dna “melting” proteins · manifested by a shift of the thermally induced helix-coil transition...

THE JOURNAI. OF BIOLO(:ICAI. CHEMI~THY Vol. 251, No. 22, Issue of November 25, pp. 7198-7214, 1976

Printed in U.S.A.

DNA “Melting” Proteins

I. EFFECTS OF BOVINE PANCREATIC RIBONUCLEASE BINDING ON THE CONFORMATION AND STABILITY OF DNA*

(Received for publication, March 11, 1976)

DAVID E. JENSEN+ AND PETER H. VON HIPPEL

From the Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403

Bovine pancreatic ribonuclease is a DNA “melting” protein, since it binds with greater overall affinity to the single-stranded than to the double-stranded form of natural and synthetic deoxyribose-containing polynucleotides. As such, the DNA-RNase system provides a simple model for the more complex and biologically relevant melting protein-nucleic acid systems. Aspects of the DNA-RNase interactions which are related to the quantitative assessment of this system as a melting protein model are investigated here.

A boundary sedimentation velocity technique is used to measure thermodynamic parameters of the interaction; association constants (K,, and K,) and site sizes (n,, and n,) are determined for the interaction of ribonuclease with native (double helical) and denatured (random coil) DNA. It is shown that log K, and log K, are linear functions of log l?Ia+], binding decreasing with increasing Na+ concentration, with K, about 2 orders of magnitude smaller than K, at the ionic strengths studied. n,, and n, are -8 and -11 nucleotide residues, respectively, indicating that potential binding sites overlap. Binding to both forms of DNA is non-cooperative.

It is shown by CD and ultraviolet spectroscopy that the binding of RNase to single- and double- stranded DNA perturbs the conformations of these polynucleotide conformations very little relative to the unliganded structures. Hydrodynamic methods are used to show that RNase binds to native DNA with- out altering the overall solution structure of the latter; however conditions which permit binding to, and stabilization of, transiently exposed single-stranded sequences result in a collapse of the stiff native DNA structure.

We demonstrate by melting transition studies that ribonuclease does bring about an equilibrium destabilization of native DNA and poly [d(A-T) ] and, by applying a ligand-perturbed helix;icoil theory developed by McGhee (McGhee, J. D. (1976) Biopolymers l&1345-1375), it is shown that the extent of the observed destabilization is in semiquantitative accord with expectations based on the measured affinity constants and site sizes for RNase binding to both DNA conformations.

Spectral methods are used to show that the relative stability of native DNA sequences of varying base composition is the same in the presence and absence of ribonuclease, strongly arguing that this “melting” ligand “traps” single-stranded sequences transiently exposed by thermal fluctuations. RNase also undergoes an ordersdisorder conformational transition as a function of temperature (the denatured form of RNase stabilizes native DNA, while native RNase destabilizes the native double helix), and the coupled equilibria involved in these interacting conformational changes are interpreted and discussed as possible models of genome regulatory interactions.

Some years ago, in the course of an investigation of the use of protein probes to examine the relative stability of double

*This research was supported in part by United States Public Health Service Research Grants GM-15792 and GM-15423, as well as by Predoctoral Traineeship (to D. E. J.) from United States Public Health Service Training Grant GM-00444. This work was submitted (by D. E. J.) to the Graduate School of the University of Oregon in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

$ Present address, Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331

helical DNA sequences of varying base composition, Felsenfeld et al. (1) discovered that bovine pancreatic ribonuclease can destabilize the native DNA conformation (i.e. lower the temperature of the double helixscoil melting transition) and suggested that this effect was due to preferential binding to the single-stranded form of the polynucleotide chain. This property established ribonuclease as the first DNA “melting” protein to be so identified, although no one thought this property to be more than a serendipitous consequence of the fact that, as a ribose-specific nuclease, RNase might be ex-

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Conformation & Stability of DNA Complexed with Ribonuclease 7199

petted to show a preferential (although catalytically ineffec- tive) binding affinity for any single-stranded polynucleotide chain.

In 1968 such preferential affinity of proteins for single- stranded nucleic acid conformations became more than an academic curiosity, when Alberts and co-workers (2) reported the isolation and partial characterization of the gene 32-protein coded by T4 and synthesized in large quantities during lytic T4 bacteriophage infection of Escherichia coli. This protein had been shown to be essential for the DNA replication, recombina- tion, and perhaps repair aspects of T4 phage biosynthesis (for references, see Ref. 3), and its major in vitro “activity” appeared to be preferential (and cooperative) binding to single-stranded DNA sequences. This discovery of a biologically relevant DNA melting (or “unwinding;” Ref. 4) protein, for which physiological function appeared to reside in the melting property, together with the simultaneous development of DNA-cellulose chromatography (2) as a tool for the isolation and purification of such proteins, triggered a flood of reports of the isolation of melting proteins from both prokaryotic and eukaryotic cells (see Ref. 4). At present, it appears that such proteins may play a central role in chromosomal expression and manipulation in a wide range of organisms.

To avoid confusion, we will use the following definition of a melting protein in this and the following papers: a protein is a DNA (or RNA) melting protein if, at equilibrium under the conditions of the experiment, it can achieve a higher binding density on the single-stranded conformation than on the double-stranded conformation of nucleic acid. Experimentally, this differential binding density brings about an equilibrium destabilization of the double helical conformation which is manifested by a shift of the thermally induced helix-coil transition of the nucleic acid to lower temperatures.’

In order to help elucidate the molecular and thermodynamic bases of the function of DNA (or RNA) melting proteins in more detail, we have undertaken a series of studies on proteins and protein models of this class. In approaching this problem, it has been useful to consider simple organic molecules, capable of forming reversible adducts with functional groups of polynucleotides exposed to reaction only in the single-stranded conformation, as “primordial” melting protein models. For this reason (in part), we have conducted an extensive series of

’ Several aspects of melting protein experiments may prevent the actual observation of the displacement to lower temperatures of the nucleic acid helixscoil transition, and thus require this careful definition of a melting protein. These situations, all of the following of which are, in fact, displayed in this series of papers, include: (a) for proteins which bind cooperatively to single strands, the effective affinity for single-stranded polynucleotides may only be greater than that for the double helical form when binding to single strands is contiguous (that is, when cooperativity is in effect). In this case the ligand may only meet the definition of a melting protein at concentrations sufficient to ensure cooperative binding. (b) The melting protein may itself denature at temperatures below that of the “test” melting experiment. If the relative binding affinities of the protein for the two nucleic acid conformations are appropriate at temperatures at which the protein is stable, we consider the native protein to be a melting protein under these conditions. (c) Kinetic effects may prevent realization of the equilibrium displacement of the melting profile under some conditions. For example, low probabilities of exposure, in predominantly double helical nucleic acids, of single-stranded loops of length or conformation adequate to permit equilibrium (often cooperative) binding of melting proteins may prevent binding during experiments of finite duration, even when binding is thermodynamically favored. This situation results in effectively hysteretic melting transitions, indicating that the equilibrium state is not equally accessible from low and high temperatures.

mechanistic studies of the chemical and conformational aspects of the interaction of formaldehyde with nucleic acids and nucleic acid components (5-7 and Footnote 2). The effects on nucleic acids of real melting proteins differ from, and are more complex than, those of formaldehyde primarily in that: (a) real proteins bind to (and cover) more than one functional group onthe polynucleotide lattice and (b) the binding may be cooperative in protein concentration. Both of these physico- chemical features may have significant physiological conse-

quences, and in this and the following papers we attempt to analyze and dissect two melting protein systems of increasing complexity which illustrate these features. First (this paper), we present a detailed study of the effects on DNA structure and stability of the non-cooperative binding of ribonuclease. This is followed (3, 8, 9) by a comparable examination of the effects of the cooperative binding of T4-coded gene 32.protein to nucleic acids and nucleic acid models.3

Since the initial identification of ribonuclease as a DNA melting protein (l), the following features of the DNA-RNase interaction have been elucidated. (a) RNase has been shown to bind preferentially to single-stranded DNA, resulting in a pronounced destabilization of the double helical DNA conformation (1, 10, 11). The magnitude of this destabilization has been shown to be markedly dependent on pH and ionic strength. (b) RNase has been shown to act as a melting protein only in its native form (1); the denatured “polycationic” form of RNase (12) functioning as a DNA double helix stabilizer (13). (c) The enzymic “active site” of ribonuclease has been implicated in DNA binding; denatured DNA, and to a lesser extent native DNA, serving as effective competitive inhibitors of enzyme action on RNA substrates (14).

In this paper we complete the characterization of ribonu-

clease as a model nucleic acid melting protein by establishing the binding constants and site sizes associated with the binding of ribonuclease to native and denatured DNA, the effects of ribonuclease binding on the solution properties and “long range” structure of native DNA, the effects of ribonuclease binding on the secondary structure of native and denatured DNA, and the effects of ribonuclease binding on the thermal melting behavior (helix-coil transition) of DNA.

MATERIALS AND METHODS

Reagents and Buffers-All reagents were American Chemical Soci- ety certified or reagent grade, spectrally pure. Solutions were made with double-distilled water. The buffer salt used in this work was 1 rnM Na,HPO,; all solutions also contained 0.1 mM Na,EDTA to complex possible contaminating metal ions. Sodium chloride was added to bring the solutions to the desired ionic strength and the pH at room temperature was adjusted with HCl.

Ribonuclease-Crystalline pancreatic ribonuclease A (phosphate and salt-free) was purchased from Worthington. RNase concentrations were routinely measured by absorbance at 277.5 nm using an extinction coefficient of 9.56 x lo3 M ~’ cm-‘, based on a molecular weight of 13,700 (15) and an extinction coefficient (per mg/ml) of 0.698 at 277.5 nm (16).

DNA and Polynucleotides-Samples of highly polymerized native DNA prepared from calf thymus were obtained from Worthington. DNA (approximately 2 mg/ml) was dissolved in 0.1 M NaCl, 1 mM Na,HPO,, 0.1 mM Na,EDTA, pH 7.7, buffer with very slow stirring at

‘5. D. McGhee and P. H. von Hippel (1976) submitted to Biochem- istry.

‘We note here that some melting proteins found in cells appear to bind non-cooperatively to single-stranded nucleic acids (e.g. the calf thymus melting protein, Herrick and Alberts (51)). Thus, this study of ribonuclease binding serves not only as a transition to the more complex cooperatively binding melting proteins of the gene 32-protein type, but also as a self-contained model for such “real” systems.


