Printed in Sweden Copyright @ 1577 by Academic Press, Inc. All righfs of reproduction in any formreserved ISSN 00144827
Experimental
Cell Research 104 (1977) 287-292
QUANTITATIVE ASPECTS OF THE BINDING OF NUCLEAR NON-HISTONE PROTEINS TO DNA AS DETERMINED BY CENTRIFUGATION IN METRIZAMIDE GRADIENTS D. RICKWOOD’ and A. J. MacGILLIVRAY Beatson Institute for Cancer Research, Glasgow, G3 6UD, Scotland
SUMMARY Density-gradient centrifugation in metrizamide gradients has been used to study the binding of nuclear proteins to DNA. The unique advantage of this method is that the nucleoprotein complexes can be isolated free of non-complexed DNA and proteins. The chromatin non-histone proteins bound to native DNA in a non-random manner. The extent of binding was dependent on the ionic strength of the medium and was decreased in the presence of RNA.
We have previously shown that, although the nuclear proteins are extremely complex, only a few of the major protein species are tissue specific [l-3]. Therefore, there is a need to develop preparative procedures by which the proteins can be selected according to their functional properties, especially since the concentration of the important regulatory proteins is likely to be extremely low. One such functional property is their ability to bind to and form specific complexes with DNA. The original work of Alberts and co-workers [4] on the binding of prokaryotic proteins to DNA using DNA immobilised on cellulose has stimulated much work on the interaction of eukaryotic proteins, particularly nuclear proteins, with DNA. This original method has been modified by using ultraviolet light to bind the DNA to the matrix [5], while 1 Present address: Department of Biology, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ, UK.
other techniques include immobilising the DNA by trapping it in polyacrylamide [6] or agarose [7] gels or by covalent linkage [g-lo]. However, non-covalently bound DNA tends to leach out from the trapping medium [ll] and, in addition, the nuclear proteins tend to bind non-specifically to the matrix onto which the DNA is bound (D. Rickwood & A. J. MacGillivray , unpublished data). Although rat-zonal sedimentation methods have been used to study the interaction of proteins with DNA [12], such methods are extremely limited because of the insoluble nature of both the free proteins and the protein-DNA complexes and in low ionic strength they both aggregate and rapidly pellet in sucrose gradients. However, with the introduction of metrizamide, a non-ionic gradient medium, it is now possible to separate nucleoprotein complexes according to their relative amounts of protein and nucleic acid [13] Exp
Cell
Res 104(1977)
288
Rickwood and MacGillivray
PH 3
PI-l10
Fig. I. Two-dimensional gel electrophoresis of nuclear proteins. The Hap 2 protein fraction was prepared as described in the text, dialysed and separated in the first dimension by isoelectric focusing and in the second by electrophoresis in the presence of sodium dodecylsulphate (SDS) as described previously [22].
and moreover the nucleoprotein complexes can be isolated free of non-complexed nucleic acids and proteins [14, 151. Protein aggregation does not affect the fractionation of the complexes and, moreover, the unique advantage of this technique is that it is possible to study not only the proteins bound to the DNA but also the DNA sequences involved in the binding. The results presented here demonstrate the utility of this approach for studying the interaction of nuclear proteins with DNA. MATERIALS
AND METHODS
All reagents were of the purest grade available. AnalaR grade urea was prepared as an 8 M stock solution, filtered and deionised immediately prior to use by passage through a column of AGSOI-X8(D) mixed-bed resin @ho&d Laboratories Ltd., Bromlky: Kent, UK). All isotopically-labelled compounds were purchased from the Radiochemical Centre, Amersham, UK. Metrizamide was a gift from Nyegaard & Co. A/S, Oslo. Friend virus-transformed cells, clone M2 (M2 cells) a culture derived from mouse stem cells, were grown in culture as previously described [16]. The M2 cells were labelled with either [3H]tryptophan (1 &i/ml, Exp Cell Res 104 (1977)
