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Fluorometric assays in the study of nucleic acid-protein interactions
4NALYTICAL
BIOCHEMISTRY
90, 543-550 (1978)
Fluorometric Assays in the Study of Nucleic Acid-Protein Interactions I. The Use of Diaminobenzoic ANGEL
Institute
PESTARIA,
Acid as a Reagent of DNA
RICARDO CASTRO, Jo& AND ROBERTO MARCO
V. CASTELL,
de Enzimologia del CSIC, Facultad dr Medicina de la Universidad Autcinoma de Madrid, Madrid-34, Spain
Received November 27. 1977 The microfluorometric determination of DNA with diaminobenzoic acid in combination with a filter binding assay offers an easy and accurate procedure to study the interaction of proteins with any source of DNA. Using a highly polymerized commercial preparation of calf thymus DNA, the binding curve of histones or protamines changes from hyperbolic to increasingly sigmoidal depending on the length and temperature of incubation. The presence in the DNA preparation of small amounts of contaminating proteases, undetectable by conventional methods, is responsible for this change in the binding curve, since the presence of phenylmethylsulfonylfluoride in the reaction mixture or the removal of the proteases from the DNA produces only hyperbolic curves.
Methods for studying interactions between nucleic acids and proteins are important for an understanding of the structure of chromatin and the physicochemical basis for the control of gene expression, Of the many published procedures (I ,2), most rely upon the use of radioactively labeled molecules to increase sensitivity. For certain purposes it is technically more convenient to use unlabelled molecules and increase sensitivity by using a technique that relies upon fluorescent emission. Such a technique is also valuable when used in conjunction with radiochemical methods (3-5). We have applied diaminobenzoic acid, a DNA-specific reagent (6-8), to study the interaction between basic proteins and DNA, using filters to collect the DNA-protein complex (9,10). The method can be used in the microgram range. In a following paper (11) the application of fluorescamine to the detection of the protein behavior in the interaction is presented.
543
0003-269717810902.0543$02.OOiO Copyright 0 1978 by Academic Press. Inc All rights of reproduction in any form reserved.
544
PESTANA
MATERIALS
ET AL.
AND METHODS
Diaminobenzoic acid (DABA)’ obtained from Merck was prepared as the hydrochloric acid salt as described (7). DABA HCI was dissolved in double-distilled water (0.2 g/ml) and extracted once with 5 mg/ml of Norit A (6). Cellulose filters used in this study were mixed cellulose esters from Millipore (HAW P/O.45 pm pore size), nitrocellulose from Sartorius (SM11308, 0.15 pm pore size) and pretreated cellulose from Gelman (Metricel cy -8,0.20 pm pore size). The filters were cut in circles of 0.9 cm in diameter and pretreated with 0.5 M KOH as described (9). Phenylmethylsulfonylfluoride (PMSF) from Sigma was dissolved in dimethyl sulfoxide. The assay contained highly polymerized calf thymus DNA (Worthington, 5 pg) and protamine (salmon sperm from Calbiochem) or unfractionated histones (Sigma II A) in TME buffer (10 mM Tris-HCl, pH 7, 1 mM MgC&, and 0.1 mM ethylendiaminotetracetic acid) in the presence of 40 pg of bovine serum albumin and a final volume of 1 ml. Protamine or histone were added last and the solution was thoroughly vortexed in a cyclomixer and allowed to stand at room temperature for different periods of time before filtration under controlled vacuum. A manifold filtration unit from Hoepfner Scientific Instruments allowing the simultaneous handling of 10 samples, was adapted to the small filter size by inserting a cylindrical plexiglass piece into the steel wells. The hollow Plexiglas adaptors of 0.7 cm inner diameter have a 0.5-mm lip at the lower end, which keeps the filter in position and prevents fluid leakage. After adding the sample the filters were washed twice with 1 ml of TME buffer, followed by two washes with 66% cold ethanol (6). The filters were then placed in small cylindrical glass vials and allowed to dry overnight in a desiccator over concentrated sulfuric acid or for 30 min in an oven at 60°C. One hundred microliters of DABA reagent was added to each vial and the capped vials were incubated for 30 min at 60°C. After cooling at 20°C 2 ml of 1 N HCl was added and the solution was transferred to a 3-ml cuvette to measure the fluorescence in a Perkin-Elmer spectrofluorometer 204, with excitation wavelength at 280 nm and emission wavelength at 500 nm. When indicated, DNA was treated with NaCl as described (12). The DNA was recovered by precipitation with 2.5 vol of cold ethanol. The supematant was dialyzed against 50 vol of water to remove ethanol and NaCl. Proteolytic activity was determined with an extremely sensitive fluorescamine based method (11). The reaction mixture contained per milliliter: 25 Fg of protamine, 10 pmol of potassium phosphate buffer, pH 7, 6.5 pg ’ Abbreviations used: DABA, TME buffer, 10 mM Tris-HCI,
diaminobenzoic acid: PMSF. pH 7. 1 mM MgC&. 0.1 mM
phenylmethylsulfonyfluoride; EDTA.
