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Model nucleoprotein complexes: Studies on the interaction of cationic homopolypeptides with DNA
J. Mol. Biol.
(1967) 24, 157-176
Model Nucleoprotein Complexes: Studies on the Interaction of Cationic Homopolypeptides with DNA DONALD E. OLINS, ADA L. OLWS AND PETER H. VON HIPPEL Department
of Biochemistry,
Dartmouth
Medical U.S.A.
School, Hanover,
New Hampshire
(Received 29 August 1966, and in revised form 24 October 1966) Complexes of native calf thymus DNA with the cationic polypeptides poly-Lornithine, poly-L-lysine, poly-n-arginine and poly-L-homoarginine, have been prepared and their solubility, stoichiometry, absorption spectra and thermal denaturation studied. Increasing the peptide cation/DNA phosphate ratio, up to electrostatic equivalence, yielded progressively more insoluble products and increased the turbidity of the “soluble” fraction. Certain spectral changes were observed which may be largely attributed to anomalous scattering in the absorbing region. Addition of the polypeptides to DNA resulted in a marked stabilization of the helix against thermal denaturation. At peptide cation/DKA phosphate ratios less than electrostatic equivalence, thermal denaturation monitored at 260 and 280 rnp revealed a biphasic transition profile: the first transition had a melting temperature similar to DNA under the same solvent conditions; the second melting temperature was characteristic for the type of polypeptide in the complex. Thermal denaturation monitored at 350 rnp (i.e., turbidity transitions) showed a monophasic profile at the higher melting temperature of the DNA-polypeptide complex. The different polypeptides stabilized DNA against melting to different extents. In order of decreasing degree of stabilization they are: poly-L-ornithine > poly-L-lysine > poly-n-arginine > poly-n-homoarginine. Analysis of the dispersion of hyperchromicity demonstrated that poly-L-ornithine and poly-L-lysine preferentially stabilize A-T rich regions, whereas poly-L-arginine and poly-I,homoarginine appear less discriminating. The soluble DNA-polypeptide complexes could be subfractionated by ultracentrifugation into a fraction which melted like “naked” DNA, and a fraction which melted at the higher temperature characteristic of the complex and showed a peptide cation/DNA phosphate ratio close to electrostatic equivalence. The experimental data imply that, under proper conditions of annealing, the basic polypeptides form definable molecular structures with DNA; the bin.ding reaction is stoichiometric and co-operative. Model-building suggests that the polypeptides could interact with either the large or small groove of DNA.
1. Introduction The DNA of eucaryotic nuclei is characteristically associated with a wide variety of basic proteins and polypeptides. This fact has suggested that these histones and protamines may have an important role in modulating and directing the genetic function of DNA in vivo, and has led many workers to carry out detailed chemical and physical investigations on the isolated proteins and the nucleoprotein complex. Fractionation and chemical study have shown that histones may contain appreciable 157
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HIPPEL
amounts of lysine and arginine, while protamines may contain as much as 70 mole ‘; ,, arginine and little or no lysine (Busch, 1965). X-Raycliffractionstuclies havesuggested that the nucleoprotamines of sperm heads are compact crystalline structures with the protamine moiety running helically in one of the grooves of the DNA (Feughelman et al., 1955; Wilkins, 1956; Zubay & Wilkins, 1962); whereas nucleohistones from interphase nuclei are considerably less crystalline with the histones in, as yet, undefined positions (Wilkins, 1956; Zubay & Wilkins, 1962; Wilkins, Zubay 85 Wilson, 1959; Luzzati & Nicolayeff, 1963; Richards, 1964). Physico-chemical studies on solutions of native nucleoproteins have been made more difficult by the propensity of these materials to aggregate. As a step toward studies of nucleoprotein in solution, we have been conducting investigations on complexes of DNA with a variety of basic synthetic homopolypeptides. Studies of the interaction of DNA with poly-L-ornithine, poly-L-lysine, poly-L-arginine and poly-L-homoarginine are reported here. It is demonstrated that all these polypeptides form specific complexes with DNA after appropriate annealing, and that all markedly stabilize DNA against thermal clenaturation. In addition, evidence is presented that varying the type of basic side chain affects the magnitude of the stabilization, as well as the differential stability of A-T versus G-C-rich regions of the DNA.
2. Materials and Methods (a) Reagents Highly polymerized calf thymus DNA was obtained from the Worthington Biochemical Corporation (lot 626), put into solution by gentle stirring overnight at room temperature, and used without further treatment. Previous lots of Worthington calf thymus DNA have been extensively tested, and have been found to be fully native and completely equivalent, by all criteria tested, to calf thymus DNA prepared in this laboratory by standard procedures (von Hippel & Felsenfeld, 1964). (mol. wt Poly-L-ornithine.HBr (mol. wt ~45,000; lot OR15), poly-L-lysme.HBr N 75,000, lot LYM), and poly-L-arginine. &H2S04 (mol. wt ~28,000, lot AR14) were
Wavelength (mp)
FIG. 1. Absorbance spectra of basic homopolypeptides. Solvent: 0.001 M-sodium buffer (pH 7.0). Concentrations of the polypeptides: 1 mg/ml. and poly-L-homoerginine (-A--A-). Curve A, spectra of poly~arginine (-.--.--) Curve B, spectra of poly-L-ornithine (-v-v-) and poly-L-lysine (-m-m-).
cacodylate
MODEL
NUCLEOPROTEIN
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169
synthesized by the Yeda Research and Development Co., Israel, and obtained from the New England Nuclear Corp. 0.Methylisourea.HzS04 was purchased from the Aldrich Chemical Co. Double glass-distilled water was used throughout. Methanol was of spectral grade. Polyarginine sulfate was converted to the hydrochloride and thus rendered watersoluble by mixing with an equimolar amount of BaCl, in aqueous solution. The resulting insoluble BaSO, was removed by centrifugation. Poly-r>-homoarginine.HCI was prepared by guanidination of poly-L-lysine with 0-methylisourea.HCl (Hettinger & Harbury, 1965). 0-methylisourea.HCl was prepared from the sulfate form by reaction with BaCl, as described above. Amino acid analysis of the product confirmed the conversion of > 99.5”: lysine to homoarginine. Ultraviolet absorption spectra were determined on the Cary 14 recording spectrophotometer for all the polypeptides used in these studies (Fig. 1). All showed negligible absorbance a,t wavelengths greater than 240 rnp. (b) Preparation
of the DNA-polypeptide
complexes
DNA dissolved at a concentration of 5 x 10m4 moles P/l. (A,,, N 3.25) in 0.001 M-sodium cacodylate buffer (pH 7.0) was made 4 M in NaCI. To a constant volume of DNA in concentrated salt solution varying amounts of polypeptide (dissolved in 0.001 M-cacodylate buffer at a peptide cation concentration of N 5 x 10m4 M) were added with vigorous mixing, followed by sufficient O*OOl M-cacodylate to make the final DNA concentration N 2.5 x 10m4 moles P/l. The resultant mixtures were at ratios of peptide cation/DNA phosphate of 0, 0.17, 0.33, 0.60, 0.67 and 1.00. We refer to these as peptide/DNA ratios. Annealing of the complexes was performed by dialysis at 0°C against 0.001 M-cacodylate buffer containing decreasing amounts of NaCI, a routine similar to that employed by Huang, Bonner & Murray (1964) for the reconstitution of nucleohistones. The dialysis steps were : 4 hr each against 0.4 M, 0.3 M, 0.15 M-NaCl; overnight against cacodylate buffer (pH 7.0) made 0.0025 M in sodium EDTA; followed by 2 to 3 daily changes of 0.001 M-cacodylate buffer (pH 7.0). Total volumes of the resultant dialysates were measured, and precipitates were removed by centrifugation for 15 min at about 1700 g. The supernatant fractions from this low-speed centrifugation, which were still noticeably opalescent at the higher peptide/DNA ratios but produced no further precipitate on standing, were saved and employed as the “soluble” DNA-polypeptide complexes.
(c) Chemical analysis DNA. concentrations were measured by a diphenylamine method (Giles & Myers, 1965). The presence of polypeptide wss found not to interfere with color development in this test. DNA samples of known concentration were run in each batch as internal standards. Concentrations of DNA were expressed as moles P/l., assuming a molar phosphate extinction coefficient for native calf thymus DNA at 260 ml* of 6500. Concentrations of the polypeptide stock solutions were measured using a Zeiss laboratory interferometer, assuming that the typical protein value of (dn/dc)550 = 1.87 x 10-l ml.,lg could be applied to all the polypeptides. The peptide content of “soluble” complexes was measured by a modification of the method of Lowry, Rosebrough, Farr & Randall (1951). Color yields were weak, especially for polyarginine and polyhomoarginine, and a small correction for DNA content was required. A,,,, for peptides at a concentration of 60 pg/ml. were as follows : polyornithine and polylysine, - 0.22 ; polyarginine and polyhomoarginine, - 0.06. (d) Melting
projibs
Melting experiments were performed in a Gilford model 2000 recording spectrophotometer equipped for the automatic measurement and recording of temperature. The platinum resistance thermometer in this instrument is located within the sample chamber controlled by thermostat and is directly below the cuvettes; control experiments using a thermistor located within the cuvettes themselves showed that the temperatures sensed by the resistance thermometer differed by less than 0.2’C from those within the cuvettes throughout the entire melting curve. Prior to melting, the samples were bubbled with helium gas (to remove other dissolved gases in favor of helium, which haa a positive temperature
I80
D. E. OLINS, lO8-
A. L. OLINS I
AND
P. H. VON
HIPPEI,
----7--T-T----T-.-
I
196 -
.J 50% (v/v)
t/
: .I
Methanol
Temperature
(‘C )
FIG. 2. Relative thermal expansion of 60% (v/v) methanol and of water. The data for 50% methanol comprise two sets of determinations. The data for water are compared with values calculated from the Handbook of Cherniatry am? Physics (A). IJ~o/v~~~= volume of solvent at tW/volume of solvent at 3O’C.
coefficient of solubility) and sealed with General Eleotric silicone rubber RTV-88 as described by Felsenfeld & Sandeen (1962). The samples were heated from 25 to 100°C at a constant rate of l”C/min, using a Northeast Scientific Co. automatic temperature programmer driving the thermoregulator of a model T-9 Tamson constant temperature bath. The raw melting data were corrected for thermal expansion of the particuIar solvent used. The relevant thermal expansion data for water and 60% (v/v) methanol-water were obtained by direct measurements of volume change in a sealed calibrated dilatometer (Fig. 2). Data correction and reduction to relative hyperchromicities (i.e. absorbance at temperature T/absorbance at 30°C) were accomplished using a simple program on the GE-235 computer. Melting temperatures of biphasic transitions (T, and T,‘) were calculated by extrapolating the first and second plateau regions through the linear approximations of each transition. The resultant intersections defined the initial and final hyperehromicities of a particular transition; the melting temperature in each case was the temperature at A hyperchromicity/2. For biphasic transitions, we designate the fist melting temperature by T, and the second by T,‘. As a measure of the co-operativity of the transition, we use a quantity (r, defbmd as the temperature interval over which the transition goes from 25% to 76% of oompletion.