5 Cilmmol) for the last generation or with [“C]TdR (0.01 &i/ml, 59 mCi/mmol) for the whole of the culture neriod (34 nenerations). Total mouse cytoplasmic RNA labelled with 32P’was a gift from Dr S. Humphries, and M2-cell nuclear RNA was a gift from Dr G: D. Birnie. Chromatin was prepared as nreviouslv described 131 by extracting purified MZcell-nuclei three times with 0.14 M NaCI, 5 mM EDTA, 50 mM Tris-HCI, pH 7.5. The chromatin was dissociated in 2 M NaCl, 5 M urea, 1 mM sodium phosphate, PH 6.8 and the chromatin proteins were ‘fractionated on hydroxyapatite [3]. More than 40% of the total chromatin non-histone proteins (fraction Hap 2) were eluted with 50 mM sodium phosphate, this fraction contained no DNA and only 1% (w/w) or less of RNA [3]. The complexity of this protein fraction was similar to those of other mouse cells [3] as shown in fig. 1. Spectrophotometric analysis of this protein fraction for the presence of cytochrome contamination showed that it contained no contaminating nuclear membrane material (D. Rickwood, W. Franke & A. J. MacGillivray, unpublished data). DNA, labelled with [l¶C]TdR, was prepared from M2 cells and E. co/i r171: when assaved with sinalestrand specific nuclease -[ 131 both preparations were greater than 95 % double-stranded. For binding studies, the Hap 2 proteins were dialysed into 2 M NaCI, 5 M urea, 1 mM EDTA, 1 mM dithioerythritol, 10 mM Tris-HCI, pH 7.5 and 200 pg of each protein fraction were mixed with 75 pg of DNA in the same medium and reconstituted by stepwise dialysis as described previously [14] except that all solutions contained 1OV M phenylmethylsulphonylfluoride to inhibit proteases. in order to obtain maximum resolution of the nucleoprotein complexes the samples were underlayered into the bottom of the gradient [15]. The procedure involved mixing the reconstituted mixtures of DNA and protein with stock metrizamide of the same ionic composition to uive a final concentration of 38 % (w/v) and 1.Oml 07 this sample was underlayered under 4.0 ml of 35 % (w/v) metrizamide in the same ionic medium. The gradients were then centrifuged in an MSE 10X10 ml aluminium rotor at 27000 rpm (45000 g) for 42 h at 2°C. The gradients were fractionated by upward displacement with Fluorochemical FC43 [18] (3M Chemical Co., London). The density of each fraction was determined from the refractive index after correction for the salt present [19]. The distribution of [Y]DNA and [3H]protein was determined by diluting samples of each fraction to 1 ml and adding 9 ml of Triton X-100 scintillator [20].
RESULTS Factors affecting the interaction of proteins with DNA Zonic strength. When the Hap 2 proteins were reconstituted with homologous DNA into dilute buffer in the absence of salt and
Binding of non-h&one proteins to DNA
289
creasing the ionic strength to 0.2 M NaCl reduced the amounts of DNA and protein in the complex by almost 50%, though the density of the complex remained unchanged (fig. 2C). When reconstitution mixtures were dialysed into 0.3 M NaCl no stable complexes were formed (fig. 20). The effects of other solutes in the gradient medium have also been studied. However, it was found that when proteins and DNA were banded separately in 4 M urea or 3 M guanidine hydrochloride the typical banding patterns observed in low concentrations of salt did not occur. Therefore such solutes cannot be used in these studies. Fig. 2. Abscissa: fraction no.; ordinate: (left) % total ilsCjyA (03); (right) % total [;H]protein
‘-Theeffect of ionic strength on the binding of Hap 2 proteins to MZ-cell DNA. The Hap 2 proteins were reconstituted to DNA from 2 M NaCl, 5 M urea into a solution containing either (A) no salt; (J3)0.14 M NaCI; (C) 0.2 M NaCi and (0) 0.3 M NaCl and separated on metrizamide gradients as described in the text. The gradients were fractionated and the density and distribution of DNA (0-O) and protein (W-U) in each fraction were determined [14]. The density of each complex is shown in each case.
separated on metrizamide gradients of the same ionic strength, almost all of protein was bound to the DNA to give a broad heterogeneous peak (fig. 2A), the density of which depends only on the input ratio of protein to DNA (D. Rickwood, unpublished data). These data are in agreement with previous results [15]. When the ionic strength of the medium was increased such that the complexes were dialysed into and separated on gradients containing 0.14 M NaCl then only about 15% of the DNA remained complexed with protein (fig. 2B). The complex banded at 1.21 g/cm3, corresponding to a protein to DNA ratio of 1.6 : 1.0 [14]. In-
5
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!