DABA ASSAY OF DNA-PROTEIN TABLE EFFECT
OF KOH PRETREATMENT ON THE RESISTANCE RETENTION OF CELLULOSE MEMBRANES
membrane, in 0.5 M KOH (membrane). in 0.5 M KOH in 0.5 M KOH treatment
545
I
Fluorometric
Sartorius 45 min Millipore 45 min I5 min No KOH
INTERACTIONS
AND
DNA
reading
Buffer”
DNA”
Histone-DNA’
Handling
0.50
0.47
4.4
Good
0.57 0.43 0.45
0.53 0.40 0.46
4.6 4.6 4.8
Bad Bad Good
‘I Filters were washed with TME buffer and 66% ethanol. ” Filters were loaded with 5 pg of DNA and washed as above. c Filters were loaded with a mixture of 5 pg of DNA and 15 Kg of histone and washed as above.
of DNA or equivalent amounts (on volume basis) of the salt extract (see above). At the appropriate time, aliquots of 100 ~1 were transferred to small glass vials, diluted with 100 ,ul of 0.05 M borate buffer, pH 8.5 and allowed to react with 100 ~1 of fluorescamine (Fluka), 0.03% in acetone. After dilution with 2 ml of double-distilled water, the resulting fluorescence was read in a Perkin-Elmer spectrofluorometer at 375 and 475 nm for the excitation and emission wavelengths, respectively. DNA concentration was estimated spectrophometrically assuming that a solution of 1 mg/ml in water gives 24 absorbance units at 260 nm. Histone concentration was also monitored spectrophotometrically assuming that a solution of 1 mg/ml in water gives 3.5 absorbance units at 230 nm. Protamine solutions were prepared by direct weighing of the vacuum-dried product. RESULTS AND DISCUSSION
Fluorometric
Filter Assay of DNA Nucleoprotein
The microfluorometric assay of DNA with DABA reagent described here is a modification of the filter binding method developed by Cattolico and Gibbs (7) for the measurement of total cell DNA. The small amount of nonspecific binding of free DNA to cellulose membranes produced a low background fluorescence (Table 1). All of the brands of filters tested produced comparable backgrounds but Millipore filters were found to be the most convenient for routine use because they produced the highest filtration rates. The recommended pretreatment of the filters with KOH (9) was found to be destructive to some batches of Millipore membranes, and therefore.
PESTANA
546
ET AL.
7-
6-
I I
I 2
I 3
DNA
1 4
I 5
/ 6
(vg)
FIG. 1. Fluorometric estimation of DNA with DABA reagent. The standard curve for free DNA (0) was obtained by pipetting aliquots of a DNA solution directly onto glass vials containing filters. The corresponding standard curve for nucleoprotein complexes was determined by using different aliquots of a nucleoprotein solution (6.8 pg of DNA and 15 pg of protamine/ml). After filtering through Millipore filters (A). The reaction with DABA was carried out as described in Methods.
the requirement for such pretreatment was investigated. In Table 1 it is shown that untreated Millipore filters gave fluorescence backgrounds and unspecific DNA binding as low as the pretreated ones, while the consistency and handling of the filters was substantially improved. Fluorescence emission was a linear function of amount of DNA and both free DNA and DNA present in nucleoprotein complexes produced essentially identical calibration curves (Fig. I). Factors Affwring thr Mrasuremrnt by thr Filter Binding Assay
of DNA-Protein
Interac~tions
To illustrate the applicability of the assay, the interaction of protamines and histones with a highly polymerized calf thymus DNA was studied. The shape of the DNA retention curves was found dependent on the time, temperature of incubation and the ionic strength of the medium in which the interaction reaction takes place. Hyperbolic curves of DNA re-
DABA ASSAY OF DNA-PROTEIN
Histone
/
INTERACTIONS
DNA
( pg /pg
547
)
FIG. 2. Time and temperature dependence of the histone-DNA interaction. Interactions were allowed to proceed for 1 h (squares) and 20 h (circles), either at 0°C (open symbols) or at room temperature (solid symbols). Solid triangles correspond to values obtained with 5 min of incubation at room temperature which served as control.
