is much more effective in light-scattering than the original ones. It should be emphasized that all the arguments brought forward are valid if only scattering centers do not exceed in size the wavelength of probing light (400 nm). Since fragments of naked DNA have no affinity for each other in aqueous solution, the stable binding of DNA strands in DNA protein complexes can be achieved only by virtue of direct protein participation. Therefore an increase in light-scattering in this kind of systems has to be interpreted in terms of protein-mediated crosslinks. Salt-de2endent condensation of Hl*DNA comp2lexes. 2593

Nucleic Acids Research On increasing NaCl concentration turbidity of HlDNA complexes in solution first sharply rose and reached a maximum at ~ 0.15 K Na+ (a "specific" peak) and then even more sharply 0.18 M Na . After this one could observe a dropped at plateau or an "unspecific" peak with a maximum at s 0.3 X Na+, and after all the solution turned almost transparent in 0.5 X Na+ (Pig. 2). A position of the "specific" peak falls within the range of the so called "physiological" ionic strengths. With deionized urea added (6 M) a "specific" peak completely disappeared, whereas a plateau or an "unspecific" peak remained almost unchanged (Fig. 2). The dependence of the light-scattering pattern on the concentration of H1-DNA complexes is shown in Fig. 3. Although the "specific" peak was lowered at decreased complex concentrations, it did not completely disappeared. The "specific" peak could in principle be an artifact due to the "end effects" i.e. to the particular nature of histone Hi

A400

02

0.4

0.6 M Na

Fig. 2. Turbidity (apparent absorbance at 400 nm) of the H1I)NA complexes as a function of NaCl concentration. 0 5 mM Na-phosphate buffer, pH 7.0, 0.1 mM Na2EDTA, 20 . - H1*DNA, CDNA 100 pg/ml, R§= 0.5 0 .0 A-A - HI-DNA in 6 M urea, CDNA= 10 )g/ml, R = 0.5 - H1-(denatured DNA), CDNAA 110 pg/ml, R = 0.5 2594

Nucleic Acids Research binding to the ends of DNA fragments. To check this possibility we tested complexes of histone Hi with DNA fragments more than 1000 base pairs in length. Provided that concentrations of DNA and histone/DNA ratios were the same, the lengthening of DNA fragments had no effect on light-scattering by Hi-DNA complexes except for a slight quantitative difference. In the electron micrograph of fixed samples of the H1-DNA complex at low ionic strength one can see unlinked DNA fragments covered by histone Hi molecules (Fig. 4). The frequency of superpositions of the fragments was rather low even at high concentrations of the complex. This is indicative of negligible if any interaction between different DNA strands bound to histone Hi in 0.025 1 Na+. The frequent end loops (or may be doughnuts?) in the HlDNA preparations in low salt point to intramolecular crosslinking interactions. A noteworthy feature of the loops is their diameter sometimes close to about 100-150 A. This means that simple binding of histone Hi to DNA permits the latter to be spontaneously bent similar to the situation in a nucleosome. In contrast, micrographs made at 0.15 M Na+ revealed a gel0

1.0

0-

0.1

M 0a 3 Nal v

Fig. 3.- Changes in turbidity (apparent absorbance at 400 nm) for various Hl-DNA concentrations. 0 5 mM Na-phosphate buffer, pH 7.0, 0.1 mM Na2 EDTA, 20. in DNA, R§= 0.5 160,g/ml i80 g/ml in DNA 40 pg/ml in DNA -

2595

Nucleic Acids Research

42

t-4 -*

4.Electron micrographs of the Hl*DNA complexes. Bufris the same as in the legend+to Fig. 2, R =0.5g A - low ionic strength, 0.025 M [IJa I B - moderate ionic strength, 0 150 M LNa] -like network which seemed to consist of crosslinked Hll DNA aggregates (Fig. 4). The macroappearance of such aggregates in a cuvette was like a uniform, sometimes slightly detached from the walls, cloud having apparently no tendency to

rapidly precipitate. An abrupt decrease of turbidity at ca. 0.18 M Na+ was evidently due to a cooperative transition of the crosslinked Hl*DiiA network to a dispersed system of large compact particles visible with naked eye. These particles contain a lot of potentially scattering material which does not interact with light on account of the shielding by surface layers. Besides the effect of screening, light-scattering by macroscopic 2596