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7200 Conformation & Stability of DNA Complexed with Ribonuclease

4-6”. Dissolved DNA was centrifuged to remove a light precipitate and routinely deproteinized by phenol and chloroform extraction. The DNA was then extensively dialyzed against buffer, and stored at 4”. T7 bacteriophage DNA was isolated and purified in this lahoratory using standard techniques. DNA concentrations were determined by absorbance at 260 nm (at room temperature). The following extinction coefficients (in terms of nucleotide residues) were used: calf thymus DNA, 6.5 x lo3 Mm’ cm-‘; T7 DNA, 6.6 x lo3 M I cm I. Denatured DNA was prepared by heating DNA samples in sealed tubes at 100” for 10 min.

obtained on a Cary 14 spectrophotometer, interfaced to a Varian 620i computer. This apparatus permits the measurement of digitized spectra to high accuracy, as well as the automatic subtraction of baselines. A detailed description of this system is in preparation.” Temperatures were measured with a thermocouple inserted in a dummy cuvette. All ultraviolet spectra were collected at 24.0 i 0.2”.

Poly [d(A-T)] was obtained from P-L Biochemicals, and dissolved and deproteinized as described above. Concentrations were determined using a molar extinction coefficient (per mol of phosphate) of 6.65 x lo3 M ’ cm ’ at 262 nm (17).

Determination of Rind& Parameters-A boundary sedimentation velocity technique developed in this laboratory for the determination of nonsequence-specific protein-DNA binding parameters was used to obtain binding constants and site sizes for the ribonuclease-DNA interaction. A detailed description of this technique appears elsewhere (18). In brief, the desired concentrations of calf thymus DNA and protein are placed in a nitrocellulose centrifuge tube (along with a shallow sucrose gradient to retard convection and facilitate the collection of fractions) and spun in a Beckman SW 50.1 swinging bucket rotor at 30,000 rpm for 1 to 3 h. During this time the boundary of the relatively low molecular weight unbound protein moves very little, while, due particularly to the molecular weight heterogeneity of the calf thymus DNA, the DNA .protein complex forms a broad concentration gradient down the tube. The contents of the centrifuge tube are then fractionated, and each fraction in the unbound protein plateau region assayed for total protein and total DNA. The bound protein concentration will be proportional to the DNA concentration in each fraction and the unbound protein concentration will be a constant. Thus, a plot of total protein versus total DNA in the several fractions describes a straight line with a slope of u (binding density in moles of protein bound per mole of DNA nucleotide) and an intercept corresponding to L,,,, (the unbound protein concentration in moles/ liter). These parameters provide one point on a binding isotherm. A binding curve is constructed using data gathered from several such runs in which the input protein concentration is varied, yielding (as discussed under “Results”) an estimate of the affinity constant and binding site size for the protein interacting with the DNA lattice.

In this study, protein and DNA concentrations in the centrifuge tube fractions were determined by ultraviolet absorption. An aliquot of each fraction was quantitatively diluted to bring the NaCl concentration to about 1 M and the optical density was determined at two wavelengths, 230 and 260 nm. These data were used, along with estimated extinction coefficients for each component at these wavelengths, to solve two simultaneous equations and thus obtain concentrations in each fraction. The extinction coefficients (in 1 M NaCl) used were: for native DNA, 2.80 x lo3 and 6.50 x lo3 Mm’ cm ’ (per mol of phosphate); and for heat-denatured DNA (at room temperature), 3.17 x lo3 and 7.10 x lo3 M-’ cm-’ at 230 and 260 nm, respectively. The ribonuclease extinction coefficients at 230 and 260 nm were 75.0 x lo3 and 4.60 x lo3 M I cm ‘, respectively.

Ultraviolet Spectral An&is-The melting profiles of some DNA and DNA’RNase complex samples were studied using the two-term analysis of the hyperchromic spectra developed by Hirshman and Felsenfeld (20). This technique requires the collection of spectral data at four wavelengths (250, 260, 270, and 280 nm) before and during the helix-coil transition, and yields an estimate of the concentration of total phosphates, dA .dT base pairs and dG .dC base pairs melted as the transition proceeds. Spectral information as a function of temperature was gathered using the Cary 14 system described above. DNA sample absorbances were determined relative to buffer, and each DNA. ribonuclease complex sample absorbance relative to a cuvette containing the equivalent protein concentration. During the melting experiments the temperature was increased at a rate of about 1.5”/min using a temperature programmer identical to that used on the Gilford model 2000. A program written for the PDP-10 computer was used to analyze the data utilizing the Hirshman and Felsenfeld spectral parameters (20) after correction for thermal volume expansion. These spectral parameters have been determined so that the analysis yields a good estimate of the moles of dA .dT and dG .dC base pairs melted when the transition is complete (defined as the point at which there is less than a 0.5% change in the magnitude of the absorbance at 260 nm over a 3” temperature increment), based on the input DNA concentration and the base composition. However, as mentioned above, there is a continuing temperature-dependent hyperchromicity beyond the helix-coil transition, due to the optical properties of single-stranded DNA. Since this coil hyperchromicity is observed to be linear with temperature in the post-transition region, it has been customary to assume that the same linearity exists over the temperature range of the transition itself and the values of “fraction melted” determined relative to a back extrapolation of the post-transition coil hyperchromicity. Thus, even though the spectral analysis closely predicts the correct concentration of melted components at the defined end-point of the transition, a coil hyperchromicity correction is required when determining the fractions melted at points in the transition region. (Such a correction is required since the DNA.RNase complex helix-coil transitions are usually broad; see “Results”). Therefore, when working up the results of the spectral analysis, the calculated molarity of dA.dT and dG .dC base pairs melted was plotted against temperature, and the fraction melted of each determined by extrapolation from the linear post-transition region. Corrections have not been made for the pretransition increase in hyperchromicity, which is generally quite small.

Thermal Melting Profiles-DNA, protein, and DNA .protein complex melting transitions were monitored using a Gilford model 2000 automatic recording spectrophotometer, modified for melting experiments as described elsewhere (19). The cell compartment temperature was controlled by a model T-9 Tamson constant temperature circulating bath connected to an external refrigeration unit. During the melting experiments the temperature was increased at a constant rate of -0.6” per min, using a Northeast Scientific Co. automatic temperature programmer to drive the thermoregulator of the bath. All melting profiles were determined at 260 nm. The raw melting data were corrected for thermal expansion, and in some cases reduced to relative hyperchromicities (i.e. absorbance at temperature Tlabsorbancqat the base temperature), using a simple PDP-10 computer program.

Circular Dichroic Absorption Spectra-Ultraviolet circular dichroic absorption (CD) spectra were measured on a Cary model 60 spec- tropolarimeter with a model 6001 CD accessory. In this study, circular dichroism is expressed either as 0, the measured ellipticity in degrees, orasAe(= eL cH = 0/331c, where 1 is the path length in centimeters and c is the concentration in moles of nucleotide residues or moles of protein). All CD spectra were obtained in l-cm path length cells on samples with an absorbance at 260 nm of less than 1.20 A units. Unless otherwise indicated, the spectra were obtained at room temperature.

In CD melting experiments a water-jacketed cell was used, and the temperature maintained with a thermostated circulating bath. The bath thermoregulator was incremented manually, and the temperature monitored using a thermistor probe inserted directly into the cell. Samples were allowed to equilibrate for 15 min at each temperature before spectra were taken; melting experiments were terminated at 50”.

In some experiments the melting data are presented as the “fraction melted” as a function of temperature. Here the data, previously corrected for thermal expansion, are further corrected for small increases in hyperchromicity which appear to be linear with temperature and occur before and after the major change in hyperchromicity due to the helix-coil transition. The volume-corrected optical densities are plotted, and straight lines drawn through the pre- and post-transition points. Points in the helix-coil transition region are then established as fraction-melted relative to these 0 and 100% limiting lines.

CD spectra were recorded from 340 to 230 nm, at a scan rate of about 50 A/min and a full range scale of 0.0440.1”. Spectra were digitized by hand in 2.5 nm increments after a smooth curve had been drawn through the data. Melting transition runs were not corrected for temperature-dependent volume changes.

Sedimentation Coefficient Determinations-Sedimentation coefficients were measured with a Spinco model E Analytical Ultracentri- fuge, employing the Photoelectric Scanning System and ultraviolet optics. A 12.mm path length double sector cell with an aluminum- filled Epon centerpiece was used. All runs were monitored at 265 nm. Generally, a scan was taken once every 4 min. Scanner traces were

Ultraoiolet Absorption Spectra-Ultraviolet spectral data were ’ W. A. Melchior and C. Klopfenstein, manuscript to be submitted.


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analyzed in the standard manner, and sedimentation coefficients calculated from the motion of the 50% concentration point in the boundary region. Routine runs for the determination of approximate double helical polynucleotide molecular weights were made at 40,000 rpm and 20” in buffer, the polynucleotide concentrations ranging between 25 and 50 pg/ml. Estimates of the molecular weights of double helical DNA samples were made using the empirical formulas of Eigner and Doty (21), which relate s’~~,~, values to molecular weights.

RESULTS

A. Binding of Ribonuclease to Native and Denatured DNA

Binding parameters for the interaction between DNA and

protein have generally been determined by techniques which

physically separate the bound from the unbound species after

binding equilibrium has been attained. Techniques such as

nitrocellulose filter binding (22, 23) and band sucrose gradient

sedimentation (24) physically separate the complex from the free ligand. Inherent in these techniques, however, is the

requirement that DNA-protein binding affinity be very high or

that the separation be very rapid, or both, if the concentrations

of isolated bound and unbound species are to be representative

of those which existed in the actual equilibrium mixture. Thus,

these methods are not well suited for DNA-protein binding

systems characterized by association constants lower than

approximately 10” Mm’, the range characteristic of the ribonu-

clease-DNA interaction, as well as most melting protein.nu-

cleic acid complexes.

Raju and Davidson (25) utilized a gel filtration procedure,

modified after that developed by Hummel and Dreyer (26), to

estimate binding parameters of ribonuclease to native and

denatured DNA (ignoring, however, the effects of potential

“overlap” of binding sites, see below). This approach does not

involve separating the complex from the supporting free

);gand, but has the disadvantage of requiring considerable

amounts of protein since the entire gel filtration column is pre-equilibrated with ligand. The boundary sedimentation

velocity technique used in this study is rapid, general, accu-

rate, and, particularly in its “micro” version (3, 18), requires

very small amounts of DNA and protein.