tention were obtained with 5 min of incubation at room temperature or with 1 h of incubation at 0°C. Longer incubations resulted in a progressive sigmoidal transformation of the curves of DNA.retention, a transformation which was faster at room temperature (Fig. 2). After a 2-h incubation at room temperature, it is found that 0.15 M NaCl is the minimum salt concentration required for reproducible hyperbolic curves of DNA retention, lower concentrations of NaCl gave consistently sigmoidal shaped curves (Fig. 3). Nevertheless, data in Fig. 3 also show that hyperbolic shaped curves can also be obtained even at low ionic strength and room temperature when the interaction reaction takes place in the presence of the serine protease inhibitor PMSF at 1 mM concentration. The above set of conditions was also shown to produce similar variations in the case of the interaction of protamine and DNA (data not presented). The effects of PMSF, temperature, and length of incubation on the shapes of the DNA retention curves suggested the involvement of a protease. Although commercial brands of eukaryotic DNA appeared protease-free when assayed by normally used methods (12,13), proteolytic activity could be easily detected in the DNA (Fig. 4) with the fluorescamine based microfluorometric method particularly in the case of high
548
PESTANA
Hlstone
ET AL.
/
DNA
fug
/pg
1
FIG. 3. Influence of the composition of the incubation medium on the interaction of histone with DNA. Incubations were for 2 h at room temperature and the reaction mixture was supplemented with NaCl at a final molar concentration of 0.025 (O), 0.05 (A), 0.1 (0) and 0.15 (m) or with PMSF at a concentration of 2.5 mM (x __ X) in the absence of salt.
molecular weight DNA preparations (14). The involvement of this proteolytic activity present in DNA in the origination of the sigmoidal shape of the binding curve was confirmed using the same DNA preparation previously extracted with 2.5 M NaCl(12,13). The resulting salt-washed DNA was almost completely free of proteolytic activity which was recovered in the DNA-free salt extract (Fig. 4). In this case, the curve representing DNA retention in the cellulose filters was hyperbolic even after 2 h of incubation at room temperature and low salt (Fig. 5) (compare with the results obtained with untreated DNA shown in Fig. 2). It is clear, though, that additional factors, besides the variable amounts of protease contamination in the DNA, affect the shape of the binding curve. For instance, the salt effect described in Fig. 3 cannot be explained simply by a change in proteolytic activity since the DNA-bound protease was not inhibited by NaCl up to 1 M (data not shown). In summary, a convenient simple nonradioactive based method to follow the interaction between proteins and DNA is described in this paper. This method, as any other based on filter retention in the complex, measures the DNA interacting with proteins. In the following paper (1 l), complementary results for measuring the amount of protein interacting with DNA are presented. Protease contamination of commercial high molecular weight preparation of eukaryotic DNAs, undetectable by conventional methods, are shown here to affect significantly the shape of the binding curve. We
DABA ASSAY OF DNA-PROTEIN
INTERACTIONS
549
120
60
Time
(min)
FIG. 4. Proteases content of DNA and its removal with high salt washing. DNA was extracted with 2.5 M NaCI. Proteolytic activity was determined (see Methods) in the native DNA preparation (0) and the salt-washed DNA (0) at a final concentrations of 6.8 &ml. Proteolytic activity was also determined in the dialyzed salt extract (W).
Histone
/
DNA
( pg / pg )
FIG. 5. Interaction of histone with salt washed DNA. The procedure was as described in Methods. Salt-washed DNA. devoid of proteolytic activity, was used in these experiments. Interaction was for 5 min (0) and 2 h (A).