Fig. 3. Abscissa: fraction no.; ordinate: (left) % total [W]DNA (O-O); (right) % total [3ZP]RNA (O---O)
or [3H]protein (CD). Effect of RNA on the interaction of Hap 2 proteins with MZcell DNA. The Hap 2 proteins, rRNA and DNA were either (A) centrifuged separately or alternatively reconstitution mixtures were centrifuged in 0.14 M NaCl containing (B) DNA (75 rg) and Hap 2 proteins (200 pg); (C) DNA (75 pg) together with 200 pg each of the Hap 2 proteins and unlabelled HnRNA and (D) unlabelled DNA (75 pg) and 200 pg each of labelled Hap 2 proteins and rRNA. The distributions of radioactive DNA (O-O), RNA (0- - -0) and protein (LW) were determined in each case. Exp Cell Res 104 (1977)
290
Rickwood and MacGillivray
-
IS
Fig. 4. Abscissa: fraction no.; ordinate: (lefr) % total PClDNA (O-0): (right) % total PHIprotein - -~
+-A).
.
-
The specificity of the binding of the Hap 2 proteins to homologous and bacterial DNA. Hap 2 proteins were reconstituted to either homologous MZ-cell DNA (A, C) or E. coli DNA (B, D) and the complexes were fractionated in gradients containing either 0.14 M NaCl (A, B) or 0.2 M NaCl (C, D). The distribution of DNA (0-O) and nrotein (Cm) were determined as described in the text.
Specificity of binding For the techniques described in this paper to be of use it is important that the binding conditions are adjusted such that the interaction of the proteins with DNA is specific. Therefore we have studied the specificity of binding of the Hap 2 proteins to homologous and bacterial DNA. As shown in fig. 4A, B, in 0.14 M NaCl the Hap 2 proteins bound preferentially to homologous DNA to give a defined complex, while the complex with E. coli DNA reproducibly appeared as a smaller, more heterogeneous peak which banded at 1.18 g/ cm3. A peak of protein at 1.20 glcm3, not associated with a peak of DNA, was also present. On the other hand, although the Hap 2 proteins still remained bound to homologous DNA in 0.2 M NaCl (fig. 4C), at this ionic strength there was no peak of E. cofi DNA associated with protein, though a small peak of protein at 1.20 g/cm3 was still present (fig. 40). DISCUSSION
RNA. We have studied the effect of the presence of RNA on the interaction of the Hap 2 proteins with DNA in 0.14 M NaCl. The samples contained either DNA and Hap 2 proteins alone (fig. 3B) or DNA and Hap 2 proteins together with an equal amount of either nuclear RNA (fig. 3C) or 32P-labelled rRNA (fig. 30). It is clear that the presence of the RNA inhibited the formation of a stable complex between DNA and the Hap 2 proteins. Moreover, it can be seen from fig. 30, that in 0.14 M NaCl and in the presence of DNA the proteins appeared to interact preferentially with the RNA. Exp Cell Res 104 (W77)
Previous work has shown that, in contrast to the cytoplasmic proteins, the nuclear proteins have a high affinity for DNA 114, 151. The data in fig. 2 indicate that the pattern of binding of the Hap 2 proteins to DNA is dependent on the final ionic strength of the medium. In 10 mM buffer all of the DNA banded as a broad heterogeneous complex, frequently in a bimodal manner. Such a result can be explained in terms of a random binding or aggregation of proteins with the DNA. However, at low ionic strength free DNA can bind to nucleoprotein complexes [14] and this precludes precise analysis of the binding reaction. On the other hand, in 0.14 M and 0.2 M NaCl free protein and DNA were present, together with a nucleoprotein complex