Measurement of DNA-Protein Binding Constants-The boundary sedimentation velocity technique was used to deter-

mine the parameters controlling the binding of ribonuclease to

DNA. The binding equilibrium established before the run is not perturbed during the run while a concentration gradient of

the DNA’protein complex is established. The equilibrium

binding density (u, protein molecules bound per DNA phos-

phate residue) is independent of complex concentration, but

the total protein concentration (L,,,,,) will vary through the

gradient according to the equation:

L total = L rree + vPNAI (1)

where Lr,,, is the unbound protein concentration. (DNA

concentration is expressed in moles of phosphate groups/liter.)

When the contents of the centrifuge tube are fractionated, and

each fraction assayed for total protein and total DNA, a plot of L tota, uersus PNA] yields a straight line of slope v and

intercept (at zero DNA concentration) equal to Lrree. These

parameters establish one point on a binding isotherm. Several such runs, varying the input concentration of protein are re-

quired to generate the complete binding curve.

Range of Measurable Binding Constants and Sources of Error-In this study, the total DNA and total protein in each

centrifuge tube fraction were assayed by ultraviolet absorption.

This procedure, however, places definite limits on the range of

affinity constants which can be determined. The free protein

concentration must make a detectable contribution to the total

protein absorbance, well outside the error in the absorption

measurement itself, if reliable data are to be obtained. This

places the lower limit to the detectable free protein concentration at about 10m6 M and thus places an upper limit of

approximately lo6 Mm’ on the binding constants which can be

determined. At the other extreme, the upper limit of the free protein concentration is that concentration at which dilutions

can be made for the ultraviolet assay which still permit the

determination of the change in total protein concentration with

changing DNA concentration across the DNA.protein complex

gradient. Experimentally, this places a lower limit of about lo3

Me’ on the measured affinity constant.

The range of binding constants which can be determined

by the sedimentation velocity technique can be increased by using different DNA and protein assay methods. It is noted,

however, that in order to obtain a reasonable distribution of

points across the range of binding densities, the DNA and

protein concentrations must be increased or decreased more or

less in concert so that the magnitude of either of the terms on

the right side of Equation 1 does not overwhelm the other.

For tight binding systems this requirement reduces the assay options to those techniques which can detect very low levels

of both DNA and protein; i.e. sensitive chemical or optical

methods and/or radioisotope techniques.

In the binding studies reported here, the range of binding

densities which could be measured under a given set of solvent

conditions was dictated at low values of binding density by

the lowest detectable free protein concentration, and at high

v values by the tendency of the DNA .ribonuclease complex to

precipitate out of solution. Precipitation in this system occurs at binding densities in excess of 0.06 to 0.07 for native DNA

binding and at slightly higher values of v for denatured DNA.

RNase complexes.

Data have been gathered for the binding of ribonuclease to native calf thymus DNA, and to heat-denatured and retooled

calf thymus DNA, in a low phosphate buffer (1 mM Na,HPO,,

0.1 mM Na, EDTA, pH 7.7). Ionic strengths were varied by adjusting the concentration of added sodium chloride. Repre- sentative binding plots are shown in Fig. 1 for the binding of

ribonuclease to native and denatured DNA under the indicated

solvent conditions. The size of the data points approximates the standard error in the assay of u and v/Lt,,,. The probable

sources of data scatter when using the sedimentation velocity

binding technique are discussed elsewhere (18). The binding

isotherms fitt,ed to these points were calculated using an

equation for the binding of large ligands to DNA developed by

McGhee and von Hippel (27).

Binding Model and Theory-Pancreatic ribonuclease has an

active site which will accommodate two phosphodiester-linked

ribonucleosides (15). Since single-stranded DNA carries all the

structural features of single-stranded RNA except the 2’-OH

group on the ribose ring necessary for the enzymic cleavage of

diester bonds, it is very likely that the protein binds to

single-stranded DNA via similar active site interactions. In

support of this view, Walz (28) has found that ribonuclease has

the same (if not a slightly greater) affinity for deoxyribonucleo-

tides as for ribonucleotides. Further, it has been shown by

Sekine et al. (14) that denatured DNA competitively inhibits

the enzymic reaction of ribonuclease with an RNA substrate,

suggesting that binding to the DNA single strand sequesters

the active site. These workers have also observed competitive


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Ribonuclease - Native DNA

0.02 M Na+ pH 7.7 24OC

002 004 006 006 010

u

60-

- KASSOC. ‘\\

- \ \

T I I I I’

B. -

Ribonuclsase - Denatured DNA

005 M No+ pH 77 24OC

0 I 1 I 002 004 006 008 010

v

FIG. 1. Experimental binding plot and theoretical binding isotherm for the ribonuclease-DNA interaction. Basic buffer: 1 mM Na,HPO,, 0.1 mM Na,EDTA, pH 7.7. Sodium chloride added to bring solution up to indicated ionic strength. A, native ribonuclease-native calf thymus DNA; 0.022 M Na+, 24”. Calculated binding constant, 2.89 x 10’ Mm’, calculated site size, 9.0 nucleotides (4.5 nucleotide pairs). B, native ribonuclease-heat-denatured calf thymus DNA; 0.050 M Na’, 24”. Calculated binding constant, 5.68 x 10’ M ‘. calculated site size, 10.1 nucleotides.

inhibition of ribonuclease-mediated hydrolysis of RNA when

native DNA is included in the reaction mixture. Several

experimental approaches have indicated that ribonuclease can specifically bind a phosphate anion in its active center (see

Ref. 15), so it is quite probable that the interaction between

native DNA and ribonuclease involves a similar active site

interaction with at least one phosphate group on the outside of

the double helical structure.

If DNA binding does utilize primarily the ribonuclease

active site, each binding event will involve only a very few

DNA residues. As a consequence, this model predicts, to a first

approximation, that binding to DNA will be non-nucleotide sequence-specific. The good fit of the binding data to a

homogeneous lattice model (see below) strongly supports this

assumption. However, the physical bulk of the ribonuclease molecule may cover a number of residues along the DNA chain,

making these regions inaccessible to further protein binding

events. Thus, even though only a very few residues may be

directly involved in the binding interaction, the actual binding

(occlusion) site size on native or denatured DNA may be

considerably larger (see Ref. 9 for a further discussion of this

point). Also, since binding is assumed to be nonspecific, these

DNA binding sites will not be discrete, i.e. the translation of a bound protein molecule 1 residue in either direction along the

DNA chain will place the protein in another binding site. Thus,

potential DNA binding sites are “overlapping.”

The binding theory cited above (Ref. 27; for other ap-

proaches to this theoretical problem, see also Refs. 29 to 31)

deals with the problem of overlapping binding of large ligands

to a one-dimensional, homogeneous lattice. In the present

context the lattice is the DNA chain, assumed to be infinitely

long so that “end effects” can be ignored. Since nonspecific

binding is assumed, the DNA chain can be considered to be

homogeneous with respect to protein binding even though it

may, in fact, be heterogeneous in base composition. This

one-dimensional lattice model is very suitable for treating

ribonuclease binding to single-stranded DNA.

Matters may become more complex when applying this

model to the binding of proteins to native double helical DNA.

Thus, for proteins binding primarily through electrostatic

interactions with backbone phosphates, binding may be of the

“cross-groove” type, involving elements of both chains to take

advantage of the higher charge-density of the native structure,

in which case the entire double helical DNA model can be

considered a single one-dimensional lattice. On the other hand,

binding of such proteins might instead involve interactions

with only a single backbone, leaving the other chain free for

additional ligand binding. Then, the double helical native

DNA could be treated as two independent lattices.

For non-cooperative ligand binding to a one-dimensional,

infinitely long lattice with overlapping binding sites, the

binding equation (Ref. 27) becomes:

v = n-l

K(l-nv) (2)

Lfree

where v is the binding density as defined above, L,,,, is the

unbound ligand concentration (an approximation to the ligand

activity), K is the association constant for the binding of the

first ligand to an otherwise ligand-free lattice, and n is the

(occlusion) site size in units of nucleotide residues.

For present purposes we will make calculations in terms of

both extreme classes of independent binding possibilities cited

above. If, for calculations of association constants and site sizes

for the binding of ribonuclease to native DNA we assume

independent binding of the protein to a single backbone, then

we define our lattice in terms of nucleotide residues and interpret site size as nucleotide residues covered. If instead we

treat the entire double helix as a single lattice, using the

“croSsgroove” interaction model, then the lattice (and the site

size) is defined (and reported) in terms of nucleotide base

pairs. The shapes of the theoretical binding isotherms (for

non-cooperative binding) for the two cases are sufficiently

similar (27) that the experimental data (Fig. 1A) cannot be

used to discriminate these models; thus, site sizes for the native DNA lattice are reported either as nucleotide residues or

as one-half as many nucleotide base pairs. Again, in a subsequent section in which we attempt to fit the

calculated melting profiles of double helical polynucleotides in

the presence of melting proteins to the experimentally ob-

served transitions, a specific model of protein binding to native

DNA must be assumed. In the initial calculations we have


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Conformation & Stability of DNA Complexed with Ribonuclease

utilized the cross-groove binding model which treats the double helical structure as a single one-dimensional lattice. This model can easily be replaced in the calculations (see Ref. 32 for the general approach) if experimental findings make another model seem more appropriate; the independent backbone lattice model has also been tested and the differences between the two theoretical melting profiles are too small to be discriminated on the basis of the experimental results.

Other, more complex models, in which two or more mutually interfering modes of protein binding to the DNA double helix are postulated, can also be developed within the framework of a one-dimensional lattice model, but the accuracy of the experimental data do not presently justify such theoretical exercises.

Binding of RNase to Native and Denatured DNA-A binding isotherm described by Equation 2 has been fitted to each set of experimental data gathered for native and denatured DNA binding. The calculations were carried out using a computer program (written by Dr. James McGhee, University of Oregon) in which trial values of K and n are alternately varied until the best least squares fit to the v/L,,,, uersus Y plot is obtained. Representative binding isotherms are shown in Fig. 1, and the binding constants and site sizes obtained under several solvent conditions tabulated in Table I. The fit of the calculated curves to the binding data over the range of binding densities accessible to the assay is quite satisfactory. This result, coupled with the trends we find in the calculated binding parameters as a function of ionic strength (considered below), gives us some confidence in the model and the assumptions used in obtaining binding parameters. It is noted that these calculations are carried out assuming protein binding is non-cooperative; consideration of the shape of binding isotherms when cooperativity factors are inserted into the binding equation (Ref. 27) strongly indicate that protein-protein cooperativity is not involved in ribonuclease binding to native or to denatured DNA.