550
PESTANA
ET AL.
would like to emphasize the need for proper controls of the type presented here, in particular the direct assay of proteolytic activities in the DNA preparations, before attempting any functional interpretation based on the shape of the DNA-protein binding curves. ACKNOWLEDGMENTS The typing by F. de Luchi and the critical reading of the manuscripts by Drs. Cl. F. de Heredia and J. Avila are gratefully acknowledged. This work has been supported by grants from the J. March Foundation and Fondo National para el Desarrollo de la Investigation Cientitica. R.C. and J.V.C. arc fellows of the Plan de Formation de1 Personal Investigador.
REFERENCES 1. Parish, J. H. (1972) Principles and Practice of Experiments with Nucleic Acids, Wiley, New York. 2. Colowick, S. P., and Kaplan, N. 0. (1971) in Methods in Enzymology, Vols. 20 and 21, Academic Press, New York/London. 3. Kelly, R. B., Cozarelli, N. R., Deutscher, M. P., Lehman, I. R., and Kornberg, A. (1970) J. Biol. Chem. 245, 39. 4. Lillehaug, J. R., Kepple, R. K., and Kepple, K. (1976) Biochemistry 15, 1858-1865. 5. Rigby, P. W. J., Dieckmann, M., Rhodes, C., and Berg, P. (1977) J. Mol. Biol. 113, 237-251. 6. Kissane, J. M., and Robbins, E. J. (1958) J. Bio/. Chem. 233, 184- 188. 7. Cattohco, R. A., and Gibbs, S. P. (1975) Anal. B&hem. 69, 572-582. 8. Hinegardner. R. T. (1971) Anal. Biochem. 39, 197-201. 9. Lin, S. Y., and Riggs, A. D. (1972) J. Mol. Biol. 72, 671-690. 10. Lin, S. Y., Lin, D., and Riggs, A. D. (1976) Nucl. Acid Res. 3, 2183-2191. 11. Castell, J. V., Pestana, A., Castro, R., and Marco, R. (1978) Anul. Biochem. 90, 551-560. 12. Furlan, M., Jerico, M., and Suhar, A. (1968) Biochim. Biophys. Acfa 167, 154-160. 13. Ta Chong, M., Garrad, W. T., and Bonner, J. (1974) Biochemistry 13. 5128-5134. 14. Pestatia, A. (1978) Biochim. Biophys. Acta, in press.
BIOCHEMISTRY
90, 543-550 (1978)
Fluorometric Assays in the Study of Nucleic Acid-Protein Interactions I. The Use of Diaminobenzoic ANGEL
Institute
PESTARIA,
Acid as a Reagent of DNA
RICARDO CASTRO, Jo& AND ROBERTO MARCO
V. CASTELL,
de Enzimologia del CSIC, Facultad dr Medicina de la Universidad Autcinoma de Madrid, Madrid-34, Spain
Received November 27. 1977 The microfluorometric determination of DNA with diaminobenzoic acid in combination with a filter binding assay offers an easy and accurate procedure to study the interaction of proteins with any source of DNA. Using a highly polymerized commercial preparation of calf thymus DNA, the binding curve of histones or protamines changes from hyperbolic to increasingly sigmoidal depending on the length and temperature of incubation. The presence in the DNA preparation of small amounts of contaminating proteases, undetectable by conventional methods, is responsible for this change in the binding curve, since the presence of phenylmethylsulfonylfluoride in the reaction mixture or the removal of the proteases from the DNA produces only hyperbolic curves.
Methods for studying interactions between nucleic acids and proteins are important for an understanding of the structure of chromatin and the physicochemical basis for the control of gene expression, Of the many published procedures (I ,2), most rely upon the use of radioactively labeled molecules to increase sensitivity. For certain purposes it is technically more convenient to use unlabelled molecules and increase sensitivity by using a technique that relies upon fluorescent emission. Such a technique is also valuable when used in conjunction with radiochemical methods (3-5). We have applied diaminobenzoic acid, a DNA-specific reagent (6-8), to study the interaction between basic proteins and DNA, using filters to collect the DNA-protein complex (9,10). The method can be used in the microgram range. In a following paper (11) the application of fluorescamine to the detection of the protein behavior in the interaction is presented.
543
0003-269717810902.0543$02.OOiO Copyright 0 1978 by Academic Press. Inc All rights of reproduction in any form reserved.
544
PESTANA
MATERIALS
ET AL.