Binding of non-h&one proteins to DNA which banded at 1.21 g/cm3. This shows that, in this case, the binding of these proteins to DNA was a non-random process and it can be interpreted in terms of a cooperative binding of the proteins to DNA. The density of the complex indicates that it had a protein to DNA ratio of 1.6: 1.0 [14] and its symmetrical shape suggests that it was relatively homogeneous. The protein to DNA ratio of the complex calculated from the specific activities of the DNA and protein was in close agreement with the value deduced from its buoyant density, hence the specific activity of the bound proteins was the same as the nonbound fraction. Thus it appears unlikely that the complex contained significant amounts of unlabelled protein, particularly histones which also complex to DNA [14]. The lack of histone contamination is shown also by the fact that the complex between the proteins and DNA was less stable to increases in the ionic strength as compared with histone-DNA complexes. The integrity of the complex was shown by the fact that, when it was rebanded in 0.14 M NaCl, less than IO % of the DNA was not complexed with protein. It is perhaps surprising that the complex formed contained such a relatively large amount of protein. However, some DNA fractions in nuclease-digested chromatin are associated with large amounts of nonhistone protein [21]. Another possible explanation is that only a fraction of these proteins are bound to the DNA itself, while the remainder attach themselves to these bound proteins, perhaps in a fairly nonspecific manner. The preferential binding of the Hap 2 proteins to RNA in the presence of native DNA is reminiscent of their high affinity for denatured DNA [15], though a direct comparison has yet to be made. In the case
291
of native DNA, in 0.14 M and 0.2 M NaCl the Hap 2 proteins bound preferentially to homologous DNA. The presence of a significant protein peak (indicated by the arrow) in fig. 4B, D which, calculation shows, was associated with insufficient DNA to justify it banding at 1.20 g/cm”, can be interpreted in terms of a weak interaction between the proteins and E. coli DNA, but that the complex formed dissociates during centrifugation. Therefore the Hap 2 proteins have a higher affinity for homologous native DNA suggesting that their interaction with DNA is sequence specific. CONCLUSIONS The techniques described in this paper can be used for both analytical and preparative work and offer a novel approach to the study of the interaction of proteins with nucleic acids. The data show that, in the absence of contaminating RNA, the nonhistone proteins can bind specifically to homologous DNA. However, further work is still necessary to analyse the species of proteins that bind to the DNA and the DNA sequences involved in the binding reaction. This work was supported by grants from the Cancer Research Campaign and the Medical Research Council. Metrizamide was a gift from Nyegaard & Co., Oslo. We wish to thank Drs G. D. Bimie and J. Paul for advice and encouragement; Mr A. C. Mackirdy and Mrs M. Freshney for providing cultures of M2 cells and Mr R. McFarlane for skilled technical assistance.
REFERENCES 1. MacGillivray, A J, Cameron, A, Krauze, R J, Rickwood, D 62 Paul J, Biochim biophys acta 277(1972)384. 2. Rickwood, D, Riches, P G & MacGillivray, A J, Biochim biophys acta 299 (1973) 162. 3. Rickwood, D & MacGillivray, A J, Eur j biochem 51 (1975) 593. 4. Alberts, B M, Amodio, F J, Jenkins, M, Gutman, E D & Ferris, F L, Cold Spring Harbor symp quant biol33 (1968) 289. 5. Litman, R M, J biol them 243 (1%8) 6222. Exp Cell Res 104 (1977)
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6. Cavalieri, L F & Carroll, E, Proc natl acad sci US 67 (1970) 807. 7. Schaller, H, Nusslein, C, Bonhoeffer, F J, Kurz, C & Nietzschmann, I, Eur j biochem 26 (1972) 474. 8. Poonian. M S. Schlabach. A J & Weissbach. A. Biochemistry 10 (1971) 424. 9. Rickwood. D. Biochim bionhvs _ _ acta 269 (1972) 47. 10. Allfrev. V G. Inoue. A, Karn. J. Johnson. E M & Vidali, G, Cold Spring Harbor symp quant biol38 (1973) 785. 11. Johnson, .I D, St. John, T & Bonner, J, Biochim biophys acta 378 (1975) 424. 12. Teng, C S, Teng, C T & Allfrey, V G, J biol them 246 (1971) 3597. 13. Bimie G D, Rickwood, D & Hell, A, Biochim biophys acta 331 (1973) 283. 14. Rickwood, D, Birnie, G D & MacGillivray, A J, Nucleic acids res 2 (1975) 723. 15. Rickwood, D, Biological separations in iodinated