A linear relation between sodium ion concentration and the binding constant data of Table I is obtained when the logarithm of the binding constant is plotted against log [Na+]. Such a plot is presented in Fig. 2, in which least squares best fit lines have been drawn through the data for native and denatured DNA binding. The pronouced sodium ion concentration dependence suggests that charge-charge interactions contribute markedly to the free energy of the binding interaction. These interactions doubtless involve the binding of one or more negatively charged DNA phosphates in and near the active site of ribonuclease, with ionic strength effects on the binding constant reflecting the weakening of these interactions as a consequence of the direct binding competition of buffer components for the interacting charged groups. This component of the binding free energy, according to simple ligand binding theory (e.g. see Ref. 29) should show a linear dependence of log K on log [Na+] (or on the concentrations of both buffer cations and anions if both components are involved in competitive binding). Recently Record et al. (33) have developed a general thermodynamic treatment of the binding of charged ligands to nucleic acids, and have shown (after making certain assumptions) that the slopes of plots of log K versus (e.g.) log [Nat] can be analyzed to determine the number of charge-charge interactions involved in binding the protein to the nucleic acid lattice; the greater the slope (for a given lattice), the more interactions of this type are involved in the binding. (The data presented here have, in fact, been

TABLE I

7203

Ribonuclease-DNA binding parameters

Basic buffer: 1 mu Na,HPO,, 0.1 mM Na,EDTA, pH 7.7. The ionic strength was adjusted with sodium chloride. The indicated sodium ion concentration is the sum of the sodium chloride and basic buffer contributions.

Lattice Na’ Temper- Affinity Rindin~ ature constant site size

M

Native calf thymus 0.010 DNA 0.012

0.022 0.032

0.052

Heat-denatured and 0.030 24 5.6 x lo5

retooled calf thymus 0.050 24 5.7 x lo5 DNA 0.070 24 1.2 x 10’

24"

5

24

30 24

24

24

M ’

3.4 x 105 2.5 x lo5 1.5 x 105 1.1 x 105 2.9 x 10’ 6.8 x lo3 1.4 x 103

mrrkofidcs

9.4

7.4

7.5

7.3

9.0 8.3

9.2

8.3"

11.0 10.1 12.5 11.2”

n Average site size.

I06

IOJ

7

r Y

104

103

I , I I I

Log K ys Log [No+] DNA: RNose

ptl 7.7

q Dtmotured DNA 0 Native DNA

I I I I I

IO-’ 10-Z

[Na+l(M)

FIG. 2. Association constants for native ribonuclease binding to native and denatured calf thymus DNA as a function of sodium ion concentration. Basic buffer: 1mM Na,HPO,, 0.1 rnM Na,EDTA, pH 7.7, 24’; sodium chloride added to bring solution up to indicated ionic strength. Native DNA, 0; denatured DNA, IZI.

analysed in just these terms by Record and co-workers (33).) We note that the RNase-denatured DNA plot is steeper than that

for the RNase-native DNA system (Fig. 2), indicating the involvement of more charge-charge interactions in the former type of complex (see Ref. 33).5

Some measurements for ribonuclease binding to native DNA has been carried out at higher and lower temperatures (Table

‘A similar, but considerably less steep, linear dependence of binding free energy on log [Nat] is seen for the gene 32-protein-native DNA interaction and is also interpreted in terms of direct phosphate binding to positively charged groups in the active site of gene 32-protein (3, 9, 33).


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I), in order to afford an estimate of the enthalpy change involved in the binding process. The results (data not shown) indicate that AH = -5 (+l) kcal/mol for this interaction.

The ribonuclease binding site sizes listed in Table I average -8 nucleotide residues (or -4 nucleotide base pairs) per protein molecule for binding to the native DNA lattice, and -11 nucleotide residues per protein molecule for single- stranded DNA binding, and these values have been used in subsequent calculations. There is no apparent trend in site size with ionic strength. As seen in Fig. 1, the theoretical isotherm becomes very shallow at high binding densities due to the very high free protein concentrations required to approach complete lattice saturation (27). As a consequence, small changes in the positioning of the binding isotherm may result in rather large changes in the abscissa intercept (l/n), accounting for the observed scatter in the determination of site size.

In general (Fig. 2), we see that RNase binds to single- stranded DNA at the various ionic strengths examined with an affinity which is about 2 orders of magnitude greater than its binding affinity for native DNA. Since the site sizes for binding to the two lattices are similar, we conclude that ribonuclease meets the definition of a melting protein and should bring about a net destabilization of the double helix as, indeed, is observed (1). After considering the effects of this ribonuclease binding on the optical and hydrodynamic properties of DNA (sections R and C), we use the binding constant and site size parameters measured here to predict the shapes of the equilibrium melting profiles of DNA (section D).

Possible Errors Associated with Using Denatured DNA as “Pure” Single-stranded Lattice-When natural DNA is heat- denatured and retooled, there is some hypochromicity regained relative to the high temperature state. For calf thymus DNA, ll,ost of this hypochromicity is due to base stacking in short, probably imperfectly base-paired regions of secondary structure (34). Over the range of ionic strengths considered in these binding studies, protein-free calf thymus DNA, heat-denatured and slowly cooled to 25”, recovers approximately 10% of its hypochromicity; addition of ribonuclease to the denatured DNA preparation removes most of this hypochromcity by melting these hairpin structures.

In the binding calculations it has been assumed that all the nucleotides in denatured DNA are available for the single strand mode of ribonuclease binding. While this is qualitatively correct, there will be some loss in protein binding density due to the favorable free energy of formation of base-paired regions along the DNA strand. Since the average length and distribution of this secondary structure is unknown, and the influence of ribonuclease binding on these base-paired regions is not understood in detail, it is not possible to calculate definitive correction factors to arrive at “pure” single strand binding parameters. However, we have gone through a calculation to determine the maximum error which could be expected from this source when using denatured and retooled DNA as a “single strand” model (for details see Ref. 35). It is apparent that applying such a correction to the experimental binding densities will move the binding isotherm (see Fig. 1) upwards and to the right. The result will be a higher value of K and a higher value of l/n, and therefore a decrease in the estimated site size. For an extreme model the values of K and n, based on observed parameters and treating heat-denatured and retooled DNA as an ideal single-stranded lattice, are, at most, about 30% too low and about 20% too high, respectively.

B. Effects of Hibonuclease Binding on Secondary Structure of Native and Denatured DNA

In order to determine whether the interaction of ribonuclease with DNA perturbs the secondary structure (backbone conformation and vicinal base-base interactions) of DNA, we have conducted careful spectral examinations of native and denatured DNA complexed with ribonuclease.

Circular Dichroism of the Native DNA .RNase Complex- Native DNA, complexed with ribonuclease up to about 40% of saturation, shows no changes in its ultraviolet absorption spectrum (data not shown; see Jensen, Ref. 35). This suggests that the binding of ribonuclease to native DNA does not measurably unstack the bases in this conformation.

Optical activity, as measured by circular dichroic absorption (CD) spectroscopy, is more sensitive than ultraviolet spectroscopy to details of local conformational change. Therefore, CD studies were undertaken to determine whether the association between ribonuclease and native DNA stabilizes unique local conformations in the double-stranded structure. The accumu- lation of such conformations at high protein binding densities, or perhaps a cooperative transition of the DNA helix to a different structure which is induced by some critical level of binding density, should be readily detectable as changes in the CD spectrum.

Fig. 3 shows the CD spectrum of a native DNA.ribonuclease complex, along with control spectra measured on equivalent concentrations of DNA and protein alone. Using the native binding constant appropriate for the solvent conditions of this experiment (obtained by linear extrapolation of the data of Fig. 2), it is calculated that the binding density of the protein on the DNA helix is at about 62% of total saturation. The sum of the control DNA and ribonuclease ellipticities has been calculated at 2.5 nm intervals across the spectrum and is also shown in Fig. 3. It is seen that the complex spectrum closely approximates the sum of the ellipticities contributed by protein and DNA alone, suggesting that the binding of ribonuclease, even at very high binding density, does not induce any significant conformational deformation of the native DNA molecule under these solvent conditions.

The temperature dependence of the DNA.RNase complex CD spectrum was investigated using the synthetic polymer poly [d(A-T) ] as a model for DNA. Under appropriate salt conditions, poly [d(A-T) ] denatures at a temperature below that at which ribonuclease itself is heat-inactivated, thus allowing observation of the single-stranded DNA .native ribonuclease complex CD spectrum. Fig. 4 shows the CD spectra of the poly [d(A-T) 1. ribonuclease complex and the poly [d(A-T) ] control, at several temperatures. The insets in Fig. 4 show the relative ultraviolet hyperchromicity of separate aliquots of these samples as a function of temperature, giving an indica- tion of the extent of denaturation of the poly[d(A-T)] double helical structure at the temperatures at which spectra were taken. In the control (Fig. 4B), the increase with increasing temperature in the magnitude of the positive CD band at 261 nm, and the decrease in the negative band at 246 nm, has been found by Gennis and Cantor (36) to be characteristic of a wide variety of synthetic and natural DNAs in the double helical conformation. The molecular basis of this temperature- dependent effect is presently unknown.

The same temperature-dependent trend is observed in the poly[d(A-T)]-ribonuclease spectrum at 10 and 20” (Fig. 4A).


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Native DNA: RNose -----.

240 260 280 300

Jy(nm)

FIG. 3. Circular dichroic absorption spectra of native calf thymus DNA, RNase, and native DNA.ribonuclease complex; 0.008 M NaCl, 1 rn~ Na,HPO,, 0.1 mM Na,EDTA, pH 7.7, 24”. Complex and control spectra are 1.37 x lo-” M DNA and 1.30 x 10 ’ M ribonuclease. With K "allYe = 3.2 x lo5 Me’ and n = 8, the calculated binding density is 0.078 or 62% of total saturation. Native DNA .ribonuclease complex, -; native DNA control, - - -; ribonuclease control, -----; the sum of the control DNA and protein ellipticities calculated at 2.5 nm intervals, 0.

1 I

40 A. Polvd(AT): RNose - i

I I I I I 6 I I I

240 260 200 300 X (nm)

\I, a

L _...*

I I I 240 260 280 300

x (nm) FIG. 4. Circular dichroic absorption spectra of poly[d(A-T)] and

poly[d(A-T)].ribonuclease complex as a function of temperature; 0.01 M NaCl, 1 rn~ Na,HPO,, 0.1 rn~ Na,EDTA, pH 7.7. A, complex spectra. Input poly [d(A-T)], 1.24 x lo-” M, input ribonuclease, 7.22 x lo-’ M. Under these solvent conditions the native binding constant is 1.46 x lo5 M- ’ and the site size is 8 nucleotides. The calculated binding density on native DNA is 0.05, about 40% of total saturation. The denatured DNA binding constant is 8.0 x 10’ M-‘, the site size 11 nucleotides, and the estimated binding density about 64% of total saturation. Inset, relative hyperchromicity at 260 nm [(Acomplex,T - A pmte,n. ,oo)lA ,>“lY ,I#( A-T, I, 10 01 as a function of temperature of an aliquot of the same poly[d(A-T) ] ribonuclease complex. Arrows indicate temperatures at which CD spectra were obtained. B, control spectra. Input poly [d(A-T)], 1.24 x lo-” M. Inset, relative hyperchromicity as a function of temperature of an aliquot of the same poly[d(A-T)] sample. Arrows indicate temperature at which CD spectra were obtained.