3. Results (a) LYolubiZity, stoichionzetry and spectra
The addition of basic homopolypeptides to DNA resulted in complexes the solubility of which, after dialysis and low-speed centrifugation, varied with the ratios of the reactants (Table 1). Increasing the ratio of peptide to DNA yielded more insoluble products. At an input ratio of peptide cations to DNA phosphates of 1: 1, complexes of polyornithine and polylysine with DNA appeared to be less soluble than those of polyarginine and polyhomoarginine (Table 1). The amounts of polypeptide actually complexed with DNA in the soluble products
MODEL
NUCLEOPROTEIN
101
COMPLEXES
1
TABLE
DNA recovered as “soluble” complex at various peptide/DNA Polypeptide : Arginine Lysine
Ornithine
( + M - 1 groups
100% 107 102 79 68 36
100% 105 99 91 75 0
100% 93 100 100 71 2
o-00
0.17 0.33 0.50 0.67 1.00
input ratiosf
Homoarginine 100% 105 90 82 71 40
t DNA as measured by the diphenylamine method; estimated mean error in total determination, f 5 %. Th.e input DNA concentration in each sample was 5 x 10T4 moles P/l.
were also measured and compared with the input ratios. Polyornithine and polylysine yielded peptide/DNA ratios similar to their input ratios; polyarginine and polyhomoarginine showed somewhat lower ratios. Ultraviolet absorption spectra were obtained for all the “soluble” complexes at room temperature. The spectra for DNA-poly-L-lysine at different peptide/DNA ratios are typical and are shown in Fig. 3. Two properties of these spectra may be noted which correlate generally with increased peptide/DNA ratios : (1) a progressive increase in turbidity at wavelengths greater than 300 rnp (a region in which neither DNA nor the polypeptides alone show any absorbance) ; and (2) an apparent Cacodylate I?
i
1
I
I
I
I
I
I
I
I
I
Methanol-cacodylate ! I I
I
I
I
.
I
60 80 4
I
220 40 60
Wavelength
1
80 300 20 40 60 80 1
(mp)
FIG. 3. Absorbance spectra of DNA-poly-L-lysine complexes. The curves have been normalized to the same amounts of DNA, as determined by diphenylamine. Input peptide cation/DNA phosphate ratios: curve A, DNA alone; B, 0.17; C, 0.33; D, O-50; E, 0.67. Solvents: “cacodylate”, 0.001 M-cacodylate buffer (pH 7.0); “methanol-cacodylate”, 50% (v/v) methanol made 0.001 M with cacodylate buffer (pH 7.0). Insert tables: values of -%mnp/O~D~~~omp for each of the samples.
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VOX
HIPPEL
hypsochromic (towards the red) shift of the approximately 260 ml* DNA absorytiull peak. This shift was also accompanied by a progressive decrease in tht: app~~~~t 0.D.260(0.D.2Bo ratio. Similar spectral effects were seen for complexes containing ~~11 type of basic homopolypeptide. However, complexes containing poly-L-ornithine and poly-L-lysine showed greater turbidity and shift in the absorption peak than those of poly-L-arginine and poly-L-homoarginine. In every case similar spectra were also obtained with the “soluble” complexes in cacodylate buffer made 50% (v;‘v) in methanol (e.g. see Fig. 3). These spectra are of considerable potential interest, since a small conformational change in DNA structure which might occur as a consequence of DNA-polypeptide complex formation might then be reflected as a spectral change in this region. Before
0.01-300
20
’
40
’
’
60
’
’
60
’
”
400
Wavelength (mp) (b) FIQ. 4. (a) Light-scattering by DNA-polylysine complexes plotted a8 log (apparent absorbance) VWSUB log A. Peptide cation/DNA phosphate ratios: curve A, 0.17; B, 0.33; C, 0.50; D, 0.67. Solvent: 0.001 a6-cacodylate buffer (pH 7.0). The error limits shown around the points on the curve A correspond to a deviation of f 0.003 absorbance unit. (b) Difference spectra of DNA-poly-L-lysine complexes measured against DNA at the smne concentration. Peptide c&ion/DNA phosphate ratios. . curve A, 0.17; B, O-33; C, 0.50; D, 0.67. Solvent: 0.001 mn-cacodylate buffer (pH 7.0).
MODEL
NUCLEOPROTEIN
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163
looking for such effects, however, we must attempt to dissect out the contributions of light-scattering from the spectra. The apparent absorbance of the complexes at wavelengths greater than 300 rnp can easily be shown to be due to scattering, since in regions far from absorption peaks an apparent absorbance due to scattering should show a wavelength dependence of the following form : OJ’xpp = const. (A-“)
(1)
where a may range from 4 for particles small relative to h (i.e. Rayleigh’s law) to 0 for macroscopic particles. Figure 4(a) shows that at X > 300 rnp, the apparent absorbance does indeed obey a relationship of this form. The values of a for different mixtures of DNA-polylysine were determined from Fig. 4(a) and were as follows: the sample with peptide cation/DNA phosphate of 0.17 had a coefficient of N 3.8; with a ratio of 0.33, a = 2.9; ratio 0.50, a = 2.9; and ratio 0.67, a = 2.8. Two other polylysine-DNA complexes were analyzed : ratio O-17, a = 2.5 ; and ratio O-33, a = 2.8. Such coefficients in the scattering law are not unreasonable for particles of intermediate size, and the relative constancy of the coefficient for complexes of differing peptide/DNA ratio suggests that the aggregates formed by the procedures used here are all of comparable size. It is unfortunate that scattering in the vicinity of a real absorption peak cannot, be corrected by a simple linear extrapolation of the wavelength dependence found in the transparent regions into the absorbing region, since in absorbing regions one expects anomalous dispersion of the index of refraction and thus also of scattering. Such anomalous scattering in the peak region would lead (for an isolated absorption peak) to a positive contribution to the apparent absorbance on the long wavelength side of the peak, a cross-over point (i.e. no scattering at a point close to the center of the peak.) and a negative contribution to the short wavelength side. Such behavior would, of course, result in a hypsochromic shift of the peak and an apparent decrease in the o.D.~~,,/o.D.~~,, ratio. As pointed out above, these expectations are realized quantitatively in the raw absorbance data (e.g. see Fig. 3). These qualitative indications, coupled with the adherence to simple scattering behavior of the apparent absorbance in the long wavelength region of the spect’ra, suggest that the simplest interpretation is that at X > 230 rnp these spectra represent the sum of the normal DNA spectrum plus a scattering contribution which increases in magnitude as the peptide/DNA ratio is increased. To test this supposition, a set of difference spectra for a series of DNA-polylysine complexes is plotted in Fig. 4(b). These difference spectra were obtained from the absorbance data presented in Fig. 3(a) by subtracting the absorbance values for DNA from those of a complex containing the same concentration of DNA. They show qualitatively the appearance one would expect if the excess apparent absorbance (beyond that due to DNA itself) were all due to scattering. The strong positive scattering on the short wavelength sides of the 260 rnp absorption peaks, and the fact that a cross-over to negative apparent absorbance values is not actually observed at h < 260 rnp, may be attributed to the superimposition of positive scattering from the long-wavelength arm of the anomalous dispersion curve centered about the strong DNA absorption peak located below 200 mp. Very similar difference spectra have recently been obtained by Eisinger (personal communication, 1966), who compared the apparent absorbance of suspensions of T4 bacteriophage with the absorbance of osmot’ically shocked phage.
164
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AND
P. H.
VON
HIPPEL
DNA-Polyornithine t
I
I
30
40
50
Cacodylate I I
60
70
I
I
I I
80
90
100 30
Temperature
I
40
Methanol-cacodylate I I 1
50
60
70
I
I
80
90
(“C 1
DNA-Polylysine I
I
40
50
Cacodylate I I
I
I
80
90
/
1
Methanol I I
-cacodylate I 1
I
1.6I.5 -
30
60
70
100 30
Temperature
40
50
60
70
80
90
1 IO
(“c)
Fro. 5. (a) and (b) Thermal denaturatian profiles of DNA-polypeptide complexes monitored at 260 mp. at 260 mp. H,,, = relative hyperchromioity Peptide cation/DNA phosphate ratios: curve A, DNA alone; B, 0.17; C, 0.33; D, 0.50; E, 0.67; 0.001 rd-sodium cacodylate buffer (pH 7.0); “methanolF, 1.00. Solvent conditions: “cacodylate”, cacodylate”, 60% (v/v) methanol made O*OOl M-sodium cacodylate (pH 7.0).
MODEL
NUCLEOPROTEIN
DNA-
Polyarginine Methanol-cacodylate
Cacodylate
30
40
50
60
165
COMPLEXES
70
80
90 100 30 40 Temperature
Cacodylate
DNA-
50
60
70
80
90
100
80
90
IO0
(“C)
Polyhomoarginine Methanol-cacodylate
1
I
I
I
I
40
50
60
70
80
I
I
I.6 1.5
I.0
30
90 100 30 40 Temperature (b) Fm.
12
t7
(‘C)
50
60
70
16G
D. E. OLINS,
A. L. OLINS
AND
I’. H.
VOX
HIPPEL
DNA-Polyornithine
30
40
50
60
70
80
90
100 30
Temperature
40
50
60
70
80
90
I
I
100
(“C)
DNA-Polylysine I
I
Cacodylate I I
I
I
I
I
Methanol-cacodylate I / I
0 Temperature
(“C)
(a)
Fro. 6. (a) and (b) Thermal denaturation proties of DNA-polypeptide complexes monitored at hyperchromicity at 280 mp. Peptide cation/DNA phosphate ratios: curve A, DNA alone; B, 0.17; C, 0.33; D, 0.50; E, 0.67; F, 1.00. Solvent conditions: “cacodylate”, 0.001 M-sodium cacodylate buffer (pH 7.0); “methanolcacodylate”, 50% (v/v) methanol made 0.001 M-sodium cacodylate (pH 7.0).
280 mp. H,,, = relative
MODEL
NUCLEOPROTEIN
COMPLEXES
167
DNA - Polyarginine I
30
I
I
40
50
Cacodylote I 1
60
70
I
I
80
90
I
I( 30 Temperature
Methanol I
I
I
40 (“C )
50
60
-cacodylate I I
70
80
90
DNA - Polyhomoarginine Methanol
Cacodylate l/1111
30
40
50
60
70
80
90
IC
30
Temperature (b)
FIQ. 6
-cacodylate
’
’
1
I
40
50
60
70
(“C )
’
k-&-i
’
1
b30
168
D. E. OLINS,
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OLINS
AND
P. H.
Cacodylote
VON
Methonol I------
HIPPEL - cacodylate ’
’
’
70
60
90
I.0 30
40
50
60
70
80
90
100 30 Temperature
40
50
I.0 60
100
(“C)
FIQ. 7. Thermal denaturation profiles of [DNA-polypeptide complexes monitored at 350 rnp. Rel A,,, = relative apparent absorbance at 360 mp. All the complexes were mixed at peptide cation/DNA phosphate = 0.60. Samples: A, DNApoly-L-homoarginine; B, DNA-poly-L-arginine; C, DNA-poly-L-lysine; D, DNA-poly-Lornithine. The right-hand co-ordinate scales refer to DNA-poly-L-arginine; the left-hand scales apply to the other complexes. Solvent conditions: “cacodylate”, 0.001 M-sodium cacodylate 60% (v/v) methanol made O-001 M-sodium ‘cacodylate buffer (pH ‘7.0); “methanol-cacodylate”, (pH 7.0).