Exp Cell Res 104 (1977)
density-gradient media (ed D Rickwood) pp. 2740. Information Retrieval Ltd, London (1976). 16. Paul, J & Hickey, I, Exp cell res 87 (1974) 20. 17. Hell. A. Birnie. G D. Slimmine. T K & Paul. J. Anal b&hem 48 (1972) 369 -’ 18. Rickwood, D, Hell, A & Birnie, G D, FEBS lett 33 (1973) 221. 19. Rickwood, D & Birnie. G D. FEBS lett 50 (1975) 102. 20. Rickwood, D & Klemperer, H G, Biochem j 123 (1971) 731. 21. Malcolm, S & Paul, J, Biological separations in iodinated density-gradient media (ed D Rickwood) pp. 41-49. Information Retrieval Ltd, London (1976). 22. MacGillivray, A J & Rickwood, D, Eur j biochem 41 (1974) 181. Received June 3, 1976 Accepted July 27, 1976
Experimental
Cell Research 104 (1977) 287-292
QUANTITATIVE ASPECTS OF THE BINDING OF NUCLEAR NON-HISTONE PROTEINS TO DNA AS DETERMINED BY CENTRIFUGATION IN METRIZAMIDE GRADIENTS D. RICKWOOD’ and A. J. MacGILLIVRAY Beatson Institute for Cancer Research, Glasgow, G3 6UD, Scotland
SUMMARY Density-gradient centrifugation in metrizamide gradients has been used to study the binding of nuclear proteins to DNA. The unique advantage of this method is that the nucleoprotein complexes can be isolated free of non-complexed DNA and proteins. The chromatin non-histone proteins bound to native DNA in a non-random manner. The extent of binding was dependent on the ionic strength of the medium and was decreased in the presence of RNA.
We have previously shown that, although the nuclear proteins are extremely complex, only a few of the major protein species are tissue specific [l-3]. Therefore, there is a need to develop preparative procedures by which the proteins can be selected according to their functional properties, especially since the concentration of the important regulatory proteins is likely to be extremely low. One such functional property is their ability to bind to and form specific complexes with DNA. The original work of Alberts and co-workers [4] on the binding of prokaryotic proteins to DNA using DNA immobilised on cellulose has stimulated much work on the interaction of eukaryotic proteins, particularly nuclear proteins, with DNA. This original method has been modified by using ultraviolet light to bind the DNA to the matrix [5], while 1 Present address: Department of Biology, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ, UK.
other techniques include immobilising the DNA by trapping it in polyacrylamide [6] or agarose [7] gels or by covalent linkage [g-lo]. However, non-covalently bound DNA tends to leach out from the trapping medium [ll] and, in addition, the nuclear proteins tend to bind non-specifically to the matrix onto which the DNA is bound (D. Rickwood & A. J. MacGillivray , unpublished data). Although rat-zonal sedimentation methods have been used to study the interaction of proteins with DNA [12], such methods are extremely limited because of the insoluble nature of both the free proteins and the protein-DNA complexes and in low ionic strength they both aggregate and rapidly pellet in sucrose gradients. However, with the introduction of metrizamide, a non-ionic gradient medium, it is now possible to separate nucleoprotein complexes according to their relative amounts of protein and nucleic acid [13] Exp
Cell
Res 104(1977)
288
Rickwood and MacGillivray
PH 3
PI-l10
Fig. I. Two-dimensional gel electrophoresis of nuclear proteins. The Hap 2 protein fraction was prepared as described in the text, dialysed and separated in the first dimension by isoelectric focusing and in the second by electrophoresis in the presence of sodium dodecylsulphate (SDS) as described previously [22].