Nucleic Acids Research

*'uu

1.0

4.0

0.8 3.0

0.6 2.0

~~~~~~~~~~~~~1.0

0.2

02

0

0

0(1

0.2

03

OA M NaCl

ig 5. Comparative dependence of the turbidity (lef5ordinate)

an exponent a (right ordinate) in the expression O.D. app const. (.AJa) on NaCl concentration for the H1-DNA complexes; A the wavelength of probing light. 5 mM Na-phosphate§buffer, pH 7.0, 0.1 mM Na2EDTA, 20 100 jg/ml,R = 0.5. CDNA= The values of a were determined from the logarithmic plots of the apparent a'Esorbances against A in the range from 400 to 750 nm. The values of a may vary from 4 for particles small relative to A (i.e. Rayleigh's law) to 0 for macroscopic particles.

an

,

particles is comparatively insensitive to their size. The conversion of scattering centers mainly into large macroscopic particles following the transition was supported by a weaker wavelength dependence of turbidity (Fig. 5). In either case, the pattern of DNA crosslinking by histone Hi was essentially altered beyond ca. 0.18 M NaCl under conditions employed. The effect of 6 M urea indicates the possible role of hydrogen and/or hydrophobic bonds in inducing aggregation and concomitant turbidity of the solutions of H1-DNA complexes at moderate ionic strengths. Although the above bonds could arise from the interaction between the globular of neighbouring histones Hi, as previously suggested by Bradbury et al. 23, there is.an evidence that this is not the case (see below). It is also possible that urea perturbs the structured parts of Hi or certain specific contacts between them and one or more

parts26

2597

Nucleic Acids Research DNA threads by destroying the relevant regions of the histone Hi molecule. There is still a less likely possibility that urea effects are a consequence of urea-induced variations in the structure of DNA. The second, "unspecific", peak was almost uneffected by urea and therefore could be due to entirely ionic interactions. This is consistent with the finding that the C-terminal fragments of the HI molecule, C, and C2 (see Materials), comprising a lot of lysine residues were capable of DNA strands crosslinking similar to histone Hi in the presence of urea (Fig. 6). As the C-terminal fragments do not self-associate in neutral aqueous solution17 owing to the heavy electric charges, the protein croselinks formed by the fragments between DNA strands should involve polyeationic "bridge" structures in which one and the same polypeptide chain would be bound to at least two different DNA helices. This is especially the case for the C2*DNA complexes where protein-protein interaction in crosslinks is extremely unlikely. Crosslinkin& "activity" of different fragments of histone Hl. In contrast to the C-terminal fragments of the Hi, none of the examined N-terminal ones (residues 1-72 and 1-106) was able to appreciably condense DNA under similar conditions (the same concentrations of the N1l2'DNA complexes at the protein/DNA weight ratios from 0.3 to 0.6). It is worth mentioning that up to 0.2 Ml NaCl both the El and particularly N2 fragments do bind to DNA and their dissociation completes at only ca. 0.5 M NaCl, slightly earlier then for the intact histone H117. Since the fragment N2, when bound to DNA, is likely to at least partly retain its hydrophobic and H-bond-forming potential, this finding seems to contradict the importance of histone-histone interactions in DNA crosslink-

ing 23

Mechanisms of DNIA

crosslinking by histone Hi. From the above data one may suggest that the molecular mechanism of DNA condensation by histone Hl in vitro is as follows. At low ionic strength (ca. 0-0.02) DNA fragments covered by the Hi molecules (H1*DNA complexes) do not inter2598

Nucleic Acids Research

0

0.2

0.4

0.6 MNaI

Fig. 6. Turbidity (apparent absorbance at 400 nm) of the fragmented DNA complexed with the C-terminal fragments of the Hi polypeptide chain. 5 ml Na-phosphate buffer, pH 7.0, 0.1 mM Na EDTA, 200 C- the fragment comprising residues C2, 107-213 - C1 DNA, 150 g/ml in DNA, R§= 0.3 - C2DNA, 150 pig/ml in DNA, R = 0.3

73-213,