The ultraviolet hyperchromicity indicates that there is some denaturation of poly[d(A-T)] at low temperatures, due to the

RNase melting protein activity. At 20”, approximately 11% of

the polynucleotide double helical structure is denatured.

Nevertheless, at this temperature the double helical structure

which remains is still covered with protein to about 36% of

total saturation. This level of binding density apparently has

little effect on the temperature-dependent changes in the

positive CD band at 261 nm. The small decrease in the

magnitude of this band is accounted for by the negative

ellipticity contributed by the protein in this region (see Fig. 3).

Thus, we conclude that ribonuclease binding has little or no

effect on the conformation of double helical DNA or DNA

models.

Optical Properties of Denatured DNA. RNase Complen- The relative hyperchromicity curve shown in Fig. 4 indicates

that nearly all of the double helical structure in poly [d(A-T) ] is

“melted out” at 46-48”. Ribonuclease is still (for the most

part) native at these temperatures, with thermal inactivation

becoming apparent at around 52” (see below). The binding

density on the single strands is calculated to be about 64% of

total saturation. The main features of the complex CD curve at

48” are very similar to those observed for the control at 46”.

The long wavelength positive band peaks at 270 nm, the

crossover is at 261 nm, and the negative band occurs at 250 nm.

The amplitude of the positive band of the complex centered at

270 nm is, however, lower (by about 12%) than can be

accounted for by the negative ellipticity of the protein.

Although these results suggest that, overall, ribonuclease

binding to single-stranded DNA does not appreciably perturb

either the conformation of the sugar-phosphate backbone, or

the relative orientations of vicinal nucleotide bases, the fact that at least two bases of the polynucleotide substrate bind

directly in the RNase active site (15) suggests that at least 2 of

the 11 bases occluded in the binding reaction should be

somewhat perturbed as a consequence of the interaction. To

test this, we have examined the behavior of poly(dA) (in which

the bases are largely stacked at 10”) in the presence and

absence of ribonuclease. Fig. 5A shows a slight hyperchromic

change of the poly(dA) spectrum on binding increasing concen-

trations of ribonuclease under these conditions; comparison

with poly(dA) totally unstacked by heating (Fig. 5B) indicates

that saturating concentrations of ribonuclease unstack less

than 20% of the bases, as expected from the estimates made

above. Fig. 5B shows that the remainder of the poly(dA) “melts” (thermally unstacks) similarly to the uncomplexed

chain, until at -55” the curves merge as the ribonuclease

denatures. (The apparent hypochroism of poly(dA) at high

temperature may be due to the stabilization of stacking

interactions by the denatured protein.)

The data of this section show that ribonuclease binding does

not alter the stacking of bases along the single chain, except

perhaps for the bases which actually articulate with the active

site. This is in marked contrast to the behavior of gene

32.protein .poly(dA) complexes (3), where the bases are totally unstacked, presumably as a consequence of the extension of

the polynucleotide backbone.

C. Effects of Ribonuclease Binding on Hydrodynamic (Long Range) Structure of DNA

It has been shown that at salt concentrations below about

0.05 M, DNA undergoes a pronounced expansion with decreas-

ing ionic strength, as evidenced by a large and continuous


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7206 Conformation & Stability of DN A Complexed with Ribonucleose

FE. 5. Ultraviolet absorption spectra and ultraviolet melting curves of polg,dl\,.ribonuclease complexes: 0.018 H NaCl, 1 m” Na,HPO,, 0.1 mu Na.EDTA. pH 7.7. Input poly(dA), 2.25 x lWB M. Input ribonuelease: rurue I, poly(dAleontrol; curve 2. 3.21 x 10~‘ M; CUP-UP 3. 6.42 x to-’ M. A. “1tra”iolet spectra at KP, (protein absorbance subtracted out). B. hyperehmmicity at 2M) nm as a function of temperature relative to the po,y(dA,absorbance at W.

decrease in sedimentation coefficient (21, 37, 38) and a corresponding increase in intrinsic viscosity (39). These changes are generally attributed to increased repulsion between the phosphate groups of the DNA backbone, which results in expansion of the macromolecular domain, and thus in the observed changes in hydrodynamic properties.

As discussed in an earlier section, the affinity of ribonuclease for native DNA appears to be strongly influenced by the polyelectrolyte character of DNA and by the extent of counterion binding to phosphate groups along the chain. Thus, the RNase binding interaction could, in turn, perturb counterion association with the charged phosphates in the vicinity of the binding site and induce some rearrangement in the local ionic atmosphere. The cumulative effect of such perturbations could cause a detectable change in the tertiary structure (i.e. flexibility) of the molecule. It is also possible that, upon binding, the physical bulk of the protein could decrease the local DNA chain flexibility. In addition, protein association could promote or inhibit interactions between segments of DNA chains by protein-protein interactions or by protein-DNA interactions which are qualitatively different from those considered above. To determine whether large scale changes in tertiary structure do accompany RNase binding to native DNA, we have examined the sedimentation velocity properties of the DNA.RNase complex as a function of binding density.

Sedimentation Properties of DNA. RNase Complex-h these studies it was necessary to determine the sedimentation coefficient of the complex as a function of DNA concentration

to permit extrapolation to zero DNA concentration. However, simple dilutions were not experimentally feasible since, in order to maintain a given protein binding density on the DNA, the dilution buffer must contain the exact concentration of free protein necessary to support the binding equilibrium. To cir- cumvent this difficulty, sedimentation coefficients for DNA, ribonuclease complexes were determined at several input protein concentrations for each of four input DNA concentrations. The change in sedimentation coefficient with DNA concentration for each level of binding density could then be determined by extrapolation from such experimental data.

In Fig. 6A the experimental sedimentation coefficients are shown plotted against binding density, with smooth curves drawn through the data points obtained at each of the four input DNA concentrations tested. Values of the sedimentation co&cient have been determined from these curves at several binding densities and used to construct the l/s uersus DNA concentration plots shown in Fig. 6B. Least squares best fit lines were calculated, using the points for each binding density, and limiting sedimentation coefficient values determined fmm the intercepts of these lines with the ordinate. It is seen that the limiting sedimentation coeffXent increases with binding density.

The molecular weight of the DNA.protein complex is expected to increase with increasing prot$in binding density, countered to some extent by an increase in the buoyancy of the complex. To determine whether other factors play a role in the observed increase in s value with binding density, the experimental results have been compared with sedimentation coefficients calculated on the assumption that only the molecular weight and the buoyancy of the complex change with increased binding density. To simplify the analysis, these sedimentation coefficients have been normalized by taking the ratio of the s-value of the complex to that of protein-free DNA.

Using the standard definition of the sedimentation coefficient, the ratio of the s values becomes:

where i subscripts indicate DNA -RNase complex values and o subscripts the values for protein-free DNA. It is assumed that the only unknown factor in this equation is the ratio of the frictional coefficients, fJf,. I f the observed increase in s value with binding density is due entirely to the molecular weight and buoyancy terms, the ratio of the frictional coefficients will be equal to 1.

A numerical value of the molecular weight term is estimated by taking M., the molecular weight of the calf thymus DNA residue, to be -306. The molecular weight of a residue complexed with ribonuclease will be a function of the binding density (v) and is calculated from the relationship:

M, = dMW...,) + M. (4)

with the molecular weight of ribonuclease taken as 13,700. To determine the buoyancy term, we used iinN* = 0.55 ml/g (Ref. 40). c”Naw = 0.70 ml/g (Ref. 41). and a solvent density (pl equal to that of water at 24’. The partial specific volume of the DNA.ribonuclease complex is also a function of the binding density; if the partial specific volumes of DNA and protein are assumed to be unchanging and additive, then 6 of the complex


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70

60 S

50

40

B I I I

Nalive CT DNA: RNose Complexes

+- 0 40 C DNA1 (ughl)

FIG. 6 (left and center). A, experimental sedimentation coefficients for native calf thymus DNA as a function of ribonuclease binding density; 0.01 M NaCl, 1 mM Na,HPO,, 0.1 mM Na,EDTA, pH 7.7, 24”. Under these conditions the native binding constant is 1.8 x lo5 Mu’ and the site size is 8 nucleotides. An aliquot of each DNA-ribonuclease sedimentation mixture was also assayed for total DNA and total protein, utilizing the same two wavelength analysis used in the binding assay (see “Materials and Methods”). The binding density was calculated using eqs. (1) and (2) (see text), and the binding parameters listed above. Curue A, input DNA = 12.2 x 10m5 M (37.5 pg/ml); curue B, input DNA = 9.16 x 10m5 M (28.2 pg/ml); curve C, input DNA = 6.18 x 10m5 M (19.0 ag/ml); curue D, input DNA = 3.08 x lo-’ M

can be calculated as follows:

v(MW vi =

RNAse)(‘RNAse) + (MWo)(VDNA) (5)

v(MW RNAse) + MWo

The ratios of the s values (s,/so) at several binding densities

were thus calculated for frictional coefficient ratios of 0.9, 1.0,

and 1.1 and the results shown as dashed lines in Fig. 7B. Also shown are the experimental s values, extrapolated to limiting DNA concentration and normalized with respect to the limiting protein-free DNA sedimentation coefficient (the size of the data points approximate the estimated experimental error). It is seen that the experimental sedimentation coefficient ratios agree well, up to a binding density of about 0.05 (40% of total saturation), with the calculated ratios in which the frictional coefficient was assumed to be unchanged (fO/fZ = I).

The magnitude of the frictional coefficient is determined by many factors, among them empirical parameters which depend on the extent of polymer-polymer and polymer-solvent interactions and on the stiffness of the overall double helical DNA

molecule. The apparent insensitivity of the frictional coefficient to increasing ribonuclease binding indicates that ribonuclease binding to native DNA, at least up to -40% coverage of the DNA lattice, does not perturb the flexibility of the DNA as reflected in its hydrodynamic properties.