and moreover the nucleoprotein complexes can be isolated free of non-complexed nucleic acids and proteins [14, 151. Protein aggregation does not affect the fractionation of the complexes and, moreover, the unique advantage of this technique is that it is possible to study not only the proteins bound to the DNA but also the DNA sequences involved in the binding. The results presented here demonstrate the utility of this approach for studying the interaction of nuclear proteins with DNA. MATERIALS
AND METHODS
All reagents were of the purest grade available. AnalaR grade urea was prepared as an 8 M stock solution, filtered and deionised immediately prior to use by passage through a column of AGSOI-X8(D) mixed-bed resin @ho&d Laboratories Ltd., Bromlky: Kent, UK). All isotopically-labelled compounds were purchased from the Radiochemical Centre, Amersham, UK. Metrizamide was a gift from Nyegaard & Co. A/S, Oslo. Friend virus-transformed cells, clone M2 (M2 cells) a culture derived from mouse stem cells, were grown in culture as previously described [16]. The M2 cells were labelled with either [3H]tryptophan (1 &i/ml, Exp Cell Res 104 (1977)
5 Cilmmol) for the last generation or with [“C]TdR (0.01 &i/ml, 59 mCi/mmol) for the whole of the culture neriod (34 nenerations). Total mouse cytoplasmic RNA labelled with 32P’was a gift from Dr S. Humphries, and M2-cell nuclear RNA was a gift from Dr G: D. Birnie. Chromatin was prepared as nreviouslv described 131 by extracting purified MZcell-nuclei three times with 0.14 M NaCI, 5 mM EDTA, 50 mM Tris-HCI, pH 7.5. The chromatin was dissociated in 2 M NaCl, 5 M urea, 1 mM sodium phosphate, PH 6.8 and the chromatin proteins were ‘fractionated on hydroxyapatite [3]. More than 40% of the total chromatin non-histone proteins (fraction Hap 2) were eluted with 50 mM sodium phosphate, this fraction contained no DNA and only 1% (w/w) or less of RNA [3]. The complexity of this protein fraction was similar to those of other mouse cells [3] as shown in fig. 1. Spectrophotometric analysis of this protein fraction for the presence of cytochrome contamination showed that it contained no contaminating nuclear membrane material (D. Rickwood, W. Franke & A. J. MacGillivray, unpublished data). DNA, labelled with [l¶C]TdR, was prepared from M2 cells and E. co/i r171: when assaved with sinalestrand specific nuclease -[ 131 both preparations were greater than 95 % double-stranded. For binding studies, the Hap 2 proteins were dialysed into 2 M NaCI, 5 M urea, 1 mM EDTA, 1 mM dithioerythritol, 10 mM Tris-HCI, pH 7.5 and 200 pg of each protein fraction were mixed with 75 pg of DNA in the same medium and reconstituted by stepwise dialysis as described previously [14] except that all solutions contained 1OV M phenylmethylsulphonylfluoride to inhibit proteases. in order to obtain maximum resolution of the nucleoprotein complexes the samples were underlayered into the bottom of the gradient [15]. The procedure involved mixing the reconstituted mixtures of DNA and protein with stock metrizamide of the same ionic composition to uive a final concentration of 38 % (w/v) and 1.Oml 07 this sample was underlayered under 4.0 ml of 35 % (w/v) metrizamide in the same ionic medium. The gradients were then centrifuged in an MSE 10X10 ml aluminium rotor at 27000 rpm (45000 g) for 42 h at 2°C. The gradients were fractionated by upward displacement with Fluorochemical FC43 [18] (3M Chemical Co., London). The density of each fraction was determined from the refractive index after correction for the salt present [19]. The distribution of [Y]DNA and [3H]protein was determined by diluting samples of each fraction to 1 ml and adding 9 ml of Triton X-100 scintillator [20].