A more quantitative analysis of these spectra is probably pointless at this stage, due to the complexities introduced by overlapping absorption peaks, each with its associated anomalous dispersion curve. Thus, while on the basis of this analysis we cannot rule out the presence of small, but real, spectral changes accompanying complex formation, there is no indication from these data that such changes do exist. (b) Thermal melting stwEies Under the solvent conditions employed in these studies, the addition of basic polypeptides resulted in a marked stabilization of the DNA structure (Figs 5, 6, 7). As Figs 5 and 6 show, for ratios of peptide to DNA less than 1: 1, biphasic melting transitions are seen at both 260 and 280 rnp. The lower of the two thermal transitions shows approximately the same T, as native DNA alone under the same solvent conditions, while T,’ for the higher melting transition appears to be characteristic of the polypeptide-DNA complex. T, and T,’ values derived from these data are assembled in Table 2. Figures 5 and 6 show that the biphasic meltings are only incompletely visualized in cacodylate buffer. Especially in the higher melting polyL-ornithine and poly-L-lysine complexes, only a small ii-action of the second transition has taken place when the temperature has reached 100°C. In order to visualize the entire transition in all the complexes, we have also run melting curves in 50% methanol-cacodylate. Note that this solvent modification reduces the melting temperature of both the DNA and the complex transitions, though the melting temperatures of the latter are reduced more (see Table 2). The gross appearance of the melting transitions in 50% methanol, like the spectra (Fig. 3), are relatively unchanged in comparison to the companion transitions in aqueous
MODEL
NUCLEOPROTEIN
169
COMPLEXES
TABLE 2 Melting temperatures of DNA and DNA-polypeptide
DNA Cacodylatc 260 52.23 280 53.0 0.8 ATroll 350 Methanol--cacodylate 260 45-g 280 46.4 1.2 AT,,II 350 -
Ornithine T, Tm’
complexest
Polypeptide : Lysine Arginine T,’ Tm Tm’ Tm
Homoarginine T, T,’
49.3 50.1 0.9 -
>lOO >I00 >lOO
50.5 51.1 0.6 -
>lOO > 100 >lOO
54.6 55.6 1.0 -
93.3 93.8 0.5 95.4
53.9 54.7 0.8 -
87.4 88.2 0.7 90.3
46.0 46.7 0.7 -
87.1 87.0 -0.1 86.9
47.1 48.0 0.8 -
84.5 84.8 0.3 84.3
44-5 45.2 0.8 -
81.7 82.2 0.8 82.3
45.7 46.1 0.7 -
76.5 77.5 1.0 80.0
t The melting temperatures at 260 and 280 rnp represent averages of from 6 to 13 independent determinations. Melting temperatures at 350 rnp are based on a single series of determinations. $ This value agrees reasonably well with that calculated for calf thymus DNA under the same ionic strength conditions from equation (2) of Schildkraut & Lifson (1965). Employing their equation and assuming 42% GC, one obtains Tm,aB,, = 48~7°C. .$Herskovits et al. (1961) obtained T,.zeo = 45.5’C for calf thymus DNA in 51.5% volume methanol at similar ionic strength. II AT, is defined as (T,.28~-T,.~gd or (Tm’.280 -T,‘.260 ). The values of AT, were averaged separately.
solution, though we may note that in addition to the T, shifts, the methanol transitions Z&O show a slightly larger apparent hyperchromicity for all the complexes and for ‘DNA itself. Figure 7 shows melting transitions for the various polypeptide-DNA complexes at 350 mp, where only turbidity changes are observed. (Neither DNA nor the polypeptides heated singly showed any detectable turbidity or turbidity change at this wavelength.) Note that even complexes formed at peptide/DNA ratios which exhibit well-developed biphasic transitions at 260 and 280 rnp show only one transition at 350 mp, and that transition parallels the higher of the two transitions seen at 260 or 280 mp (Table 2). The above results suggest the following picture. (1) In the biphasic melting transitions, the first melting represents the helix --f coil transition of free DNA molecules (or portions of molecules), while the second meltings are characteristic of the particular DNA-polypeptide complex involved. The elevation in T,’ above the T, for free DNA in each case represents the degree to which that particular polypeptide has stabilized DNA against thermal denaturation. (2) As the ratio of amino (or guanidino) groups to DNA phosphates increases toward unity, the lower thermal transition disappears, suggesting a saturation of the free DNA by the added pepbide. (3) Herskovits, Singer & Geiduschek (1961) had previously shown that the T, of native DNA is lowered by added methanol. We have demonstrated that the thermal stability of DNA-polypeptide complexes may be reduced in a similar way by added methanol. (4) The 350 rnp melting results show that the turbidity which develops on EDNA-polypeptide complex formation is due to the complex alone, and is not affected by the melting of free DNA molecules (or portions of molecules) present in the same solution.
I;(1
I).
I’,.
or,1ss,
.A.
r,.
C)T,ISS
ASI) ‘L’ABLE
DNA-polylysine
(+)i(-) groups
mixed at diflerent
input
Tlt2
T,’
t1.
\‘OS
ti11’1’1~1,
3
ratios and melted itc. ,Ilethlrtrlol-ctr~c,dyltrt~ /
260 mp
,
I’.
280 m/J o
o’
’
’
Tm
T Ill ’
o
a’ ’
AT ,n
~‘l’ Ll,
--
f AT, $ This
is defined as (Tm,zso-TTm,zao); first transition was too small
AT,’
as T,,,‘~zso-TT,‘,,,,).
to be evaluated
accurately.
Additional insight into the properties of the DNA-polypeptide complexes may be obtained by further study of these data. First, we may note that the various polypeptides tested stabilize the DNA transition to varying extents : in order of decreasing stabilization the complexes may be listed DNA-poly-L-ornithine>DNA-poly-~lysine>DNA-poly-L-arginine>DNA-poly-L-homoarginine. This progression is seen in the transitions monitored at 260, 280 and 350 rnp, and in both aqueous solutions and 50% methanol (Figs 5,6, and 7 and Table 2). Second, inspection of Figs 5 and 6 suggests (and Table 3 shows in detail for one set of complexes) that T,’ for the various polypeptides is invariant with peptide/DNA ratio. That is, the higher melting transition in the biphasic melting curves occurs at a fixed melting temperature, regardless of the fraction of the total DNA which is complexed. This behavior is in marked contrast to that observed with small polycations, such as Mg2+ (Dove & Davidson, 1962), spermine, spermidine and diamines (Mahler, Mehrotra & Sharp, 1961; Mandel, 1962; Tabor, 1962; Mahler & Mehrotra, 1963), and tetra-L-lysine (this laboratory, work to be published). These additives shift the entire melting curve in a monophasic fashion; even at low cation/DNA ratios. This suggests that these smaller polycations are bound to DNA loosely and reversibly, whereas the polypeptides examined in this study appear to be bound essentially irreversibly. These differences will be considered further in a subsequent paper. Comparison of the various values of T,‘,,,, with Tmf,280 obtained on the same DNA-polypeptide complexes provides additional insight into the nature of the interaction. Felsenfeld & Sandeen (1962) have shown, on the basis of extensive studies of the dispersion of DNA hyperchromism, that the hyperchromic changes which accompany melting are weighted differently with respect to base composition at different wavelengths. At 260 rnp, the hyperchromic contribution of the A-T base pairs exceeds that of the G-C pairs, whereas at 280 rnp., the converse is true. Therefore a transition monitored at 260 rnp will reflect more heavily the melting behavior of the A-T pairs, and the G-C pairs are weighted more heavily in the transition measured at 280 mp. As a consequence, the difference between Tm,2B0 and T,,zso (AT,) for a given DNA provides a crude measure of the non-randomness of the distribution of base pairs along the helix (i.e., their distribution into relatively ,4-Trich or G-C-rich regions). Differences in AT, measured in this study for the free DNA transitions in both cacodylate and methanol-cacodylate solution were about
MODEL
NUCLEOPROTEIN
171
COMPLEXES
0.9 dtrO.Z”C. In contrast, we may note (Table 2) that there is a small but definite decrease in the comparable A!!!‘, values for some of the DNA-polypeptide complexes. This digerence is particularly striking in the case of the polyornithine and polylysine complexes, where AT, ’ is close to zero, suggesting that these polypeptides preferentially stabilize A-T-rich regionsof the native DNA helix against thermal denaturation (or alternatively, destabilize G-C-rich regions). The shapes of the melting curves obtained for the “soluble” DNA-polypeptide complexes were found to be very dependent on the method of preparation. Thus the complicated routine of annealing the complexes by dialysis against decreasing concentrations of salt, as described in Materials and Methods, was found to he necessary in order to obtain complexes with sharp melting transitions. Addition of the polypeptide to DNA at low ionic strength resulted in stabilization of the DS’A. but the second transition was considerably broader than that obtained with samples carried through the regimen of salt-concentration changes. The experiments with thtx annealed complexes demonstrated that these transitions were as sharp as or sharper TABLE
Cakulnfed
values of (T (co-operatiuity)
4
for the melting
of DNA
a.nd DNA-polypeptids
com,plexest
(mcL)
DNA
(‘acodylate 260 6.3 280 6.9 Methanol-cacodylate 260 6.2 280 6.7 t The value
Ornithine [I (I’
Polypeptide: Lysine 0 u’
Arginine D cl’
Homoarginine 0 0’
6.2 7.3
-
5.9 6.8
-
8.2 8.1
4.2 4.3
-5.9 6.4
3.4 4.7
7.0 7.6
3.0 2.6
7.4 8.0
2.8 2.6
5.5 5.9
4.4 4.7
6.0 6.6
5.6 5.4
of D represents
averages
of from
6 to 13 independent
measurements.
than those of DNA alone (see Table 4). In this context it should be noted that increased ionic strength or the presence of divalent cations (at a ratio of equivalents Mg”” /DNA phosphate greater than l*O), has also been observed to lead to sharper (more co-operative) thermal DNA transitions (Dove & Davidson, 1962). The requirement for annealing, plus the different type of melting curve obtained with non-annealed complexes, also suggests that complex formation is essentially irreversible under our final conditions: i.e. that there is little or no migration of the bound polypeptide from one DNA site to another. Furthermore, the fact that T, for the first transition in the DNA polypeptide complexes is essentially identical to that in solutions containing DNA alone, suggests that the added polypeptide is bound completely and thus does not affect the ionic environment of the uncomplexed portion of the DNA (see also footnotes, Table 2). Another problem of considerable interest in these studies was to determine whether the separately melting free DNA present in complexes containing less than 1:l rabies of peptide to DNA corresponds to DNA molecules completely free of polypeptide. while other DNA molecules are completely complexed (i.e. a non-random distribution ,.)f t.ht llolppeptide on DNA), or whether all the DNA molecules in the solution
172
D. E. OLINS,
A. L.
OLINS
AND
P. H.
VON
HIPPEL
0.6 260/280 I.21
0.5n
A
-.-.-.a -.-
220 40 60 80 300 20 40
60 80 400
Wavelength (rnp) FIG. 8. Absorbance spectra of a DNA-poly-I,-lysine complex and fractions obtained by ultracentrifugation. The curves have been normalized to the same amount of DNA, as determined by diphenylamine. Samples: A, complex mixed at peptide cation/DNA phosphate ratio of 0.50; B, supernatant fraction after centrifugation; C, resuspended pellet. Inset table: values of o.D.~~~,,,~/o.D.~~~,,,~ for each of the samples. Solvent : 0.001 M-sodium cacodylate buffer (pH 7.0).
DNA-
1.6
1
I
Polylysine fractions
I
I
1
I
I
I.5-
Temperature CC 1 FIQ. 9. Thermal denaturation profiles of a DNA-poly-L-lysine complex and fractions obtained by ultracentrifugation, monitored at 260 mp. Hzao = relative hyperchromicity at 260 mp. Samples: A, complex mixed at peptide cation/DNA phosphate ratio = 0.50; B, supernatant fraction after centrifugation; C, resuspended pellet. Solvent: 50% (v/v) methanol made O*OOl Ja-sodium cacodylate (pH 7.0).