AND METHODS
Diaminobenzoic acid (DABA)’ obtained from Merck was prepared as the hydrochloric acid salt as described (7). DABA HCI was dissolved in double-distilled water (0.2 g/ml) and extracted once with 5 mg/ml of Norit A (6). Cellulose filters used in this study were mixed cellulose esters from Millipore (HAW P/O.45 pm pore size), nitrocellulose from Sartorius (SM11308, 0.15 pm pore size) and pretreated cellulose from Gelman (Metricel cy -8,0.20 pm pore size). The filters were cut in circles of 0.9 cm in diameter and pretreated with 0.5 M KOH as described (9). Phenylmethylsulfonylfluoride (PMSF) from Sigma was dissolved in dimethyl sulfoxide. The assay contained highly polymerized calf thymus DNA (Worthington, 5 pg) and protamine (salmon sperm from Calbiochem) or unfractionated histones (Sigma II A) in TME buffer (10 mM Tris-HCl, pH 7, 1 mM MgC&, and 0.1 mM ethylendiaminotetracetic acid) in the presence of 40 pg of bovine serum albumin and a final volume of 1 ml. Protamine or histone were added last and the solution was thoroughly vortexed in a cyclomixer and allowed to stand at room temperature for different periods of time before filtration under controlled vacuum. A manifold filtration unit from Hoepfner Scientific Instruments allowing the simultaneous handling of 10 samples, was adapted to the small filter size by inserting a cylindrical plexiglass piece into the steel wells. The hollow Plexiglas adaptors of 0.7 cm inner diameter have a 0.5-mm lip at the lower end, which keeps the filter in position and prevents fluid leakage. After adding the sample the filters were washed twice with 1 ml of TME buffer, followed by two washes with 66% cold ethanol (6). The filters were then placed in small cylindrical glass vials and allowed to dry overnight in a desiccator over concentrated sulfuric acid or for 30 min in an oven at 60°C. One hundred microliters of DABA reagent was added to each vial and the capped vials were incubated for 30 min at 60°C. After cooling at 20°C 2 ml of 1 N HCl was added and the solution was transferred to a 3-ml cuvette to measure the fluorescence in a Perkin-Elmer spectrofluorometer 204, with excitation wavelength at 280 nm and emission wavelength at 500 nm. When indicated, DNA was treated with NaCl as described (12). The DNA was recovered by precipitation with 2.5 vol of cold ethanol. The supematant was dialyzed against 50 vol of water to remove ethanol and NaCl. Proteolytic activity was determined with an extremely sensitive fluorescamine based method (11). The reaction mixture contained per milliliter: 25 Fg of protamine, 10 pmol of potassium phosphate buffer, pH 7, 6.5 pg ’ Abbreviations used: DABA, TME buffer, 10 mM Tris-HCI,
diaminobenzoic acid: PMSF. pH 7. 1 mM MgC&. 0.1 mM
phenylmethylsulfonyfluoride; EDTA.
DABA ASSAY OF DNA-PROTEIN TABLE EFFECT
OF KOH PRETREATMENT ON THE RESISTANCE RETENTION OF CELLULOSE MEMBRANES
membrane, in 0.5 M KOH (membrane). in 0.5 M KOH in 0.5 M KOH treatment
545
I
Fluorometric
Sartorius 45 min Millipore 45 min I5 min No KOH
INTERACTIONS
AND
DNA
reading
Buffer”
DNA”
Histone-DNA’
Handling
0.50
0.47
4.4
Good
0.57 0.43 0.45
0.53 0.40 0.46
4.6 4.6 4.8
Bad Bad Good
‘I Filters were washed with TME buffer and 66% ethanol. ” Filters were loaded with 5 pg of DNA and washed as above. c Filters were loaded with a mixture of 5 pg of DNA and 15 Kg of histone and washed as above.