RESULTS Factors affecting the interaction of proteins with DNA Zonic strength. When the Hap 2 proteins were reconstituted with homologous DNA into dilute buffer in the absence of salt and
Binding of non-h&one proteins to DNA
289
creasing the ionic strength to 0.2 M NaCl reduced the amounts of DNA and protein in the complex by almost 50%, though the density of the complex remained unchanged (fig. 2C). When reconstitution mixtures were dialysed into 0.3 M NaCl no stable complexes were formed (fig. 20). The effects of other solutes in the gradient medium have also been studied. However, it was found that when proteins and DNA were banded separately in 4 M urea or 3 M guanidine hydrochloride the typical banding patterns observed in low concentrations of salt did not occur. Therefore such solutes cannot be used in these studies. Fig. 2. Abscissa: fraction no.; ordinate: (left) % total ilsCjyA (03); (right) % total [;H]protein
‘-Theeffect of ionic strength on the binding of Hap 2 proteins to MZ-cell DNA. The Hap 2 proteins were reconstituted to DNA from 2 M NaCl, 5 M urea into a solution containing either (A) no salt; (J3)0.14 M NaCI; (C) 0.2 M NaCi and (0) 0.3 M NaCl and separated on metrizamide gradients as described in the text. The gradients were fractionated and the density and distribution of DNA (0-O) and protein (W-U) in each fraction were determined [14]. The density of each complex is shown in each case.
separated on metrizamide gradients of the same ionic strength, almost all of protein was bound to the DNA to give a broad heterogeneous peak (fig. 2A), the density of which depends only on the input ratio of protein to DNA (D. Rickwood, unpublished data). These data are in agreement with previous results [15]. When the ionic strength of the medium was increased such that the complexes were dialysed into and separated on gradients containing 0.14 M NaCl then only about 15% of the DNA remained complexed with protein (fig. 2B). The complex banded at 1.21 g/cm3, corresponding to a protein to DNA ratio of 1.6 : 1.0 [14]. In-
5
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!
Fig. 3. Abscissa: fraction no.; ordinate: (left) % total [W]DNA (O-O); (right) % total [3ZP]RNA (O---O)
or [3H]protein (CD). Effect of RNA on the interaction of Hap 2 proteins with MZcell DNA. The Hap 2 proteins, rRNA and DNA were either (A) centrifuged separately or alternatively reconstitution mixtures were centrifuged in 0.14 M NaCl containing (B) DNA (75 rg) and Hap 2 proteins (200 pg); (C) DNA (75 pg) together with 200 pg each of the Hap 2 proteins and unlabelled HnRNA and (D) unlabelled DNA (75 pg) and 200 pg each of labelled Hap 2 proteins and rRNA. The distributions of radioactive DNA (O-O), RNA (0- - -0) and protein (LW) were determined in each case. Exp Cell Res 104 (1977)
290
Rickwood and MacGillivray
-
IS
Fig. 4. Abscissa: fraction no.; ordinate: (lefr) % total PClDNA (O-0): (right) % total PHIprotein - -~
+-A).
.
-
The specificity of the binding of the Hap 2 proteins to homologous and bacterial DNA. Hap 2 proteins were reconstituted to either homologous MZ-cell DNA (A, C) or E. coli DNA (B, D) and the complexes were fractionated in gradients containing either 0.14 M NaCl (A, B) or 0.2 M NaCl (C, D). The distribution of DNA (0-O) and nrotein (Cm) were determined as described in the text.