MODEL
NUCLEOPROTEIN
COMPLEXES
173
contain regions which are complexed, alternating with regions which are essentially “naked” (a random distribution of polypeptide). To investigate this question we subjected several complexes with peptide/DNA ratios < 1 to extensive ultracentrifugation (15 minutes at 76,000 gin a no. 40 rotor, Spinco model L preparative ultracentrifuge at 5°C). After centrifugation, the complexes could be separated into a pellet and a supernatant fraction, and the pellet resuspended in fresh eacodylate buffer by gentle stirring. Both fractions were then analyzed for peptide and DNA content, ultraviolet spectra were obtained, and then both were made 50% (v/v) in methanol, and melted. Typical results, obtained with a DNA-polylysine complex mixed aft a peptide cation/DNA ratio of 0.5 are shown in Figs 8 and 9. Spectra of the unfractionated complex, plus those of the supernatant fraction and the (resuspended) pellet obtained by centrifugation, are shown in Fig. 8. The supernatant fraction yielded a spectrum (and ratio of 0.D.260 to O.D.zso) more similar t’o DNA than was the original complex (see Fig. 3 for comparison). The resuspended pellet showed even more turbidity and decrease in o.D.~~~/o.D.~~~ than observed for the unfractionatetl complex. Figure 9 demonstrates the results obtained on melting the fractions and the original DNA-polylysine complex. The supernatant fraction shows no higher melting fraction; the resuspended pellet is devoid of the lower melting fraction. Thus, from the point of view of melting behavior, the resuspended pellet represents a “purified” polypeptide-DNA complex. Similar spectral and melting data have been obtained for fractionated complexes of DNA-poly-L-ornithine, DNA-poly-L-arginine and DNA-polg-t-homoarginine. In a number of experiments, “purified” complexes showed a melting temperature several degrees lower than the T,’ of the corresponding unfractionated complex. Further studies are being directed at this point. The ratio of peptide cation to DNA phosphate was measured for a series of different DNA-peptide complexes mixed at an input ratio of peptide cation to DNA phosphate of 0.5. The “purified complex” of DNA-poly-L-ornithine had a cation/phosphate ratio of 0.9; “purified” DNA-poly-L-lysine, 1-l ; “purified” DNA-poly-L-arginine, 1.0; and “purified” DNA-poly-L-homoarginine, 1.3. Thus, these “purified” complexes contained approximately 1 peptide cation per DNA phosphate. Furthermore, they are not merely a small fraction of the total complex; between 50 and 70% of the total DKA was pelleted in these complexes. It thus appears that the annealed “soluble” comple.xes of DNA-polypeptide contain DNA molecules in two conditions : some arc complexed to approximate electrostatic equivalence with polypeptide, whereas the rest’ seem essentially free of polypeptide. A few more words might usefully be said about our use of the term “irreversible” in this paper. In our annealing system the complexes are initially formed (in high salt concentration) under “reversible” equilibrium conditions. The reaction between DNA and the polypeptides under these conditions is well defined, stoichiomet,ric and. co-operative. By progressive dialysis into a low-salt concentration milieu, WC then “freeze” the system into an “irreversible” state which, however, reflects the binding specificity characteristic of the high-salt concentration equilibrium. Presumably this state also represents rather closely the interaction specificity (and equilibrium) which applies at low salt concentration, but which cannot be attained by direct mixing of DNA and polypeptide at low ionic strength because of the kinetic barriers that greatly slow the rearrangement of such interacting polyelectrolytc
174
D.
E.
OLINS,
A.
L.
OLINS
AND
P.
H.
VOX
HIPPEL
systems under non-annealing conditions. In low salt concentration, using polypeptidcx of molecular weights comparable to those employed here, irreversibility is essentially total over the time scale involved in melting experiments (period of a few hours). However, over much longer times further rearrangements may take place. Using mixtures of radioactively-labeled DNA’s, Leng & Felsenfeld (1966) have demonstrated directly the short-time reversibility of formation of DNApolylysine complexes at high ionic strength. Tsuboi, Matsuo & Ts’o (1966), by similar methods, have shown that DNA-polylysine complex formation is essentially irreversible over substantial periods of time in low salt concentration.
4. Discussion The present studies indicate that under proper conditions of annealing, basic homopolypeptides interact with native DNA, resulting in the formation of definable molecular complexes. The following points emerge from an investigation of their properties. (1) DNA can form “soluble” complexes with basic polypeptides. (2) The DNA of such complexes exhibits a marked stabilization against thermal denaturation. (3) The extent of stabilization varies with the chemical nature of the basic polypeptide. (4) Complexes involving poly-r.-ornithine and poly-n-lysine appear to stabilize preferentially A-T-rich regions of DNA; whereas poly-L-arginine and poly-L-homoarginine stabilize more indiscriminately. (5) Under the low ionic strength conditions employed, complexing appears to be essentially irreversible. (6) The interaction of peptide with DNA shows elements of co-operativity, since, as assayed by thermal denaturation, the DNA of soluble complexes could be fractionated by ultracentrifugation into molecules melting like “naked” DNA, and molecules appearing to be totally complexed with peptide. (7) Complexing is approximately stoichiometric, since these “purified” complexes contained approximately 1 basic residue per DNA phosphate. (8) Complex formation produced several apparent spectral changes which, however, all appear to be attributable to the various manifestations of light-scattering from absorbing particles. Several of these points are in accord with results recently obtained by Tsuboi et al. (1966) in a study of the interaction of poly-r.-lysine with DNA and with poly (I + C). These authors also concluded that the binding reaction was stoichiometric and irreversible. Ohba (1966) has studied hyperchromic dispersion of DNA-poly-Llysine, DNA-protamine and DNA-histone complexes, and has concluded that polylysine and histones preferentially stabilize A-T-rich regions, whereas protamine does not appear to have this effect. The difference in degree of stabilization by the various basic polypeptides does not appear to be explainable simply on the basis of electrostatic neutralization. Examination of molecular models of the polypeptides reveals a correlation between extended side-chain length and complex stability: i.e., the shorter the side chain, the greater the stabilization, as manifested by a progressive increase in 27,‘. This differential stabilization may be a consequence of steric limitations, of differing hydrophobicity of the various side-chains, or different over-all charge densities. Earlier studies by Spitnik, Lipshitz & Chargaff (1955) have suggested that poly-nlysine interacts preferentially with A-T-rich regions of native DNA. In the present. studies, poly-L-ornithine and poly-L-lysine, in particular, brought about a more simultaneous melting of A-T- and G-C-rich regions than is observed in uncomplexect
MODEL
NUCLEOPROTEIN
COMPLEXES
I75
DNA. In view of the earlier data, it is more reasonable to assume that these polypeptides preferentially stabilize A-T-rich regions than to suggest that the observed melting effects result from a labilization of G-C-rich regions. These conclusions are also in accord with the results of some direct solubility studies on DNA-poly-L-lysine complexes recently carried out by Leng & Felsenfeld (1966). It is also pertinent to point out here that recent studies by Johns & Butler (1964) have demonstrated that, “lysine-rich” histones appear to be preferentially associated with A-T-rich regions of DNA in native nucleohistones. X-Ray diffraction studies in nucleoprotamines (Feughelman et al., 1955; Wilkins, 1956) and on a DNA-polylysine complex (Wilkins, 1956) have suggested that the peptide moiety winds around DNA in one of the grooves. Wilkins and co-workers (Feughelman et al., 1955; Wilkins, 1956) appear to favor binding in the narrow groove. Luzzatti (1963), however, has challenged their interpretations of the X-ra) data.. Tsuboi et al. (1966) present a model of polylysine winding through the large groove. On the basis of a careful examination of space-filling models, we have not beert able to eliminate on steric grounds either groove as the site for binding. A direct verification of the binding region is clearly required. Present studies in our laboratory are focused on determining, by chemical means, if basic pept,ides, protamines and histones interact with a particular groove of the DNA molecule. Thix investigation was supported in part by U.S. Public Health Service research grant AM-03412 (to P. H. v. H.) and National Science Foundation research grant GB-2060 (to S. I.); also by a Post-Doctoral Research Fellowship (F-123) from the Helen Hay Whitney Foundation (to D. E. 0.) and a Career Development Award (GM-K3-5479) from tbe U.S. Public Health Service (P. H. v. H.) We also gratefully acknowledge the skilled experimental assistance of Mrs Judith Gilbert in these studies. Two of us (D. E. 0. and A. 1,. 0.) are very grateful to Dr Shinya Ino& for providing partial support for our research activities during the time this work was in progress, as well as for many stimulating discussions and much good advice. REFERENCES Busch, H. (1965). H&ones and Other Nuclear Proteins, p. 28. New York: -4cademic Press. Dove, W. F. & Davidson, N. (1962). J. Mol. Biol. 5, 467. Felsenfeld, G. B Sandeen, G. (1962). J. Mol. BioZ. 5, 587. Feughelman, M., Langridge, R., Seeds, Vc’. E., Stokes, A. R., Wilson, H. R., Hooper, C. W., Wilkins, M. H. F., Barclay, R. D. & Hamilt’on, L. D. (1955). Nuture. 175, 834. Gile:s, K. W. dz Myers, A. (1965). Nature, 206, 93. Herskovits, T. T., Singer, S. J. & Geiduschek, E. P. (1961). Arch. Biochem. Biophys. 94, 99. Hetringer, T. P. & Harbury, H. A. (1965). Biochemistry, 4, 2585. Hua.ng, R. C. C., Bonner, J. & Murray, K. (1964). J. Mol. BioZ. 8, 54. Johns, 15. W., & Butler, J. A. V. (1964). Nature, 204, 853. Leny, M. & Felsenfeld, G. (1966). Proc. Nat. Acud. Sci., Wash. 56, 1325. Lowry, 0. H., Rosebrough, N. J., Farr, A. G. 8E Randall, R. J. (1951). J. BioZ. Glum. 193,265 Luzzati., V. (1963). J. Mol. Biol. 7, 758. Luzzati., V. & Nicolai’eff, A. (1963). J. Mol. BioZ. 7, 142. Mahler, H. R. & Mehrotra, B. D. (1963). Biochim. biophys. Acta, 68, 211. Mahler, H. R., Mehrotra, B. D. & Sharp, C. W. (1961). Biochem. Biophys. Hrs. Comm. 4. 79. Mandel, M. (1962). J. Mol. BioZ. 5, 435. Ohba, Y. (1966). Biochim. biophys. Acta, 123, 84. Richards, B. M. (1964). In The Nucleohistones, ed. by J. Bonner & P. Ts’o, p. 108. New York : Academic Press. Schildkraut. C. 8: Lifson, S. (1965). Biopolymers, 3, 196.
176
D. E. OLINS,
A. L.
OLINS
AND
P. H.
VON
HIPl’XT,
Spitnik, P., Lipshitz, R. & Chargaff, E. (1955). J. Bid. Chem. 215, 765. Tabor, H. (1962). Biochemistry 1, 496. Tsuboi, M., Matsuo, K. & Ts’o, P. 0. P. (1966). J. MOE. Biol. 15, 256. von Hippel, P. H. & Felsenfeld, G. (1964). Biochemistry, 3, 27 Wilkins, M. H. F. (1956). Cold Spr. Had. Sym;o. Quunt. Biol. 21, 75. Wilkins, M. H. F., Zubay, G. & Wilson, H. R. (1959). J. Mol. Biol. 1, 179. Zubay, G. & Wilkins, M. H. F. (1962). J. Mol. Biol. 4, 444.