of DNA or equivalent amounts (on volume basis) of the salt extract (see above). At the appropriate time, aliquots of 100 ~1 were transferred to small glass vials, diluted with 100 ,ul of 0.05 M borate buffer, pH 8.5 and allowed to react with 100 ~1 of fluorescamine (Fluka), 0.03% in acetone. After dilution with 2 ml of double-distilled water, the resulting fluorescence was read in a Perkin-Elmer spectrofluorometer at 375 and 475 nm for the excitation and emission wavelengths, respectively. DNA concentration was estimated spectrophometrically assuming that a solution of 1 mg/ml in water gives 24 absorbance units at 260 nm. Histone concentration was also monitored spectrophotometrically assuming that a solution of 1 mg/ml in water gives 3.5 absorbance units at 230 nm. Protamine solutions were prepared by direct weighing of the vacuum-dried product. RESULTS AND DISCUSSION
Fluorometric
Filter Assay of DNA Nucleoprotein
The microfluorometric assay of DNA with DABA reagent described here is a modification of the filter binding method developed by Cattolico and Gibbs (7) for the measurement of total cell DNA. The small amount of nonspecific binding of free DNA to cellulose membranes produced a low background fluorescence (Table 1). All of the brands of filters tested produced comparable backgrounds but Millipore filters were found to be the most convenient for routine use because they produced the highest filtration rates. The recommended pretreatment of the filters with KOH (9) was found to be destructive to some batches of Millipore membranes, and therefore.
PESTANA
546
ET AL.
7-
6-
I I
I 2
I 3
DNA
1 4
I 5
/ 6
(vg)
FIG. 1. Fluorometric estimation of DNA with DABA reagent. The standard curve for free DNA (0) was obtained by pipetting aliquots of a DNA solution directly onto glass vials containing filters. The corresponding standard curve for nucleoprotein complexes was determined by using different aliquots of a nucleoprotein solution (6.8 pg of DNA and 15 pg of protamine/ml). After filtering through Millipore filters (A). The reaction with DABA was carried out as described in Methods.
the requirement for such pretreatment was investigated. In Table 1 it is shown that untreated Millipore filters gave fluorescence backgrounds and unspecific DNA binding as low as the pretreated ones, while the consistency and handling of the filters was substantially improved. Fluorescence emission was a linear function of amount of DNA and both free DNA and DNA present in nucleoprotein complexes produced essentially identical calibration curves (Fig. I). Factors Affwring thr Mrasuremrnt by thr Filter Binding Assay
of DNA-Protein
Interac~tions
To illustrate the applicability of the assay, the interaction of protamines and histones with a highly polymerized calf thymus DNA was studied. The shape of the DNA retention curves was found dependent on the time, temperature of incubation and the ionic strength of the medium in which the interaction reaction takes place. Hyperbolic curves of DNA re-
DABA ASSAY OF DNA-PROTEIN
Histone
/
INTERACTIONS
DNA
( pg /pg
547
)
FIG. 2. Time and temperature dependence of the histone-DNA interaction. Interactions were allowed to proceed for 1 h (squares) and 20 h (circles), either at 0°C (open symbols) or at room temperature (solid symbols). Solid triangles correspond to values obtained with 5 min of incubation at room temperature which served as control.
tention were obtained with 5 min of incubation at room temperature or with 1 h of incubation at 0°C. Longer incubations resulted in a progressive sigmoidal transformation of the curves of DNA.retention, a transformation which was faster at room temperature (Fig. 2). After a 2-h incubation at room temperature, it is found that 0.15 M NaCl is the minimum salt concentration required for reproducible hyperbolic curves of DNA retention, lower concentrations of NaCl gave consistently sigmoidal shaped curves (Fig. 3). Nevertheless, data in Fig. 3 also show that hyperbolic shaped curves can also be obtained even at low ionic strength and room temperature when the interaction reaction takes place in the presence of the serine protease inhibitor PMSF at 1 mM concentration. The above set of conditions was also shown to produce similar variations in the case of the interaction of protamine and DNA (data not presented). The effects of PMSF, temperature, and length of incubation on the shapes of the DNA retention curves suggested the involvement of a protease. Although commercial brands of eukaryotic DNA appeared protease-free when assayed by normally used methods (12,13), proteolytic activity could be easily detected in the DNA (Fig. 4) with the fluorescamine based microfluorometric method particularly in the case of high
548
PESTANA
Hlstone
ET AL.