In Fig. 7A, the relative hyperchromicity of DNA.ribonuclease complexes at 23” is graphed as a function of the binding density. The solvent composition was identical to that used in the sedimentation runs. It is seen that the relative hyperchromicity is equal to 1.0 at binding densities below about 0.05

and, by this criterion, the DNA is assumed to be native over this range. Above this binding density the relative hyperchromicity increases, suggesting that an increasing fraction of

0 0.02 0.04 0.06 0.08

~mtive

(9.47 pg/ml). B, interpolated sedimentation coefficients plotted as l/s ueraus DNA concentration. The value of the binding density corresponding to each set of points taken from Fig. 6A is indicated.

FIG. 7 (right). A, relative hyperchromicity at 260 nm of DNA .ribonuclease complexes; 0.01 M NaCl, 1 mM Na,HPO,, 0.1 mM Na,EDTA, pH 7.7, 23”. Complex absorptions were determined against blanks containing the equivalent concentrations of protein. B, ratio of experimental s, to so as a function of binding density; the size of the data points approximate the standard errors of the ratios as calculated from the standard errors of the l/s intercepts (Fig. 6B). - - -, calculated curves for the indicated values of f,/f, (see text).

the DNA is denatured. This denaturation is probably due to the melting protein behavior of ribonuclease, becoming apparent only at high binding densities at 23”. Concomitant with this apparent denaturation is a sharp increase in the sedimentation coefficient of the complex, as indicated by the upward deviation in the data points of Fig. 7B. It is likely that locally denatured regions give the (mostly native) DNA molecule an overall greater flexibility, resulting in a faster sedimenting form.6

D. Effects of Ribonuclease Binding on Stability of Native DNA Conformation

Ribonuclease-perturbed DNA Melting Profile-The overall character of DNA.ribonuclease complex melting curves is determined in large part by the thermal denaturation of ribonuclease. This is due to the differential effect of native and denatured ribonuclease binding on DNA helix stability. Ap- parently both forms of ribonuclease (assuming, for simplicity, a two-state, nativesdenatured transition) bind to and stabilize the double helical conformation of DNA. Native DNA stabilization by native ribonuclease is implicit in the measured

affinity of the protein for this structure (see above). However, this stabilization is not directly observed under our solvent conditions, because concurrent DNA single strand stabilization by the native protein dominates the melting transition. On

‘Once ribonuclease begins to stabilize denatured regions in the complex, the binding density on the remaining double helical regions decreases. This has not been taken into account in Fig. 7B; thus, the binding density for the data points corresponding to partially denatured DNA (at the right side of the graph) are plotted along the abscissa as though all the DNA were still native. However, they do clearly show the trend of the sedimentaton coefficient values as denaturation of the double helix begins, accompanied by the “collapse” of the overall structure.


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protein denaturation the active site is lost, and with it the ability to preferentially stabilize single-stranded DNA. The denatured protein then becomes, in effect, a polycation (ribonuclease has a net charge of +3 or +4 at neutral pH; see Ref. 12), which stabilizes the higher charge-density double helical form of the DNA lattice.

These features of the ribonuclease-perturbed DNA melting profile are illustrated in Fig. 8A, which shows the equilibrum (reversible) melting behavior of T7 DNA in the presence of increasing concentrations (curves 5-2) of ribonuclease, under salt conditions where ribonuclease is tightly bound to the various forms of DNA throughout the transition. At temperatures below 55”, net destabilization of the native DNA structure by ribonuclease is observed; the melting transition (in this particular experiment) is shifted downward by 7-12” (relative to the control melt) as input protein concentration is increased, with little change in melting profile shape. However, the rate of increase of DNA hyperchromicity with temperature starts to fall off at about 50”, as the system begins to show the effects of ribonuclease denaturation. This is a consequence of the fact that denatured ribonuclease no longer serves as a native DNA destabilizer; indeed, as indicated above, because of the polycationic character of the denatured protein, it now binds preferentially to the higher charge-density native DNA double

helix, resulting in some renaturation of the DNA. However, Fig. 8A shows that, even below the transition temperature of the protein-free control, renaturation of the DNA is not complete, indicating that some native (DNA-destabilizing) ribonuclease remains. In fact, between 60 and 70”, we observe only a progressive increase in the fraction of double helix stabilized by denatured ribonuclease. This implies that over this entire temperature range the nativesdenatured protein equilibrium is piogressively shifted from the native (DNA-destabilizing) to the denatured (DNA-stabilizing) form. At temperatures above 70”, the stability of the native DNA-denatured RNase complex is overcome and the remaining native DNA melts out.

Temperature PC)

Temperature PC)

Clearly, then, melting transitions of the sort illustrated in Fig. 8A are the net result of a matrix of coupled equilibria involving interactions of native and denatured RNase with native and denatured DNA (see Fig. 10, below). For conceptual con- venience we divide the transition into two parts: a low temperature portion in which the shape and position of the DNA melting profile should be dominated by the distribution of native ribonuclease between single- and double-stranded DNA sequences and a high temperature portion in which equilibria involving denatured ribonuclease dominate the melting transition.

FIG. 8. Ribonuclease-T7 phage DNA melting curves; 1 mM Na,H- PO,, 0.1 mM Na,EDTA, pH 7.7. A, experimental melting curves; input DNA, 1.95 x 10m5M. Curue I, DNA control; curve2, input protein, 1.55 x 1Om6 M; curve 3, input protein, 1.24 x 1Om6 M; curue 4, input protein, 9.31 x 10-r M; curue 5, input protein, 7.45 x 10-r M. B, theoretical melting curves. Input DNA and ribonuclease concentrations as in A above. The input parameters for the calculation are as follows (we note that the binding parameters used in this and the following figures have been independently determined, see text); Z’,,, of the DNA = 59.4”; AH (enthalpy of helix formation per base pair) = 8 kcal/mol; nucleation parameter, 0, = 5 x lo-“; nh = 4 base pairs; n, = 11 bases; K, = 7.1 x 10’ M’; K, = 1.3 x 10” M-‘; AH (for ribonuclease helix and coil binding) = -5.0 kcal/mol; wh and wc = 1.

Native Ribonuclease Effects on DNA Stability-The low

temperature portion of the transition curve is more germane to the main thrust of these papers since it reflects primarily the melting protein behavior of native ribonuclease, presumably uncomplicated by the “antimelting protein” effects of the denatured nuclease. Assuming that this portion of the melting curve can indeed be modeled by a set of equilibria involving the binding of the native ligand to either the single-stranded or the double-stranded sequences of DNA, we have attempted to fit it by utilizing the theoretical approach to such systems developed by McGhee (32). This approach involves coupling helix- coil theory for polynucleotides (in which, as a first approximation, the helix is taken as homogeneous in base composition) to ligand binding equilibria involving the two forms of the polynucleotide. The unperturbed transition is characterized by T, (the experimentally determined midpoint temperature of

the helixscoil transition at the relevant salt concentration), by AH (the enthalpy of helix formation per base pair) for the free polynucleotide (these two parameters together define the value and temperature dependence of s, the equilibrium constant for adding a base pair to a double helical sequence), and by u (the parameter in which all cooperativity parameters are lumped in a simple nearest neighbor Ising model treatment). Ligand binding is expressed in terms of n,, K, and w, (the site size, association constant, and protein-protein interaction cooperativity parameter, respectively, for ribonuclease binding to coil sequences) and nhr K,, and wh (the same parameters relevant to ribonuclease binding to double helical sequences); for further details and definitions of these parameters see Refs. 27 and 32.

The results of such an analysis for the lower part (native ligand) of the experimental transitions of Fig. 8A, using binding parameters measured independently as described in preceding sections, are shown in Fig. 8B. The best fit he-


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lixzcoil transition parameters derived from poly [d(A-T) ] measurements (32) are used to model the unperturbed DNA, values

of n, and nh are taken from the results of Table I, values of K,

and K, from linear extrapolations of the log K uersus log ma+]

plots of Fig. 2 (we assume K, and K, are approximately

independent of nucleotide sequence and composition because

of the good fit of the data of Fig. 1, A and B to a homogeneous

lattice model), and wh and o, are taken as unity (see Fig. 1).

The actual parameters used in the calculations are listed in the

legend to Fig. 8B.

Clearly, the fit is qualitatively acceptable in that the

transitions are displaced to lower temperatures to about the

observed extent and the displaced transitions initially run approximately parallel to one another and to the control, but

the fit is quantitatively only mediocre. In light of the number of

parameters involved and the tentativeness of our estimations

of the value of several of them, as well as the semiarbitrary

nature of the model assumed for RNase binding to native DNA

(see section A, above) and the use of a homogeneous lattice in

these calculations, we have not attempted to improve the fit by

adjusting the input parameters. Rather, we consider briefly the

form of the melting profiles and the effects on the form of

modifying various of the parameters and models used. (a) The

temperature at which the perturbed transitions begin depends

on the ratio of K, to K, (for n,, s n,). Small errors in the

extrapolated values of K, and K, (as well as errors in estimates

of n, and n,J could easily account for the displacement of the

“bottoms” of the transitions from the observed (-40’) to the

calculated (-30”) values. Indeed, we have found that changes

of less than 1 order of magnitude in the input values of either

K e or K,, can match these parts of the calculated transitions to

those obtained experimentally. (b) The initial parts of the

experimental transitions may be less steep than the calculated

transitions because the denatured loops which originally appear in native DNA may expand less cooperatively, due to

the presence of higher melting dG .dC sequences, than in the assumed homogeneous lattice. A better fit of the calculated

melting curves to those observed may be achieved when a

theory describing ligand perturbation of heterogeneous DNA

helix-coil transitions becomes available. (c) Because of the

apparent breadth of the ribonuclease nativezdenatured transition in the presence of DNA (Fig. 8A), a simple division of

the DNA melting profile into a low temperature portion (pro-

tein native) and a high temperature portion (protein denatured)

may not be entirely justified when attempting to calculate DNA-ligand melting curves.

To assess the magnitude of the protein transition effect on

the DNA helix-coil equilibrium we turn now to an experimen-

tal evaluation of the perturbation of the native,?denatured

ribonuclease transition by the coupled helixscoil transition equilibrium of the DNA.

Effect of DNA Binding on the NatiuezDenatured RNase Transition-As indicated above, the unusual features at the

upper end of the experimental ligand binding-perturbed melt-

ing profiles of T7 DNA are due to RNase denaturation and the

“antimelting protein” behavior of the denatured form of this enzyme. Some of these effects are brought out in Fig. 9, A and , R, in which the behavior of the (hypochromic) ribonuclease

transition is studied in the presence and absence of native DNA (Fig. 9A) and denatured DNA (single-stranded poly(dT);

Fig. 9B).