(1967) 24, 157-176
Model Nucleoprotein Complexes: Studies on the Interaction of Cationic Homopolypeptides with DNA DONALD E. OLINS, ADA L. OLWS AND PETER H. VON HIPPEL Department
of Biochemistry,
Dartmouth
Medical U.S.A.
School, Hanover,
New Hampshire
(Received 29 August 1966, and in revised form 24 October 1966) Complexes of native calf thymus DNA with the cationic polypeptides poly-Lornithine, poly-L-lysine, poly-n-arginine and poly-L-homoarginine, have been prepared and their solubility, stoichiometry, absorption spectra and thermal denaturation studied. Increasing the peptide cation/DNA phosphate ratio, up to electrostatic equivalence, yielded progressively more insoluble products and increased the turbidity of the “soluble” fraction. Certain spectral changes were observed which may be largely attributed to anomalous scattering in the absorbing region. Addition of the polypeptides to DNA resulted in a marked stabilization of the helix against thermal denaturation. At peptide cation/DKA phosphate ratios less than electrostatic equivalence, thermal denaturation monitored at 260 and 280 rnp revealed a biphasic transition profile: the first transition had a melting temperature similar to DNA under the same solvent conditions; the second melting temperature was characteristic for the type of polypeptide in the complex. Thermal denaturation monitored at 350 rnp (i.e., turbidity transitions) showed a monophasic profile at the higher melting temperature of the DNA-polypeptide complex. The different polypeptides stabilized DNA against melting to different extents. In order of decreasing degree of stabilization they are: poly-L-ornithine > poly-L-lysine > poly-n-arginine > poly-n-homoarginine. Analysis of the dispersion of hyperchromicity demonstrated that poly-L-ornithine and poly-L-lysine preferentially stabilize A-T rich regions, whereas poly-L-arginine and poly-I,homoarginine appear less discriminating. The soluble DNA-polypeptide complexes could be subfractionated by ultracentrifugation into a fraction which melted like “naked” DNA, and a fraction which melted at the higher temperature characteristic of the complex and showed a peptide cation/DNA phosphate ratio close to electrostatic equivalence. The experimental data imply that, under proper conditions of annealing, the basic polypeptides form definable molecular structures with DNA; the bin.ding reaction is stoichiometric and co-operative. Model-building suggests that the polypeptides could interact with either the large or small groove of DNA.
1. Introduction The DNA of eucaryotic nuclei is characteristically associated with a wide variety of basic proteins and polypeptides. This fact has suggested that these histones and protamines may have an important role in modulating and directing the genetic function of DNA in vivo, and has led many workers to carry out detailed chemical and physical investigations on the isolated proteins and the nucleoprotein complex. Fractionation and chemical study have shown that histones may contain appreciable 157
158
D. E. OLINS,
a.
L.
OLINS
AND
P. H.
VON
HIPPEL
amounts of lysine and arginine, while protamines may contain as much as 70 mole ‘; ,, arginine and little or no lysine (Busch, 1965). X-Raycliffractionstuclies havesuggested that the nucleoprotamines of sperm heads are compact crystalline structures with the protamine moiety running helically in one of the grooves of the DNA (Feughelman et al., 1955; Wilkins, 1956; Zubay & Wilkins, 1962); whereas nucleohistones from interphase nuclei are considerably less crystalline with the histones in, as yet, undefined positions (Wilkins, 1956; Zubay & Wilkins, 1962; Wilkins, Zubay 85 Wilson, 1959; Luzzati & Nicolayeff, 1963; Richards, 1964). Physico-chemical studies on solutions of native nucleoproteins have been made more difficult by the propensity of these materials to aggregate. As a step toward studies of nucleoprotein in solution, we have been conducting investigations on complexes of DNA with a variety of basic synthetic homopolypeptides. Studies of the interaction of DNA with poly-L-ornithine, poly-L-lysine, poly-L-arginine and poly-L-homoarginine are reported here. It is demonstrated that all these polypeptides form specific complexes with DNA after appropriate annealing, and that all markedly stabilize DNA against thermal clenaturation. In addition, evidence is presented that varying the type of basic side chain affects the magnitude of the stabilization, as well as the differential stability of A-T versus G-C-rich regions of the DNA.
2. Materials and Methods (a) Reagents Highly polymerized calf thymus DNA was obtained from the Worthington Biochemical Corporation (lot 626), put into solution by gentle stirring overnight at room temperature, and used without further treatment. Previous lots of Worthington calf thymus DNA have been extensively tested, and have been found to be fully native and completely equivalent, by all criteria tested, to calf thymus DNA prepared in this laboratory by standard procedures (von Hippel & Felsenfeld, 1964). (mol. wt Poly-L-ornithine.HBr (mol. wt ~45,000; lot OR15), poly-L-lysme.HBr N 75,000, lot LYM), and poly-L-arginine. &H2S04 (mol. wt ~28,000, lot AR14) were
Wavelength (mp)
FIG. 1. Absorbance spectra of basic homopolypeptides. Solvent: 0.001 M-sodium buffer (pH 7.0). Concentrations of the polypeptides: 1 mg/ml. and poly-L-homoerginine (-A--A-). Curve A, spectra of poly~arginine (-.--.--) Curve B, spectra of poly-L-ornithine (-v-v-) and poly-L-lysine (-m-m-).
cacodylate
MODEL
NUCLEOPROTEIN
COMPLEXES
169
synthesized by the Yeda Research and Development Co., Israel, and obtained from the New England Nuclear Corp. 0.Methylisourea.HzS04 was purchased from the Aldrich Chemical Co. Double glass-distilled water was used throughout. Methanol was of spectral grade. Polyarginine sulfate was converted to the hydrochloride and thus rendered watersoluble by mixing with an equimolar amount of BaCl, in aqueous solution. The resulting insoluble BaSO, was removed by centrifugation. Poly-r>-homoarginine.HCI was prepared by guanidination of poly-L-lysine with 0-methylisourea.HCl (Hettinger & Harbury, 1965). 0-methylisourea.HCl was prepared from the sulfate form by reaction with BaCl, as described above. Amino acid analysis of the product confirmed the conversion of > 99.5”: lysine to homoarginine. Ultraviolet absorption spectra were determined on the Cary 14 recording spectrophotometer for all the polypeptides used in these studies (Fig. 1). All showed negligible absorbance a,t wavelengths greater than 240 rnp. (b) Preparation
of the DNA-polypeptide
complexes
DNA dissolved at a concentration of 5 x 10m4 moles P/l. (A,,, N 3.25) in 0.001 M-sodium cacodylate buffer (pH 7.0) was made 4 M in NaCI. To a constant volume of DNA in concentrated salt solution varying amounts of polypeptide (dissolved in 0.001 M-cacodylate buffer at a peptide cation concentration of N 5 x 10m4 M) were added with vigorous mixing, followed by sufficient O*OOl M-cacodylate to make the final DNA concentration N 2.5 x 10m4 moles P/l. The resultant mixtures were at ratios of peptide cation/DNA phosphate of 0, 0.17, 0.33, 0.60, 0.67 and 1.00. We refer to these as peptide/DNA ratios. Annealing of the complexes was performed by dialysis at 0°C against 0.001 M-cacodylate buffer containing decreasing amounts of NaCI, a routine similar to that employed by Huang, Bonner & Murray (1964) for the reconstitution of nucleohistones. The dialysis steps were : 4 hr each against 0.4 M, 0.3 M, 0.15 M-NaCl; overnight against cacodylate buffer (pH 7.0) made 0.0025 M in sodium EDTA; followed by 2 to 3 daily changes of 0.001 M-cacodylate buffer (pH 7.0). Total volumes of the resultant dialysates were measured, and precipitates were removed by centrifugation for 15 min at about 1700 g. The supernatant fractions from this low-speed centrifugation, which were still noticeably opalescent at the higher peptide/DNA ratios but produced no further precipitate on standing, were saved and employed as the “soluble” DNA-polypeptide complexes.
(c) Chemical analysis DNA. concentrations were measured by a diphenylamine method (Giles & Myers, 1965). The presence of polypeptide wss found not to interfere with color development in this test. DNA samples of known concentration were run in each batch as internal standards. Concentrations of DNA were expressed as moles P/l., assuming a molar phosphate extinction coefficient for native calf thymus DNA at 260 ml* of 6500. Concentrations of the polypeptide stock solutions were measured using a Zeiss laboratory interferometer, assuming that the typical protein value of (dn/dc)550 = 1.87 x 10-l ml.,lg could be applied to all the polypeptides. The peptide content of “soluble” complexes was measured by a modification of the method of Lowry, Rosebrough, Farr & Randall (1951). Color yields were weak, especially for polyarginine and polyhomoarginine, and a small correction for DNA content was required. A,,,, for peptides at a concentration of 60 pg/ml. were as follows : polyornithine and polylysine, - 0.22 ; polyarginine and polyhomoarginine, - 0.06. (d) Melting
projibs
Melting experiments were performed in a Gilford model 2000 recording spectrophotometer equipped for the automatic measurement and recording of temperature. The platinum resistance thermometer in this instrument is located within the sample chamber controlled by thermostat and is directly below the cuvettes; control experiments using a thermistor located within the cuvettes themselves showed that the temperatures sensed by the resistance thermometer differed by less than 0.2’C from those within the cuvettes throughout the entire melting curve. Prior to melting, the samples were bubbled with helium gas (to remove other dissolved gases in favor of helium, which haa a positive temperature
I80
D. E. OLINS, lO8-
A. L. OLINS I
AND
P. H. VON
HIPPEI,
----7--T-T----T-.-
I
196 -
.J 50% (v/v)
t/
: .I
Methanol
Temperature
(‘C )
FIG. 2. Relative thermal expansion of 60% (v/v) methanol and of water. The data for 50% methanol comprise two sets of determinations. The data for water are compared with values calculated from the Handbook of Cherniatry am? Physics (A). IJ~o/v~~~= volume of solvent at tW/volume of solvent at 3O’C.
coefficient of solubility) and sealed with General Eleotric silicone rubber RTV-88 as described by Felsenfeld & Sandeen (1962). The samples were heated from 25 to 100°C at a constant rate of l”C/min, using a Northeast Scientific Co. automatic temperature programmer driving the thermoregulator of a model T-9 Tamson constant temperature bath. The raw melting data were corrected for thermal expansion of the particuIar solvent used. The relevant thermal expansion data for water and 60% (v/v) methanol-water were obtained by direct measurements of volume change in a sealed calibrated dilatometer (Fig. 2). Data correction and reduction to relative hyperchromicities (i.e. absorbance at temperature T/absorbance at 30°C) were accomplished using a simple program on the GE-235 computer. Melting temperatures of biphasic transitions (T, and T,‘) were calculated by extrapolating the first and second plateau regions through the linear approximations of each transition. The resultant intersections defined the initial and final hyperehromicities of a particular transition; the melting temperature in each case was the temperature at A hyperchromicity/2. For biphasic transitions, we designate the fist melting temperature by T, and the second by T,‘. As a measure of the co-operativity of the transition, we use a quantity (r, defbmd as the temperature interval over which the transition goes from 25% to 76% of oompletion.