/
DNA
fug
/pg
1
FIG. 3. Influence of the composition of the incubation medium on the interaction of histone with DNA. Incubations were for 2 h at room temperature and the reaction mixture was supplemented with NaCl at a final molar concentration of 0.025 (O), 0.05 (A), 0.1 (0) and 0.15 (m) or with PMSF at a concentration of 2.5 mM (x __ X) in the absence of salt.
molecular weight DNA preparations (14). The involvement of this proteolytic activity present in DNA in the origination of the sigmoidal shape of the binding curve was confirmed using the same DNA preparation previously extracted with 2.5 M NaCl(12,13). The resulting salt-washed DNA was almost completely free of proteolytic activity which was recovered in the DNA-free salt extract (Fig. 4). In this case, the curve representing DNA retention in the cellulose filters was hyperbolic even after 2 h of incubation at room temperature and low salt (Fig. 5) (compare with the results obtained with untreated DNA shown in Fig. 2). It is clear, though, that additional factors, besides the variable amounts of protease contamination in the DNA, affect the shape of the binding curve. For instance, the salt effect described in Fig. 3 cannot be explained simply by a change in proteolytic activity since the DNA-bound protease was not inhibited by NaCl up to 1 M (data not shown). In summary, a convenient simple nonradioactive based method to follow the interaction between proteins and DNA is described in this paper. This method, as any other based on filter retention in the complex, measures the DNA interacting with proteins. In the following paper (1 l), complementary results for measuring the amount of protein interacting with DNA are presented. Protease contamination of commercial high molecular weight preparation of eukaryotic DNAs, undetectable by conventional methods, are shown here to affect significantly the shape of the binding curve. We
DABA ASSAY OF DNA-PROTEIN
INTERACTIONS
549
120
60
Time
(min)
FIG. 4. Proteases content of DNA and its removal with high salt washing. DNA was extracted with 2.5 M NaCI. Proteolytic activity was determined (see Methods) in the native DNA preparation (0) and the salt-washed DNA (0) at a final concentrations of 6.8 &ml. Proteolytic activity was also determined in the dialyzed salt extract (W).
Histone
/
DNA
( pg / pg )
FIG. 5. Interaction of histone with salt washed DNA. The procedure was as described in Methods. Salt-washed DNA. devoid of proteolytic activity, was used in these experiments. Interaction was for 5 min (0) and 2 h (A).
550
PESTANA
ET AL.
would like to emphasize the need for proper controls of the type presented here, in particular the direct assay of proteolytic activities in the DNA preparations, before attempting any functional interpretation based on the shape of the DNA-protein binding curves. ACKNOWLEDGMENTS The typing by F. de Luchi and the critical reading of the manuscripts by Drs. Cl. F. de Heredia and J. Avila are gratefully acknowledged. This work has been supported by grants from the J. March Foundation and Fondo National para el Desarrollo de la Investigation Cientitica. R.C. and J.V.C. arc fellows of the Plan de Formation de1 Personal Investigador.
REFERENCES 1. Parish, J. H. (1972) Principles and Practice of Experiments with Nucleic Acids, Wiley, New York. 2. Colowick, S. P., and Kaplan, N. 0. (1971) in Methods in Enzymology, Vols. 20 and 21, Academic Press, New York/London. 3. Kelly, R. B., Cozarelli, N. R., Deutscher, M. P., Lehman, I. R., and Kornberg, A. (1970) J. Biol. Chem. 245, 39. 4. Lillehaug, J. R., Kepple, R. K., and Kepple, K. (1976) Biochemistry 15, 1858-1865. 5. Rigby, P. W. J., Dieckmann, M., Rhodes, C., and Berg, P. (1977) J. Mol. Biol. 113, 237-251. 6. Kissane, J. M., and Robbins, E. J. (1958) J. Bio/. Chem. 233, 184- 188. 7. Cattohco, R. A., and Gibbs, S. P. (1975) Anal. B&hem. 69, 572-582. 8. Hinegardner. R. T. (1971) Anal. Biochem. 39, 197-201. 9. Lin, S. Y., and Riggs, A. D. (1972) J. Mol. Biol. 72, 671-690. 10. Lin, S. Y., Lin, D., and Riggs, A. D. (1976) Nucl. Acid Res. 3, 2183-2191. 11. Castell, J. V., Pestana, A., Castro, R., and Marco, R. (1978) Anul. Biochem. 90, 551-560. 12. Furlan, M., Jerico, M., and Suhar, A. (1968) Biochim. Biophys. Acfa 167, 154-160. 13. Ta Chong, M., Garrad, W. T., and Bonner, J. (1974) Biochemistry 13. 5128-5134. 14. Pestatia, A. (1978) Biochim. Biophys. Acta, in press.