The ribonuclease transition is monitored by observing

changes (decreases) in ultraviolet absorption at 287 nm, the

30 40 50 60 70

Temparalurs (“Cl

ii 8 a -0.04 -

30 40 50 60 70

Temperature (“0

FIG. 9. Ribonuclease melting curves in the presence of polynucleotide; 0.02 M NaCl, 1 mM Na,HPO,, 0.1 rn~ Na,EDTA, pH 7.7. A, ribonuclease and native calf thymus (C7’) DNA. Input protein concentration, 3.3 x 10 ’ M, input DNA concentration, 9.0 x lo-” M.

p, complex; - - -, RNase control. B, ribonuclease and poly(dT). Input protein concentration, 2.55 x 10m5 M; input poly(dT) concentration, 2.94 x 10.’ M. - -, complex; -, RNase control.

wavelength of a sharp minimum in the native-denatured

RNase difference spectrum (42). It has been found that the

midpoint (‘I’,) of this optical transition occurs at about 61” for

free ribonuclease and that this T, is rather insensitive to

changes in ionic strength over the range of salt concentrations considered in this study (16).

As Fig. 9A shows, the presence of native DNA depresses T, for the ribonuclease transition by several degrees, as expected from the increased affinity of denatured ribonuclease (relative

to native RNase) for the native DNA structure. In addition,

some RNase denaturation (involving 10 to 25% of the mole-

cules) is observed at temperatures as much as 20” below the

perturbed RNase T,. Thus, we can conclude that some denatured ribonuclease is present throughout the transitions

shown in Fig. 8A, which may account for some of the deviations

observed at low temperatures from the theoretical transitions shown in Fig. 88.

Fig. 913 shows that the situation has still another dimension,

since the preferential binding of native ribonuclease to single-

stranded DNA stabilizes the native RNase molecule against denaturation. In this experiment, poly(dT), which shows only

minor temperature-dependent changes in its optical proper-

ties, was present in excess, so that most of the RNase can be


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assumed bound. As expected, binding to single-stranded

polynucleotide sequences stabilizes the native RNase struc-

me, so in an experiment of the type illustrated in Fig. 8A, as

single-stranded sequences of DNA become available they

would be expected to antagonize the ribonuclease denaturing

effect of the double helical DNA, still further complicating the

situation.

The network of coupled equilibria which must be taken into

account in a full analysis of transitions such as those of Fig. 8A is summarized in Fig. 10. Aside from the complexity of the

necessary computations, the limitations on our knowledge of

the relevant native RNase binding parameters and our total

lack of information on the binding parameters for denatured

RNase preclude further attempts at quantitative modeling of

these transitions. However, we feel that this qualitative analy-

sis of the situation is well justified since physiologically

relevant DNA-protein interaction systems certainly do show

such mutually perturbing conformational changes in DNA-

protein interactions (e.g. the lac repressor-inducer-operator-

DNA system; see Ref. 43). Effects of RNase Binding on Poly[d(A-T)] Melting Transi-

tion-As a, perhaps, more realistic test of the ability of the

helix-coil transition theory, using the ribonuclease binding

parameters we have measured, to predict the reversible,

ligand-perturbed, DNA melting profiles, we have examined the

melting of the double helical model compound, poly[d(A-T)],

under various salt conditions in the presence of varying RNase

concentrations. This model compound is preferred over natural

DNA because it melts at a lower temperature, thus permitting

the transitions to proceed through most of the melting phase at

temperatures below the unperturbed RNase denaturation

transition and therefore avoids most of the effects of the

coupied DNA-RNase denaturation equilibria. Also, poly [d(A-T)] is a homogeneous polynucleotide and so the com-

plications of differential stability of various double helical

sequences are avoided.

Experimental transitions measured in 0.03, 0.02, and 0.01 M

Na+ are shown in Fig. 11, A, B, and D, and the comparable

theoretical curves (generated using the independently mea-

sured parameters listed in the figure legends) as shown in Fig. 11, A, C, and D. Again, the fit is qualitatively reasonable, but

quantitatively far from ideal. The major discrepancy in the

theoretical curves seems to be that they start at too high a

temperature and appear to be too sharp. Again, we are not in a

position to provide a definitive explanation of the quantitative

discrepancies. One possibility is that the double helical hairpin

loops transiently formed with poly [d(A-T) ] at temperatures

well below T, provide denatured sequences capable of binding

RNase in excess of the simple (two-state) loops predicted by

the simple helix,-coil theory used here. This would lead to an

extension of the ligand-perturbed poly [d(A-T) ] transition to

lower temperatures than predicted, in accord with observation

(and contrary to the findings with T7 DNA, which should be

incapable of making many such loops and which, indeed, starts

melting at temperatures higher than predicted by theory using

the same binding constants). This result is also in accord with

the observed effect of gene 32-protein on poly [d(A-T)], in that these transient hair-pin loops are found to provide “nuclei” for

gene 32-protein melting under conditions where natural DNAs

are impervious (3).

Thus, this explanation will account for the apparent “pre- melting” (relative to theoretical expectations) of RNase-

destabilized poly [d(A-T)]. The “top” of the experimental

nRNase~nDNA~nRNase~nRNase~dDNA

\DNA /r/ +I=

dDNf %

dRNase.nDNAe dRNaseedRNase.dDNA FIG. 10. Schematic representation of the various coupled equilibria

involved in the interactive native to denatured ribonuclease (nRNase *dRNase) and native to denatured DNA (nDNA*dDNA) transitions (see text).

transitions appear at too high a temperature (relative to

theoretical expectations) and thus may well be due to the

generation of some denatured RNase under these conditions,

which will stabilize the native DNA at the top of the transition

beyond the point expected on the basis of simple theory.

Obviously, there may be many other factors which partially

contribute to these discrepancies, again including errors in

parameter estimation, partially inappropriate models of the

interaction of the various forms of RNase with the binding

lattice(s), etc.’

In summary, the correspondence of the experimental and theoretical curves is sufficiently good to give us confidence in

the general predictive approach, and at least qualitatively

reasonable arguments can be raised to account for the direc-

tions of the deviations of theory from experiment.

DNA Base Sequences Melted by RNase Binding-One of the

questions toward which these melting protein studies are

directed is whether such melting proteins effect a destabiliza-

tion of the native helix by trapping open single-stranded loops

generated by spontaneous thermal fluctuations, or whether they

somehow force open the structure by forming some type of

intermediate complex with the closed structure and then

“isomerize” to the single-stranded ligand-bound complex. A

complete answer to this question can only be given after a

careful kinetic study of the entire reaction pathway, as has been carried out for the model “melting protein,” formalde-

hyde; for the HCHO-DNA system it has been shown unequivo-

cally that spontaneously generated loops are indeed trapped. *

Although such kinetic studies have not been done on the

RNase-DNA system or, indeed, on any melting protein-DNA

system some tentative approaches can be made.’ Thus, given

the apparent non-nucleotide sequence specificity of ribonu-

clease binding to DNA lattices, we may ask whether at

comparable “fraction melted” in the ligand-perturbed and the

uncomplexed DNA transition, the base composition of the

melted regions are the same. This question may be approached

by a spectral analysis of the hyperchromicity of the melting transitions.

It is well known that the stability of a DNA sample depends on its percentage composition of dG.dC base pairs; the melt-

7Pre1iminary calculations were also made of the ligand-perturbed poly [d(A-T)] transitions in which ribonuclease was assumed to bind indeuendentlv to each of the two sugar-ohosnhate backbones of double ., . helical DNA, instead of binding to both simultaneously in a cross- groove mode (see “Binding Mode1 and Theorv” under section A). The calculated transition curves for this model (data not show) are displaced insignificantly (by less than 1”) from those of Fig. 11.

*Some approaches to such experiments have been made with other protein-nucleic acid systems. Thus, Wingert and van Hippel (44) showed that the acid-soluble nucleotides liberated on initial digestion of native DNA by micrococcal nuclease increased progressively in adenine and thymine content as the experimental temperature is decreased below the T, of the DNA. Micrococcal nuclease has a strong preference for single-stranded regions and this experiment was interpreted in terms of “trapping” and preferential digestion by the enzyme of the intrinsically less stable, and therefore more probable, transiently open single-stranded dA .dT-rich sequences.


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Poly d(AT): RNose 0.03 M No+

-cl I 2 9, I c 0.5 -

.2 5 - Observed t ---- Colculoted

0 /

I I I IO 20 30 40 50 60

Temperature (“C)

0 LA IO 20 30 40 50 60

I

I.0 B I I

Poly d(AT) : RNase

0.020 M No+

_ Experimental Curves

1.0 c 1 r

Poly d(AT) : RNose

0.020 M No+

Theoretic01 Curves

0 I I IO 20 30 40 50 6

Temperature (“C)

10 D I

Poly d(AT). RNase

O.OlOM No+

- Observed

- -- z Calculated

_C

g 0.5 - : .- t 0 i

0 1 I 1 _ IO 20 30 40 50 60

Temperature (“C)

FIG. 11. Ribonuclease-poly [d(A-T)] melting curves; basic buffer, 1 rn~ Na,HPO,, 0.1 mM Na,EDTA, pH 7.7, sodium chloride added to bring the solution up to the indicated ionic strength. A, experimental and theoretical melting curves, 0.03 M Na +. p, experimental; - -, theoretical. Input poly[d(A-T)], 5.69 x 10m5 M; input protein, 2.61 x lo-’ M. The input parameters for the theoretical calculation are as follows: T, of poly[d(A-T)] = 51.5”; AH (enthalpy of helix formation per base pair) = 8.0 kcal/mol; c = 5 x 10 ‘; nh = 4 base pairs; n, = 11 bases; K, = 8.74 x lo3 Mm’; K, = 5.65 x lo5 Mm’; AH (for ribonuclease helix and coil binding) = -5.0 kcal/mol; wh and wc = 1. B, experimental melting curves as a function cf protein concentration, 0.02 M Na’. Input poly[d(A-T)] concentration, 5.8 x lo-” M. Input protein concentration: curue 1, poly [d(A-T)] control; curue 2, 1.36 x lo-” M; curve 3, 1.78 x 1Om6 M; curve 4, 2.09 x 10 6 M; curue 5, 2.51~ 10m6 M; curue 6, 3.14 x 1O-6 M. C, theoretical melting curves as a function of protein

ing temperature (as observed by optical density changes at 260

nm) increases nearly linearly with per cent dG .dC content (45).