3. Results (a) LYolubiZity, stoichionzetry and spectra
The addition of basic homopolypeptides to DNA resulted in complexes the solubility of which, after dialysis and low-speed centrifugation, varied with the ratios of the reactants (Table 1). Increasing the ratio of peptide to DNA yielded more insoluble products. At an input ratio of peptide cations to DNA phosphates of 1: 1, complexes of polyornithine and polylysine with DNA appeared to be less soluble than those of polyarginine and polyhomoarginine (Table 1). The amounts of polypeptide actually complexed with DNA in the soluble products
MODEL
NUCLEOPROTEIN
101
COMPLEXES
1
TABLE
DNA recovered as “soluble” complex at various peptide/DNA Polypeptide : Arginine Lysine
Ornithine
( + M - 1 groups
100% 107 102 79 68 36
100% 105 99 91 75 0
100% 93 100 100 71 2
o-00
0.17 0.33 0.50 0.67 1.00
input ratiosf
Homoarginine 100% 105 90 82 71 40
t DNA as measured by the diphenylamine method; estimated mean error in total determination, f 5 %. Th.e input DNA concentration in each sample was 5 x 10T4 moles P/l.
were also measured and compared with the input ratios. Polyornithine and polylysine yielded peptide/DNA ratios similar to their input ratios; polyarginine and polyhomoarginine showed somewhat lower ratios. Ultraviolet absorption spectra were obtained for all the “soluble” complexes at room temperature. The spectra for DNA-poly-L-lysine at different peptide/DNA ratios are typical and are shown in Fig. 3. Two properties of these spectra may be noted which correlate generally with increased peptide/DNA ratios : (1) a progressive increase in turbidity at wavelengths greater than 300 rnp (a region in which neither DNA nor the polypeptides alone show any absorbance) ; and (2) an apparent Cacodylate I?
i
1
I
I
I
I
I
I
I
I
I
Methanol-cacodylate ! I I
I
I
I
.
I
60 80 4
I
220 40 60
Wavelength
1
80 300 20 40 60 80 1
(mp)
FIG. 3. Absorbance spectra of DNA-poly-L-lysine complexes. The curves have been normalized to the same amounts of DNA, as determined by diphenylamine. Input peptide cation/DNA phosphate ratios: curve A, DNA alone; B, 0.17; C, 0.33; D, O-50; E, 0.67. Solvents: “cacodylate”, 0.001 M-cacodylate buffer (pH 7.0); “methanol-cacodylate”, 50% (v/v) methanol made 0.001 M with cacodylate buffer (pH 7.0). Insert tables: values of -%mnp/O~D~~~omp for each of the samples.
102
D. E.
OLIKS,
A. L.
OLINS
AND
P. H.
VOX
HIPPEL
hypsochromic (towards the red) shift of the approximately 260 ml* DNA absorytiull peak. This shift was also accompanied by a progressive decrease in tht: app~~~~t 0.D.260(0.D.2Bo ratio. Similar spectral effects were seen for complexes containing ~~11 type of basic homopolypeptide. However, complexes containing poly-L-ornithine and poly-L-lysine showed greater turbidity and shift in the absorption peak than those of poly-L-arginine and poly-L-homoarginine. In every case similar spectra were also obtained with the “soluble” complexes in cacodylate buffer made 50% (v;‘v) in methanol (e.g. see Fig. 3). These spectra are of considerable potential interest, since a small conformational change in DNA structure which might occur as a consequence of DNA-polypeptide complex formation might then be reflected as a spectral change in this region. Before
0.01-300
20
’
40
’
’
60
’
’
60
’
”
400
Wavelength (mp) (b) FIQ. 4. (a) Light-scattering by DNA-polylysine complexes plotted a8 log (apparent absorbance) VWSUB log A. Peptide cation/DNA phosphate ratios: curve A, 0.17; B, 0.33; C, 0.50; D, 0.67. Solvent: 0.001 a6-cacodylate buffer (pH 7.0). The error limits shown around the points on the curve A correspond to a deviation of f 0.003 absorbance unit. (b) Difference spectra of DNA-poly-L-lysine complexes measured against DNA at the smne concentration. Peptide c&ion/DNA phosphate ratios. . curve A, 0.17; B, O-33; C, 0.50; D, 0.67. Solvent: 0.001 mn-cacodylate buffer (pH 7.0).
MODEL
NUCLEOPROTEIN
COMPLEXES
163
looking for such effects, however, we must attempt to dissect out the contributions of light-scattering from the spectra. The apparent absorbance of the complexes at wavelengths greater than 300 rnp can easily be shown to be due to scattering, since in regions far from absorption peaks an apparent absorbance due to scattering should show a wavelength dependence of the following form : OJ’xpp = const. (A-“)
(1)
where a may range from 4 for particles small relative to h (i.e. Rayleigh’s law) to 0 for macroscopic particles. Figure 4(a) shows that at X > 300 rnp, the apparent absorbance does indeed obey a relationship of this form. The values of a for different mixtures of DNA-polylysine were determined from Fig. 4(a) and were as follows: the sample with peptide cation/DNA phosphate of 0.17 had a coefficient of N 3.8; with a ratio of 0.33, a = 2.9; ratio 0.50, a = 2.9; and ratio 0.67, a = 2.8. Two other polylysine-DNA complexes were analyzed : ratio O-17, a = 2.5 ; and ratio O-33, a = 2.8. Such coefficients in the scattering law are not unreasonable for particles of intermediate size, and the relative constancy of the coefficient for complexes of differing peptide/DNA ratio suggests that the aggregates formed by the procedures used here are all of comparable size. It is unfortunate that scattering in the vicinity of a real absorption peak cannot, be corrected by a simple linear extrapolation of the wavelength dependence found in the transparent regions into the absorbing region, since in absorbing regions one expects anomalous dispersion of the index of refraction and thus also of scattering. Such anomalous scattering in the peak region would lead (for an isolated absorption peak) to a positive contribution to the apparent absorbance on the long wavelength side of the peak, a cross-over point (i.e. no scattering at a point close to the center of the peak.) and a negative contribution to the short wavelength side. Such behavior would, of course, result in a hypsochromic shift of the peak and an apparent decrease in the o.D.~~,,/o.D.~~,, ratio. As pointed out above, these expectations are realized quantitatively in the raw absorbance data (e.g. see Fig. 3). These qualitative indications, coupled with the adherence to simple scattering behavior of the apparent absorbance in the long wavelength region of the spect’ra, suggest that the simplest interpretation is that at X > 230 rnp these spectra represent the sum of the normal DNA spectrum plus a scattering contribution which increases in magnitude as the peptide/DNA ratio is increased. To test this supposition, a set of difference spectra for a series of DNA-polylysine complexes is plotted in Fig. 4(b). These difference spectra were obtained from the absorbance data presented in Fig. 3(a) by subtracting the absorbance values for DNA from those of a complex containing the same concentration of DNA. They show qualitatively the appearance one would expect if the excess apparent absorbance (beyond that due to DNA itself) were all due to scattering. The strong positive scattering on the short wavelength sides of the 260 rnp absorption peaks, and the fact that a cross-over to negative apparent absorbance values is not actually observed at h < 260 rnp, may be attributed to the superimposition of positive scattering from the long-wavelength arm of the anomalous dispersion curve centered about the strong DNA absorption peak located below 200 mp. Very similar difference spectra have recently been obtained by Eisinger (personal communication, 1966), who compared the apparent absorbance of suspensions of T4 bacteriophage with the absorbance of osmot’ically shocked phage.
164
D.
E. OLINS,
A. L.
OLINS
AND
P. H.
VON
HIPPEL
DNA-Polyornithine t
I
I
30
40
50
Cacodylate I I
60
70
I
I
I I
80
90
100 30
Temperature
I
40
Methanol-cacodylate I I 1
50
60
70
I
I
80
90
(“C 1
DNA-Polylysine I
I
40
50
Cacodylate I I
I
I
80
90
/
1
Methanol I I
-cacodylate I 1
I
1.6I.5 -
30
60
70
100 30
Temperature
40
50
60
70
80
90
1 IO
(“c)
Fro. 5. (a) and (b) Thermal denaturatian profiles of DNA-polypeptide complexes monitored at 260 mp. at 260 mp. H,,, = relative hyperchromioity Peptide cation/DNA phosphate ratios: curve A, DNA alone; B, 0.17; C, 0.33; D, 0.50; E, 0.67; 0.001 rd-sodium cacodylate buffer (pH 7.0); “methanolF, 1.00. Solvent conditions: “cacodylate”, cacodylate”, 60% (v/v) methanol made O*OOl M-sodium cacodylate (pH 7.0).
MODEL
NUCLEOPROTEIN
DNA-
Polyarginine Methanol-cacodylate
Cacodylate
30
40
50
60
165
COMPLEXES
70
80
90 100 30 40 Temperature
Cacodylate
DNA-
50
60
70
80
90
100
80
90
IO0
(“C)
Polyhomoarginine Methanol-cacodylate
1
I
I
I
I
40
50
60
70
80
I
I
I.6 1.5
I.0
30
90 100 30 40 Temperature (b) Fm.
12
t7
(‘C)
50
60
70
16G
D. E. OLINS,
A. L. OLINS
AND
I’. H.
VOX
HIPPEL
DNA-Polyornithine
30
40
50
60
70
80
90
100 30
Temperature
40
50
60
70
80
90
I
I
100
(“C)
DNA-Polylysine I
I
Cacodylate I I
I
I
I
I
Methanol-cacodylate I / I
0 Temperature
(“C)
(a)
Fro. 6. (a) and (b) Thermal denaturation proties of DNA-polypeptide complexes monitored at hyperchromicity at 280 mp. Peptide cation/DNA phosphate ratios: curve A, DNA alone; B, 0.17; C, 0.33; D, 0.50; E, 0.67; F, 1.00. Solvent conditions: “cacodylate”, 0.001 M-sodium cacodylate buffer (pH 7.0); “methanolcacodylate”, 50% (v/v) methanol made 0.001 M-sodium cacodylate (pH 7.0).
280 mp. H,,, = relative
MODEL
NUCLEOPROTEIN
COMPLEXES
167
DNA - Polyarginine I
30
I
I
40
50
Cacodylote I 1
60
70
I
I
80
90
I
I( 30 Temperature
Methanol I
I
I
40 (“C )
50
60
-cacodylate I I
70
80
90
DNA - Polyhomoarginine Methanol
Cacodylate l/1111
30
40
50
60
70
80
90
IC
30
Temperature (b)
FIQ. 6
-cacodylate
’
’
1
I
40
50
60
70
(“C )
’
k-&-i
’
1
b30
168
D. E. OLINS,
A. L.
OLINS
AND
P. H.
Cacodylote
VON
Methonol I------
HIPPEL - cacodylate ’
’
’
70
60
90
I.0 30
40
50
60
70
80
90
100 30 Temperature
40
50
I.0 60
100
(“C)
FIQ. 7. Thermal denaturation profiles of [DNA-polypeptide complexes monitored at 350 rnp. Rel A,,, = relative apparent absorbance at 360 mp. All the complexes were mixed at peptide cation/DNA phosphate = 0.60. Samples: A, DNApoly-L-homoarginine; B, DNA-poly-L-arginine; C, DNA-poly-L-lysine; D, DNA-poly-Lornithine. The right-hand co-ordinate scales refer to DNA-poly-L-arginine; the left-hand scales apply to the other complexes. Solvent conditions: “cacodylate”, 0.001 M-sodium cacodylate 60% (v/v) methanol made O-001 M-sodium ‘cacodylate buffer (pH ‘7.0); “methanol-cacodylate”, (pH 7.0).