act with each other. As the concentration of NaCl in solution increases to 0.05-0.15 M the Hi molecules begin to crosslink DNA strands and the crosslinks do at least partly depend on the exact secondary and/or tertiary structure of the Hi or DNA. At this stage direct specific contacts between DNA and the structured regions of the Hi molecules could be involved. It is corroborated by the effects of urea and denaturation of DNA. Histone-histone interactions do not seem to be of importance. At 0.2 M NaCl and higher concentrations ionic crosslinks predominate, with the histone Hi presumably forming polyeationic "bridges" between DNA strands. The "bridge" structure can be formed either by the whole Hi molecule "sticking" to different DNA strands by the opposite terminal parts or by the long and flexible C-terminal fragment alone (Fig. 7). More sophisticated alternatives can not be excluded. In high salt ( > 0.5 X NaCl) the Hl-DNA complexes are completely dissociated6. A rather sharp transition from the "specific" mechanism of crosslinking to the mechanism of polycationic "bridges" at ca. 0.18 M NaCl provides great alterations in the compactness 2599

Nucleic Acids Research

0.025 MNaCl

0.15 M

0.60 M Ox30M Fig. 7. Tentative mechanisms of DNA condensation by histone Hi. At lowionic strength (0.025) the H1-DNA complexes do not interact with each other. At moderate ionic strength (ca. 0.15) the crosslinking is in part due to "specific" hisT-ne-DNA interactions. At a higher ionic strength (0.3) the main element of crosslinks is the polycationic "bridge" structure. At high ionic strength (0.5-0.6) the complexes are dissociated.

of the H1DNA system due to a subtle change in salt concentration. This effect might in principle be used for chromatin density regulation in a cell nucleus. The light-scattering curves obtained with preparations of chromatin from calf thymus23 differ from those measured herein in that they do not reveal any special change in turbidity at 0.18 M NaCl. This difference is attributable to the kinetically hindered condensation of long and high molecular weight chromatin fibers. Indeed, even for the employed short DNA fragments associated with histone Hi it takes about 30-40 min to reach an equilibrium at moderate ionic strengths (see Materials and methods). Naturally, this period should be greatly lengthened on increasing the molecular weight of deoxynucleoprotein. However, in native nuclei the kinetic obstacle could be circumvented in virtue of the preexisting ordering of the chromatin structure.

2600

Nucleic Acids Research

A400 i 113

0.6.

020

0 Q2 04 0.6 MNe 0 (12 04 0p 0 3W4 M8 (12 0.16 MM9 Fig. 8. Effects of MIgCl and pH on HlD1A condensation.

A400, an apparent

absorgance

at 400 rm;

, an ionic strength;

CDNA= 150,g/ml, R§= 0.5, 200. &,

*

- MgC12 added, 5 mM Na-phosphate buffer, pH 7.0 - NaCl added, 25 mM Na-phosphate buffer, pH 7.9 - NaCl added, 25 mM Na-acetate buffer , pH 5.1

Effects of _H and divalent cations. As well as for chromatin", MgCl2 was more effective than NaCl in promoting H1*DNA condensation (Fig. 8). With this salt added light-scattering went up and down in the "specific" peak at substantially lower ionic strength but the final transparency of the solution was achieved at almost the same ionic strength 0.4-0.5. Thus the model system of choice reflects also the property of chromatin condensation to depend upon the nature of counterions. Lowering the pH of HlDNA solutions in the range 7.9 to 5.1 was accompanied by a progressive displacement of the scattering curves towards higher salt concentrations without changing their shape (Fig. 8). Since at low pH values the overall negative charge of DNA is somewhat decreased, it clearly shows that the protein moiety rather than the net charge on DNA dominates the reaction of DNA strands crosslinking by histone HI. 260i

Nucleic Acids Research

0 0

Q2

0.4

06 MNa+

Fig. 9. Turbidity (apparent absorbance at 400 nmx) of the fragment DNA complexed with the phosphorylated on Ser-37 histone Hi as a function of NaCl concentration. 5 mM Na-phosphate buffer, pH 7.0, 0.1 mM Na2EDTA, 20 CDOA= 160 ),g/ml, R = 0.6

S!2ificit_2Lf_the

model

a8ytem and the role of Ser-37

0222tEXholation . The described system of H1*DNA complexes was rather sensitive to the states of its components. For instance, the revealed transition at 0.18 M NaCl was not detected in the solutions of histone Hi complexed with partially denatured DNA (Fig. 2). Moreover, none of the Hi fragments examined could give the pattern of light-scattering with a "specific" peak (Fig. 6). In spite of such a specificity of the system with regard to histone Hi moiety, the phosphorylation of the Hi on Ser-37 resulted in only slight quantitative change of H1-DNA light-scattering (Fig. 9). The effect was independent of whether the histone has been phosphorylated directly in complexes with DNA or the prephosphorylated histone has been used. This evidences for only weak influence of the Ser-37 phosphorylation on DNA strands crosslinking by means of histone Hi in the model system. It follows that Ser-37 is likely not the phosphorylation site to be involved in 2602