Specificity of binding For the techniques described in this paper to be of use it is important that the binding conditions are adjusted such that the interaction of the proteins with DNA is specific. Therefore we have studied the specificity of binding of the Hap 2 proteins to homologous and bacterial DNA. As shown in fig. 4A, B, in 0.14 M NaCl the Hap 2 proteins bound preferentially to homologous DNA to give a defined complex, while the complex with E. coli DNA reproducibly appeared as a smaller, more heterogeneous peak which banded at 1.18 g/ cm3. A peak of protein at 1.20 glcm3, not associated with a peak of DNA, was also present. On the other hand, although the Hap 2 proteins still remained bound to homologous DNA in 0.2 M NaCl (fig. 4C), at this ionic strength there was no peak of E. cofi DNA associated with protein, though a small peak of protein at 1.20 g/cm3 was still present (fig. 40). DISCUSSION
RNA. We have studied the effect of the presence of RNA on the interaction of the Hap 2 proteins with DNA in 0.14 M NaCl. The samples contained either DNA and Hap 2 proteins alone (fig. 3B) or DNA and Hap 2 proteins together with an equal amount of either nuclear RNA (fig. 3C) or 32P-labelled rRNA (fig. 30). It is clear that the presence of the RNA inhibited the formation of a stable complex between DNA and the Hap 2 proteins. Moreover, it can be seen from fig. 30, that in 0.14 M NaCl and in the presence of DNA the proteins appeared to interact preferentially with the RNA. Exp Cell Res 104 (W77)
Previous work has shown that, in contrast to the cytoplasmic proteins, the nuclear proteins have a high affinity for DNA 114, 151. The data in fig. 2 indicate that the pattern of binding of the Hap 2 proteins to DNA is dependent on the final ionic strength of the medium. In 10 mM buffer all of the DNA banded as a broad heterogeneous complex, frequently in a bimodal manner. Such a result can be explained in terms of a random binding or aggregation of proteins with the DNA. However, at low ionic strength free DNA can bind to nucleoprotein complexes [14] and this precludes precise analysis of the binding reaction. On the other hand, in 0.14 M and 0.2 M NaCl free protein and DNA were present, together with a nucleoprotein complex
Binding of non-h&one proteins to DNA which banded at 1.21 g/cm3. This shows that, in this case, the binding of these proteins to DNA was a non-random process and it can be interpreted in terms of a cooperative binding of the proteins to DNA. The density of the complex indicates that it had a protein to DNA ratio of 1.6: 1.0 [14] and its symmetrical shape suggests that it was relatively homogeneous. The protein to DNA ratio of the complex calculated from the specific activities of the DNA and protein was in close agreement with the value deduced from its buoyant density, hence the specific activity of the bound proteins was the same as the nonbound fraction. Thus it appears unlikely that the complex contained significant amounts of unlabelled protein, particularly histones which also complex to DNA [14]. The lack of histone contamination is shown also by the fact that the complex between the proteins and DNA was less stable to increases in the ionic strength as compared with histone-DNA complexes. The integrity of the complex was shown by the fact that, when it was rebanded in 0.14 M NaCl, less than IO % of the DNA was not complexed with protein. It is perhaps surprising that the complex formed contained such a relatively large amount of protein. However, some DNA fractions in nuclease-digested chromatin are associated with large amounts of nonhistone protein [21]. Another possible explanation is that only a fraction of these proteins are bound to the DNA itself, while the remainder attach themselves to these bound proteins, perhaps in a fairly nonspecific manner. The preferential binding of the Hap 2 proteins to RNA in the presence of native DNA is reminiscent of their high affinity for denatured DNA [15], though a direct comparison has yet to be made. In the case
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of native DNA, in 0.14 M and 0.2 M NaCl the Hap 2 proteins bound preferentially to homologous DNA. The presence of a significant protein peak (indicated by the arrow) in fig. 4B, D which, calculation shows, was associated with insufficient DNA to justify it banding at 1.20 g/cm”, can be interpreted in terms of a weak interaction between the proteins and E. coli DNA, but that the complex formed dissociates during centrifugation. Therefore the Hap 2 proteins have a higher affinity for homologous native DNA suggesting that their interaction with DNA is sequence specific. CONCLUSIONS The techniques described in this paper can be used for both analytical and preparative work and offer a novel approach to the study of the interaction of proteins with nucleic acids. The data show that, in the absence of contaminating RNA, the nonhistone proteins can bind specifically to homologous DNA. However, further work is still necessary to analyse the species of proteins that bind to the DNA and the DNA sequences involved in the binding reaction. This work was supported by grants from the Cancer Research Campaign and the Medical Research Council. Metrizamide was a gift from Nyegaard & Co., Oslo. We wish to thank Drs G. D. Bimie and J. Paul for advice and encouragement; Mr A. C. Mackirdy and Mrs M. Freshney for providing cultures of M2 cells and Mr R. McFarlane for skilled technical assistance.
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