A more quantitative analysis of these spectra is probably pointless at this stage, due to the complexities introduced by overlapping absorption peaks, each with its associated anomalous dispersion curve. Thus, while on the basis of this analysis we cannot rule out the presence of small, but real, spectral changes accompanying complex formation, there is no indication from these data that such changes do exist. (b) Thermal melting stwEies Under the solvent conditions employed in these studies, the addition of basic polypeptides resulted in a marked stabilization of the DNA structure (Figs 5, 6, 7). As Figs 5 and 6 show, for ratios of peptide to DNA less than 1: 1, biphasic melting transitions are seen at both 260 and 280 rnp. The lower of the two thermal transitions shows approximately the same T, as native DNA alone under the same solvent conditions, while T,’ for the higher melting transition appears to be characteristic of the polypeptide-DNA complex. T, and T,’ values derived from these data are assembled in Table 2. Figures 5 and 6 show that the biphasic meltings are only incompletely visualized in cacodylate buffer. Especially in the higher melting polyL-ornithine and poly-L-lysine complexes, only a small ii-action of the second transition has taken place when the temperature has reached 100°C. In order to visualize the entire transition in all the complexes, we have also run melting curves in 50% methanol-cacodylate. Note that this solvent modification reduces the melting temperature of both the DNA and the complex transitions, though the melting temperatures of the latter are reduced more (see Table 2). The gross appearance of the melting transitions in 50% methanol, like the spectra (Fig. 3), are relatively unchanged in comparison to the companion transitions in aqueous
MODEL
NUCLEOPROTEIN
169
COMPLEXES
TABLE 2 Melting temperatures of DNA and DNA-polypeptide
DNA Cacodylatc 260 52.23 280 53.0 0.8 ATroll 350 Methanol--cacodylate 260 45-g 280 46.4 1.2 AT,,II 350 -
Ornithine T, Tm’
complexest
Polypeptide : Lysine Arginine T,’ Tm Tm’ Tm
Homoarginine T, T,’
49.3 50.1 0.9 -
>lOO >I00 >lOO
50.5 51.1 0.6 -
>lOO > 100 >lOO
54.6 55.6 1.0 -
93.3 93.8 0.5 95.4
53.9 54.7 0.8 -
87.4 88.2 0.7 90.3
46.0 46.7 0.7 -
87.1 87.0 -0.1 86.9
47.1 48.0 0.8 -
84.5 84.8 0.3 84.3
44-5 45.2 0.8 -
81.7 82.2 0.8 82.3
45.7 46.1 0.7 -
76.5 77.5 1.0 80.0
t The melting temperatures at 260 and 280 rnp represent averages of from 6 to 13 independent determinations. Melting temperatures at 350 rnp are based on a single series of determinations. $ This value agrees reasonably well with that calculated for calf thymus DNA under the same ionic strength conditions from equation (2) of Schildkraut & Lifson (1965). Employing their equation and assuming 42% GC, one obtains Tm,aB,, = 48~7°C. .$Herskovits et al. (1961) obtained T,.zeo = 45.5’C for calf thymus DNA in 51.5% volume methanol at similar ionic strength. II AT, is defined as (T,.28~-T,.~gd or (Tm’.280 -T,‘.260 ). The values of AT, were averaged separately.
solution, though we may note that in addition to the T, shifts, the methanol transitions Z&O show a slightly larger apparent hyperchromicity for all the complexes and for ‘DNA itself. Figure 7 shows melting transitions for the various polypeptide-DNA complexes at 350 mp, where only turbidity changes are observed. (Neither DNA nor the polypeptides heated singly showed any detectable turbidity or turbidity change at this wavelength.) Note that even complexes formed at peptide/DNA ratios which exhibit well-developed biphasic transitions at 260 and 280 rnp show only one transition at 350 mp, and that transition parallels the higher of the two transitions seen at 260 or 280 mp (Table 2). The above results suggest the following picture. (1) In the biphasic melting transitions, the first melting represents the helix --f coil transition of free DNA molecules (or portions of molecules), while the second meltings are characteristic of the particular DNA-polypeptide complex involved. The elevation in T,’ above the T, for free DNA in each case represents the degree to which that particular polypeptide has stabilized DNA against thermal denaturation. (2) As the ratio of amino (or guanidino) groups to DNA phosphates increases toward unity, the lower thermal transition disappears, suggesting a saturation of the free DNA by the added pepbide. (3) Herskovits, Singer & Geiduschek (1961) had previously shown that the T, of native DNA is lowered by added methanol. We have demonstrated that the thermal stability of DNA-polypeptide complexes may be reduced in a similar way by added methanol. (4) The 350 rnp melting results show that the turbidity which develops on EDNA-polypeptide complex formation is due to the complex alone, and is not affected by the melting of free DNA molecules (or portions of molecules) present in the same solution.
I;(1
I).
I’,.
or,1ss,
.A.
r,.
C)T,ISS
ASI) ‘L’ABLE
DNA-polylysine
(+)i(-) groups
mixed at diflerent
input
Tlt2
T,’
t1.
\‘OS
ti11’1’1~1,
3
ratios and melted itc. ,Ilethlrtrlol-ctr~c,dyltrt~ /
260 mp
,
I’.
280 m/J o
o’
’
’
Tm
T Ill ’
o
a’ ’
AT ,n
~‘l’ Ll,
--
f AT, $ This
is defined as (Tm,zso-TTm,zao); first transition was too small
AT,’
as T,,,‘~zso-TT,‘,,,,).
to be evaluated
accurately.
Additional insight into the properties of the DNA-polypeptide complexes may be obtained by further study of these data. First, we may note that the various polypeptides tested stabilize the DNA transition to varying extents : in order of decreasing stabilization the complexes may be listed DNA-poly-L-ornithine>DNA-poly-~lysine>DNA-poly-L-arginine>DNA-poly-L-homoarginine. This progression is seen in the transitions monitored at 260, 280 and 350 rnp, and in both aqueous solutions and 50% methanol (Figs 5,6, and 7 and Table 2). Second, inspection of Figs 5 and 6 suggests (and Table 3 shows in detail for one set of complexes) that T,’ for the various polypeptides is invariant with peptide/DNA ratio. That is, the higher melting transition in the biphasic melting curves occurs at a fixed melting temperature, regardless of the fraction of the total DNA which is complexed. This behavior is in marked contrast to that observed with small polycations, such as Mg2+ (Dove & Davidson, 1962), spermine, spermidine and diamines (Mahler, Mehrotra & Sharp, 1961; Mandel, 1962; Tabor, 1962; Mahler & Mehrotra, 1963), and tetra-L-lysine (this laboratory, work to be published). These additives shift the entire melting curve in a monophasic fashion; even at low cation/DNA ratios. This suggests that these smaller polycations are bound to DNA loosely and reversibly, whereas the polypeptides examined in this study appear to be bound essentially irreversibly. These differences will be considered further in a subsequent paper. Comparison of the various values of T,‘,,,, with Tmf,280 obtained on the same DNA-polypeptide complexes provides additional insight into the nature of the interaction. Felsenfeld & Sandeen (1962) have shown, on the basis of extensive studies of the dispersion of DNA hyperchromism, that the hyperchromic changes which accompany melting are weighted differently with respect to base composition at different wavelengths. At 260 rnp, the hyperchromic contribution of the A-T base pairs exceeds that of the G-C pairs, whereas at 280 rnp., the converse is true. Therefore a transition monitored at 260 rnp will reflect more heavily the melting behavior of the A-T pairs, and the G-C pairs are weighted more heavily in the transition measured at 280 mp. As a consequence, the difference between Tm,2B0 and T,,zso (AT,) for a given DNA provides a crude measure of the non-randomness of the distribution of base pairs along the helix (i.e., their distribution into relatively ,4-Trich or G-C-rich regions). Differences in AT, measured in this study for the free DNA transitions in both cacodylate and methanol-cacodylate solution were about
MODEL
NUCLEOPROTEIN
171
COMPLEXES
0.9 dtrO.Z”C. In contrast, we may note (Table 2) that there is a small but definite decrease in the comparable A!!!‘, values for some of the DNA-polypeptide complexes. This digerence is particularly striking in the case of the polyornithine and polylysine complexes, where AT, ’ is close to zero, suggesting that these polypeptides preferentially stabilize A-T-rich regionsof the native DNA helix against thermal denaturation (or alternatively, destabilize G-C-rich regions). The shapes of the melting curves obtained for the “soluble” DNA-polypeptide complexes were found to be very dependent on the method of preparation. Thus the complicated routine of annealing the complexes by dialysis against decreasing concentrations of salt, as described in Materials and Methods, was found to he necessary in order to obtain complexes with sharp melting transitions. Addition of the polypeptide to DNA at low ionic strength resulted in stabilization of the DS’A. but the second transition was considerably broader than that obtained with samples carried through the regimen of salt-concentration changes. The experiments with thtx annealed complexes demonstrated that these transitions were as sharp as or sharper TABLE
Cakulnfed
values of (T (co-operatiuity)
4
for the melting
of DNA
a.nd DNA-polypeptids
com,plexest
(mcL)
DNA
(‘acodylate 260 6.3 280 6.9 Methanol-cacodylate 260 6.2 280 6.7 t The value
Ornithine [I (I’
Polypeptide: Lysine 0 u’
Arginine D cl’
Homoarginine 0 0’
6.2 7.3
-
5.9 6.8
-
8.2 8.1
4.2 4.3
-5.9 6.4
3.4 4.7
7.0 7.6
3.0 2.6
7.4 8.0
2.8 2.6
5.5 5.9
4.4 4.7
6.0 6.6
5.6 5.4
of D represents
averages
of from
6 to 13 independent
measurements.
than those of DNA alone (see Table 4). In this context it should be noted that increased ionic strength or the presence of divalent cations (at a ratio of equivalents Mg”” /DNA phosphate greater than l*O), has also been observed to lead to sharper (more co-operative) thermal DNA transitions (Dove & Davidson, 1962). The requirement for annealing, plus the different type of melting curve obtained with non-annealed complexes, also suggests that complex formation is essentially irreversible under our final conditions: i.e. that there is little or no migration of the bound polypeptide from one DNA site to another. Furthermore, the fact that T, for the first transition in the DNA polypeptide complexes is essentially identical to that in solutions containing DNA alone, suggests that the added polypeptide is bound completely and thus does not affect the ionic environment of the uncomplexed portion of the DNA (see also footnotes, Table 2). Another problem of considerable interest in these studies was to determine whether the separately melting free DNA present in complexes containing less than 1:l rabies of peptide to DNA corresponds to DNA molecules completely free of polypeptide. while other DNA molecules are completely complexed (i.e. a non-random distribution ,.)f t.ht llolppeptide on DNA), or whether all the DNA molecules in the solution
172
D. E. OLINS,
A. L.
OLINS
AND
P. H.
VON
HIPPEL
0.6 260/280 I.21
0.5n
A
-.-.-.a -.-
220 40 60 80 300 20 40
60 80 400
Wavelength (rnp) FIG. 8. Absorbance spectra of a DNA-poly-I,-lysine complex and fractions obtained by ultracentrifugation. The curves have been normalized to the same amount of DNA, as determined by diphenylamine. Samples: A, complex mixed at peptide cation/DNA phosphate ratio of 0.50; B, supernatant fraction after centrifugation; C, resuspended pellet. Inset table: values of o.D.~~~,,,~/o.D.~~~,,,~ for each of the samples. Solvent : 0.001 M-sodium cacodylate buffer (pH 7.0).
DNA-
1.6
1
I
Polylysine fractions
I
I
1
I
I
I.5-
Temperature CC 1 FIQ. 9. Thermal denaturation profiles of a DNA-poly-L-lysine complex and fractions obtained by ultracentrifugation, monitored at 260 mp. Hzao = relative hyperchromicity at 260 mp. Samples: A, complex mixed at peptide cation/DNA phosphate ratio = 0.50; B, supernatant fraction after centrifugation; C, resuspended pellet. Solvent: 50% (v/v) methanol made O*OOl Ja-sodium cacodylate (pH 7.0).