Nucleic Acids Research chromosome condensation in vivo. Quite the same results have been obtained on substitution of E. coli DNA for calf thymus DNA.

Conclusions 1. The crosslinking of DNA strands by histone Hi in vitro can result from both the "specific" interactions between the Hi and DNA involving hydrogen and/or hydrophobic bonds, and formation of polycationic protein "bridges" fastening distant regions of DNA. 2. The effectiveness of the crosslinks depends on a proportion between the two mechanisms which in turn can be controlled by slight changes in the ionic strength or ion composition of the medium. 3. The phosphorylation of histone Hi on Ser-37 by a specific cANP-dependent histone kinase does not appreciably affect the crosslinking of DNA strands by histone Hi in the model system although disturbs the interaction of the histone globular part with DNA6 The phosphorylation of Ser-37 seems not to be involved in chromosome condensation. NOTE

After this work had been sent to the editor, we obtained which was very closely tied in with the subject of the present communication. The data27 concerning phosphorylation of Ser-37 parallel our own conclusions in the sense that this modification has little effect on the pattern of crosslinking of DNA complexed with histone Hi. As follows from the paper, the crosslinking is greatly influenced by the phosphorylation of sites located in the C-terminal part of the histone Hi molecule. Although it seems likely, a correct interpretation of changes in turbidity requires a simultaneous evaluation of the size of light-scattering particles. Differences in shape of turbidity curves in our paper and in the article27 should be ascribed to the fragmentation of DNA used in our experiments. a

paper27

ACKNOWLEDGEiENTS We are grateful to Prof. G.P. Georgiev for

a

sample of 2603

Nucleic Acids Research E. coli DNA and Dr V.A. Kadykov for performing the electron microscopy experiments. Our thanks are also due to Dr Sergey N. Kurochkin for preparing the N-bromosuccinimide fragments of histone Hi.

§ABBREVIATIONS

AND SYMBOLS

DNP, deoxyribonucleoprotein; PXSF, phenylmethanesulfonyl fluoride; protein-DNA, reconstituted complex of the protein with linear fragmented DNA; N1, N2, C1 and C2, fragments of the Hi polypeptide chain composed of amino acid residues 1-72, 1-106, 73-213 and 107-213, respectively; cAMP, cyclic adenosine-35.5'monophosphate; R, weight ratio protein/DNA. REFERENCES 1 Olins, A.L. and Olins, D.E. (1974) Science 183, 330-333

2 Hewish, D.R. and Burgoyne, L.A. (1973) Biochem. Biophys. Res. Commun. 52, 504-510 3 Noll, M. (1974) Nature 251, 249-251 4 Kornberg, R.D. and Thomas, J.O. (1974) Science 184, 865-868 5 Thomas, J.O. and Kornberg, R.D. (1975) Proc. Nat. Acad. Sci. USA 729 2626-2630 6 Glotov, B.O., Nikolaev, L.G., Kurochkin, S.N., Severin, E.S. (1977) Nucl. Acids Res. 4, 1065-1082 7 Chalkley, R. and Hunter, C. (1975) Proc. Nat. Acad. Sci. USA 72, 1304-1308 8 Bonner, W.M. and Pollard, H.B. (1975) Biochem. Biophys. Res. Commun. 64, 282-288 9 Bram, S., Butler-Browne, G., Bradbury, E.M., Baldwin, G., Reiss, C. and Ibel, K. (1974) Biochimie 56, 987-994 10 Baldwin, G.P., Boseley, P.G. and Bradbury, E.M. (1975) Nature 253, 245-249 11 Bradbury, E.M., Danby, S.E., Rattle, H.W.E. and Giancotti, V. (1975) Eur. J. Biochem. 57, 97-105 12 Littau, V.C., Allfrey, V.G., Frenster, J.H., Mirsky, A.E. (1965) Proc. Nat. Acad. Sci. USA 54, 1204-1212 13 Hess, 0. and Meyer, G.F. (1968) Adv. Genet. 14, 171-223 14 Littau, V.C., Allfrey, V.G., Frenster, G.H., Mirsky, A.E. (1964) Proc. Nat. Acad. Sci. USA 52, 93-100 15 Berendes, H.D. (1973) Int. Rev. Cytol. 35, 61-116 16 Khesin, R.B. and Leibovitch, B.A. (1976) Mol. Biol. (Russ.) 10, 1-34 17 Glotov, B.O., Nikolaev, L.G., Kurochkin, S.N., Trakht I.N. and Severin, E.S. (1978) Studia Biophysica 69, 145-15 18 Jensen, R.H. and Chalkley, R. (1968) Biochemistry 7,

4388-4395

19 Davies, K.E. and Walker I.O. (1974) Nucl. Acids Res. 1, 129-139 20 Sluyser, M. and Snellen-Jurgens, N.H. (1970) Biochim. Biophys. Acta 199, 490-499 21 Kozlov, Yu. I., Debabov, V.G. and Sladkova, I.A. (1971) Mol. Biol. (Russ.) 5, 467-471 2604

Nucleic Acids Research 22 Billett, M.A. and Barry, J.M. (1974) Eur. J. Biochem. 49,

477-484

23 Bradbury, E.M., Carpenter, B.G. and Rattle, H.W.E. (1973) Nature, 241, 123-126 24 Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 25 Olins, D.E., Olins, A.L. and von Hippel, P.H. (1967) J. Mol. Biol. 24, 157-176 26 Hartman, P.G., Chapman, G.E., Moss, T. and Bradbury, E.M. (1977) Eur. J. Biochem. 77, 45-51 27 Matthews, H.R. and Bradbury, E.M. (1978) Exp. Cell Res. 111, 343-351

2605