MODEL
NUCLEOPROTEIN
COMPLEXES
173
contain regions which are complexed, alternating with regions which are essentially “naked” (a random distribution of polypeptide). To investigate this question we subjected several complexes with peptide/DNA ratios < 1 to extensive ultracentrifugation (15 minutes at 76,000 gin a no. 40 rotor, Spinco model L preparative ultracentrifuge at 5°C). After centrifugation, the complexes could be separated into a pellet and a supernatant fraction, and the pellet resuspended in fresh eacodylate buffer by gentle stirring. Both fractions were then analyzed for peptide and DNA content, ultraviolet spectra were obtained, and then both were made 50% (v/v) in methanol, and melted. Typical results, obtained with a DNA-polylysine complex mixed aft a peptide cation/DNA ratio of 0.5 are shown in Figs 8 and 9. Spectra of the unfractionated complex, plus those of the supernatant fraction and the (resuspended) pellet obtained by centrifugation, are shown in Fig. 8. The supernatant fraction yielded a spectrum (and ratio of 0.D.260 to O.D.zso) more similar t’o DNA than was the original complex (see Fig. 3 for comparison). The resuspended pellet showed even more turbidity and decrease in o.D.~~~/o.D.~~~ than observed for the unfractionatetl complex. Figure 9 demonstrates the results obtained on melting the fractions and the original DNA-polylysine complex. The supernatant fraction shows no higher melting fraction; the resuspended pellet is devoid of the lower melting fraction. Thus, from the point of view of melting behavior, the resuspended pellet represents a “purified” polypeptide-DNA complex. Similar spectral and melting data have been obtained for fractionated complexes of DNA-poly-L-ornithine, DNA-poly-L-arginine and DNA-polg-t-homoarginine. In a number of experiments, “purified” complexes showed a melting temperature several degrees lower than the T,’ of the corresponding unfractionated complex. Further studies are being directed at this point. The ratio of peptide cation to DNA phosphate was measured for a series of different DNA-peptide complexes mixed at an input ratio of peptide cation to DNA phosphate of 0.5. The “purified complex” of DNA-poly-L-ornithine had a cation/phosphate ratio of 0.9; “purified” DNA-poly-L-lysine, 1-l ; “purified” DNA-poly-L-arginine, 1.0; and “purified” DNA-poly-L-homoarginine, 1.3. Thus, these “purified” complexes contained approximately 1 peptide cation per DNA phosphate. Furthermore, they are not merely a small fraction of the total complex; between 50 and 70% of the total DKA was pelleted in these complexes. It thus appears that the annealed “soluble” comple.xes of DNA-polypeptide contain DNA molecules in two conditions : some arc complexed to approximate electrostatic equivalence with polypeptide, whereas the rest’ seem essentially free of polypeptide. A few more words might usefully be said about our use of the term “irreversible” in this paper. In our annealing system the complexes are initially formed (in high salt concentration) under “reversible” equilibrium conditions. The reaction between DNA and the polypeptides under these conditions is well defined, stoichiomet,ric and. co-operative. By progressive dialysis into a low-salt concentration milieu, WC then “freeze” the system into an “irreversible” state which, however, reflects the binding specificity characteristic of the high-salt concentration equilibrium. Presumably this state also represents rather closely the interaction specificity (and equilibrium) which applies at low salt concentration, but which cannot be attained by direct mixing of DNA and polypeptide at low ionic strength because of the kinetic barriers that greatly slow the rearrangement of such interacting polyelectrolytc
174
D.
E.
OLINS,
A.
L.
OLINS
AND
P.
H.
VOX
HIPPEL
systems under non-annealing conditions. In low salt concentration, using polypeptidcx of molecular weights comparable to those employed here, irreversibility is essentially total over the time scale involved in melting experiments (period of a few hours). However, over much longer times further rearrangements may take place. Using mixtures of radioactively-labeled DNA’s, Leng & Felsenfeld (1966) have demonstrated directly the short-time reversibility of formation of DNApolylysine complexes at high ionic strength. Tsuboi, Matsuo & Ts’o (1966), by similar methods, have shown that DNA-polylysine complex formation is essentially irreversible over substantial periods of time in low salt concentration.
4. Discussion The present studies indicate that under proper conditions of annealing, basic homopolypeptides interact with native DNA, resulting in the formation of definable molecular complexes. The following points emerge from an investigation of their properties. (1) DNA can form “soluble” complexes with basic polypeptides. (2) The DNA of such complexes exhibits a marked stabilization against thermal denaturation. (3) The extent of stabilization varies with the chemical nature of the basic polypeptide. (4) Complexes involving poly-r.-ornithine and poly-n-lysine appear to stabilize preferentially A-T-rich regions of DNA; whereas poly-L-arginine and poly-L-homoarginine stabilize more indiscriminately. (5) Under the low ionic strength conditions employed, complexing appears to be essentially irreversible. (6) The interaction of peptide with DNA shows elements of co-operativity, since, as assayed by thermal denaturation, the DNA of soluble complexes could be fractionated by ultracentrifugation into molecules melting like “naked” DNA, and molecules appearing to be totally complexed with peptide. (7) Complexing is approximately stoichiometric, since these “purified” complexes contained approximately 1 basic residue per DNA phosphate. (8) Complex formation produced several apparent spectral changes which, however, all appear to be attributable to the various manifestations of light-scattering from absorbing particles. Several of these points are in accord with results recently obtained by Tsuboi et al. (1966) in a study of the interaction of poly-r.-lysine with DNA and with poly (I + C). These authors also concluded that the binding reaction was stoichiometric and irreversible. Ohba (1966) has studied hyperchromic dispersion of DNA-poly-Llysine, DNA-protamine and DNA-histone complexes, and has concluded that polylysine and histones preferentially stabilize A-T-rich regions, whereas protamine does not appear to have this effect. The difference in degree of stabilization by the various basic polypeptides does not appear to be explainable simply on the basis of electrostatic neutralization. Examination of molecular models of the polypeptides reveals a correlation between extended side-chain length and complex stability: i.e., the shorter the side chain, the greater the stabilization, as manifested by a progressive increase in 27,‘. This differential stabilization may be a consequence of steric limitations, of differing hydrophobicity of the various side-chains, or different over-all charge densities. Earlier studies by Spitnik, Lipshitz & Chargaff (1955) have suggested that poly-nlysine interacts preferentially with A-T-rich regions of native DNA. In the present. studies, poly-L-ornithine and poly-L-lysine, in particular, brought about a more simultaneous melting of A-T- and G-C-rich regions than is observed in uncomplexect
MODEL
NUCLEOPROTEIN
COMPLEXES
I75
DNA. In view of the earlier data, it is more reasonable to assume that these polypeptides preferentially stabilize A-T-rich regions than to suggest that the observed melting effects result from a labilization of G-C-rich regions. These conclusions are also in accord with the results of some direct solubility studies on DNA-poly-L-lysine complexes recently carried out by Leng & Felsenfeld (1966). It is also pertinent to point out here that recent studies by Johns & Butler (1964) have demonstrated that, “lysine-rich” histones appear to be preferentially associated with A-T-rich regions of DNA in native nucleohistones. X-Ray diffraction studies in nucleoprotamines (Feughelman et al., 1955; Wilkins, 1956) and on a DNA-polylysine complex (Wilkins, 1956) have suggested that the peptide moiety winds around DNA in one of the grooves. Wilkins and co-workers (Feughelman et al., 1955; Wilkins, 1956) appear to favor binding in the narrow groove. Luzzatti (1963), however, has challenged their interpretations of the X-ra) data.. Tsuboi et al. (1966) present a model of polylysine winding through the large groove. On the basis of a careful examination of space-filling models, we have not beert able to eliminate on steric grounds either groove as the site for binding. A direct verification of the binding region is clearly required. Present studies in our laboratory are focused on determining, by chemical means, if basic pept,ides, protamines and histones interact with a particular groove of the DNA molecule. Thix investigation was supported in part by U.S. Public Health Service research grant AM-03412 (to P. H. v. H.) and National Science Foundation research grant GB-2060 (to S. I.); also by a Post-Doctoral Research Fellowship (F-123) from the Helen Hay Whitney Foundation (to D. E. 0.) and a Career Development Award (GM-K3-5479) from tbe U.S. Public Health Service (P. H. v. H.) We also gratefully acknowledge the skilled experimental assistance of Mrs Judith Gilbert in these studies. Two of us (D. E. 0. and A. 1,. 0.) are very grateful to Dr Shinya Ino& for providing partial support for our research activities during the time this work was in progress, as well as for many stimulating discussions and much good advice. REFERENCES Busch, H. (1965). H&ones and Other Nuclear Proteins, p. 28. New York: -4cademic Press. Dove, W. F. & Davidson, N. (1962). J. Mol. Biol. 5, 467. Felsenfeld, G. B Sandeen, G. (1962). J. Mol. BioZ. 5, 587. Feughelman, M., Langridge, R., Seeds, Vc’. E., Stokes, A. R., Wilson, H. R., Hooper, C. W., Wilkins, M. H. F., Barclay, R. D. & Hamilt’on, L. D. (1955). Nuture. 175, 834. Gile:s, K. W. dz Myers, A. (1965). Nature, 206, 93. Herskovits, T. T., Singer, S. J. & Geiduschek, E. P. (1961). Arch. Biochem. Biophys. 94, 99. Hetringer, T. P. & Harbury, H. A. (1965). Biochemistry, 4, 2585. Hua.ng, R. C. C., Bonner, J. & Murray, K. (1964). J. Mol. BioZ. 8, 54. Johns, 15. W., & Butler, J. A. V. (1964). Nature, 204, 853. Leny, M. & Felsenfeld, G. (1966). Proc. Nat. Acud. Sci., Wash. 56, 1325. Lowry, 0. H., Rosebrough, N. J., Farr, A. G. 8E Randall, R. J. (1951). J. BioZ. Glum. 193,265 Luzzati., V. (1963). J. Mol. Biol. 7, 758. Luzzati., V. & Nicolai’eff, A. (1963). J. Mol. BioZ. 7, 142. Mahler, H. R. & Mehrotra, B. D. (1963). Biochim. biophys. Acta, 68, 211. Mahler, H. R., Mehrotra, B. D. & Sharp, C. W. (1961). Biochem. Biophys. Hrs. Comm. 4. 79. Mandel, M. (1962). J. Mol. BioZ. 5, 435. Ohba, Y. (1966). Biochim. biophys. Acta, 123, 84. Richards, B. M. (1964). In The Nucleohistones, ed. by J. Bonner & P. Ts’o, p. 108. New York : Academic Press. Schildkraut. C. 8: Lifson, S. (1965). Biopolymers, 3, 196.
176
D. E. OLINS,
A. L.
OLINS
AND
P. H.
VON
HIPl’XT,
Spitnik, P., Lipshitz, R. & Chargaff, E. (1955). J. Bid. Chem. 215, 765. Tabor, H. (1962). Biochemistry 1, 496. Tsuboi, M., Matsuo, K. & Ts’o, P. 0. P. (1966). J. MOE. Biol. 15, 256. von Hippel, P. H. & Felsenfeld, G. (1964). Biochemistry, 3, 27 Wilkins, M. H. F. (1956). Cold Spr. Had. Sym;o. Quunt. Biol. 21, 75. Wilkins, M. H. F., Zubay, G. & Wilson, H. R. (1959). J. Mol. Biol. 1, 179. Zubay, G. & Wilkins, M. H. F. (1962). J. Mol. Biol. 4, 444.