This is due to the closely coupled denaturation behavior of dA .dT and dG .dC base pairs in heterogeneous DNA. The

interspersion of dG .dC and dA .dT pairs brings the mean melt-

ing temperature of the dA .dT pairs up, and that of the dG .dC

pairs down, from the values in the homopolymers, which differ

by approximately 40” (45). In spite of this overall base composi-

tional heterogeneity, there are regions in DNA which are rela-

tively dA .dT-rich, and other regions which are dG .dC-rich,

due to local random (though, of course, genetically based) fluctuations in nucleotide composition. The dA dT-rich sequences

Temperature (“C)

concentration, 0.02 M Na+. Input poly[d(A-T)] and protein concentrations as in B above. The input parameters for the theoretical calculation are as follows: I’,,, of poly[d(A-T)] = 47.2”; AH (enthalpy of helix formation per base pair) = 8.0 kcal/mol; c = 5 x lo-’ M; n,, = 4 base pairs; n, = 11 bases; Kh = 3.36 x 10’ Mu’; Kc = 3.58 x lo8 M-I; AH (for helix and coil binding) = -5.0 kcal/mol; w,, and W= = 1. D, experimental and theoretical melting curves, 0.01 M Nat. -, experimental; - -, theoretical. Input poly[d(A-T)] concentration, 5.8 x 10m5 M; input protein concentration, 2.72 x 1Om6 M. The input parameters for the theoretical calculation are as follows: T, of poly[d(A-T)] = 40.7”; AH (enthalpy of helix formation per base pair) = 8.0 kcal/mol; SJ = 5 x lo-‘; nh = 4 base pairs; n, = 11 bases; K, = 3.37 x lo5 Mm’; K, = 8.41 x 10’ Mm’; AH (for ribonuclease helix and coil binding) = -5.0 kcal/mol; wh and w, = 1.

age” heterogeneity, and the dG.dC-rich regions at slightly

higher temperatures thus giving an “intramolecular disper- sion” to the melting curve. It has been found that the differential melting of dA.dT-and dG.dC-rich regions in the

course of thermal denaturation can be understood via an analysis of the changing ultraviolet absorption spectrum

through the transition (20, 46).

The spectral analysis is based on the observation that the

hypochromicity of base-paired DNA can be accounted for

semiquantitatively by assuming that hypochromism arises

from interactions between neighboring base pairs. It is possible

to obtain information about the separate contributions of

melt at slightly lower temperatures than sequences of “aver- dA.dT and dG.dC pairs which have been denatured. The


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method of spectral analysis used in this study requires the coilection of data at four wavelengths (250, 260, 270, and 280 nm) for native DNA and through the helix-coil transition. These data are then applied to a series of equations for which the constants (spectral parameters) have been derived from the analysis of a large collection of bacterial DNAs (46). This type of analysis has been applied to the melting of calf thymus DNA in the presence and absence of ribonuclease (for further details of the calibration and analysis of the system, see Ref. 35).

Fig. 12A shows the helix-coil transitions of DNA and of the DNA.RNase complex, plotted as function of temperature. “Fraction melted” is the fraction of total dA.dT base pairs (solid line) or dG.dC base pairs (dashed line) melted in the course of the transition as determined by spectral analysis. It is seen that during the control DNA denaturation there are always more dA.dT base pairs melted at each temperature than dG.dC base pairs. The DNA’RNase complex melt (Fig. 12A) shows the expected net destabilization of the DNA double helix by native ribonuclease, and the collapse to the more hypochromic form of DrjA when the protein loses its active site by denaturation. The base composition of the regions successively destabilized in the complex by native RNase appears very similar to those of the DNA control. Near the end of the destabilization phase, however, some dG.dC base pair preference becomes apparent, and just before stabilization begins there appear to be more dG.dC pairs than dA.dT pairs melted. This is more easily seen when the dA .dT base pairs melted are directly compared with the dG .dC pairs melted (Fig. 12B). The DNA control curve (solid line) always lies above a diagonal between 0 and 100% melted, indicating that throughout the transition more dA.dT than dG.dC base pairs are melted. Further, through the low temperature three-quarters of the transition, the slope of the control curve is greater than 1, indicating that through this region more dA.dT than dG.dC base pairs are melting. The DNA.RNase complex curve indicates an apparent preference of native ribonuclease for dG.dC base pairs that becomes increasingly satisfied as the melt proceeds (the slope of the curve approaches 1 and then becomes less than 1). Although RNase has a general preference for pyrimidine mononucleotides, it has been observed that the cytidine monophosphates are slightly more effective than the uridine monophosphates in the inhibition of cyclic CMP hydrolysis by ribonuclease (47). Therefore dG.dC regions may be stabilized by RNase in slight preference in dA.dT regions. However, before suggesting that a weak dG.dC preference does apply here, the possibility that the observed curvature is due to an induced hypochromicity when ribonuclease binds to nucleotides must be explored.

Hummel et al. (48) observed that the binding of nucleotides by RNase resulted in negative ultraviolet difference spectra in the area of nucleotide absorption. It was later shown that these difference spectra essentially represent perturbations of the nucleotide spectrum when the nucleotide is in the more hydro- phobic environment of the ribonuclease active site (49, 50). To determine the possible influence of this RNase-induced nucleotide hypochromicity on the spectral analysis, a calculation has been carried out to estimate the maximum perturbation due to this effect. The details are presented elsewhere (Jensen (35)), and the result is shown as the corrected line in Fig. 12B. Clearly, a large fraction of the apparent preferential melting of dG .dC-rich regions by ribonuclease may be attributed to such a spectral artifact.

This spectral analysis suggests that the base compositions of

native DNA sequences successively destabilized by ribonuclease are not very different from those sequentially destabilized by thermal denaturation of uncomplexed DNA; i.e. those base pair sequences in the DNA double helix which are intrinsically less stable, and thus have a greater probability of being found in a transiently single-stranded conformation, are indeed those which are also most effectively destabilized by ribonuclease. This result strongly supports the view that ribonuclease operates as a melting protein by trapping sequences in proportion to their intrinsic probability of assuming a transient single-stranded state, rather than by forcing open the native structure on some other basis.

Effects of Helix Ends on DNA Melting Transition-To bolster further the interpretation that spontaneously opening single-stranded loops are trapped in predominantly native DNA by ribonuclease binding, it is important to assay the effects of “melting-from-the-ends” on the ribonuclease- induced T7 DNA transition. In Fig. 13, we compare control and ribonuclease-perturbed melting transitions for whole and extensively sonicated T7 DNA. The sonicated material is reduced in molecular weight to about 0.012 of its original value, representing an -180-fold increase in helix ends per molecule. Obviously (see the control DNA melts), the T, of the melting profile is decreased by -6” by sonication and the melting becomes slightly less cooperative (the transition is somewhat

less steep). The sonicated DNA-ribonuclease-perturbed transition shows a similar destabilization (-7”) relative to the un- sonicated control, and also some decrease in the steepness of the transition. Thus, to a first approximation, and taking into account the differences in extent of RNase denaturation in the two experiments, we may conclude that ribonuclease- induced melting does not involve appreciably more melting- from-the-ends than is characteristic of the DNA-only control of the same molecular weight. This finding is also in keeping with results with formaldehyde-’ and gene 3‘2-protein-induced (3) melting studies; in both of these systems also the ends of the DNA molecules seem to differ relatively little, as effective melting nucleation sites, from other regions of the double helical molecule.

DISCUSSION

In this paper we have examined the thermodynamic parameters-characterizing, and the structural consequences of, complex formation between DNA and the model melting protein, ribonuclease. The binding constants and sites sizes for the interaction of ribonuclease with native and denatured DNA have been measured for both lattices, and the data shown to fit well to a non-cooperative, non-base-sequence-specific, overlap binding model; the relative binding affinities at all ionic strengths tested show that RNase does indeed fit the criteria of a melting protein.

The conformation of both native and denatured DNA held in the RNase complex has been investigated by various techniques and it is shown that ribonuclease binding does not appreciably alter either the local or the long range conformations characteristic of unliganded DNA on either side of the melting transition. And this result, coupled with the fact that ribonuclease binding is non-cooperative, suggests the short single-stranded sequences which occur with finite probability at temperatures below the melting temperature of native DNA should be effectively trapped by ribonuclease under conditions where the free energy of binding exceeds the conformational free energy of the double helix, resulting in an equilibrium de-


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- dA.dT Base Pairs

---- dG * dC Base Pairs

0 60

Temperature

- DNA - DNA

--- DNA-RNase Complex DNA-RNase Complex

----- DNA-RNase Complex

brrected for Hypochromicily

----- DNA-RNase Complex

C,orrected for Hypochromicily

Fraction dG.dC Melted

FIG. 12. Ultraviolet spectral analysis of the ribopuclease.calf thymus DNA complex helix-coil transition by the two-term method of Hirschman and Felsenfeld (ZO), see “Materials and Methods” 1 mM Na,HPO,, 0.1 mM Na,EDTA, pH 7.7. Input calf thymus DNA, 6.42 x 10m5 M; input ribonuclease, 5.31 x 10m6 M. The absorption of the complex was determined relative to an appropriate protein blank; the optical density change upon ribonuclease denaturation contributes less than 1% to the total optical density. A, fraction of dA.dT base pairs (p) and dG dC base pairs (- - -) melted as a function of temperature for the DNA .ribonuclease complex and the DNA control. J3, plot of dA .dT uersus dG .dC base pairs melted; DNA control, -, DNA. RNase complex; ---; complex curve recalculated (see text) taking into account the induced hypochromicity when nucleotides are bound in the ribonuclease active site, -----

pression of the native DNA melting profile. The results pre-

sented here confirm this expectation, in contrast to the situa-

tion with gene 32.protein, where the unusual conformation of

the single-stranded DNA in the complex, together with the

consequences of cooperative binding, effectively prevents ob-

servation of the equilibrium destabilization of native DNA (3).

Attempts have been made to fit the equilibrium melting

profiles of native DNA and poly[d(A-T)] complexed with

ribonuclease, using the measured binding parameters for the

native protein to both single- and double-stranded lattices and the coupled large ligand-binding helixscoil transition theory

recently developed by McGhee (32). It is shown that the

predicted transitions fit the experimental melting profiles

qualitatively but that quantitative fit is not attained, probably

P f a.5 I I 6 ‘G :: t 1

0 I ---; 20 40 60 80

Temperature PC)

FIG. 13. Bacteriophage T7 DNA RNase melting curves; 1 mM Na,HPO,, 0.1 M Na*EDTA, pH 7.7, lNa+] = 0.002 M. p. 29.4 S DNA, approximate molecular weight = 17.8 x lo’, [DNA] = 1.95 x 10m5 M, [RNase] = 1.24 x 10m6 M. - -, 5.5 S DNA, approximate molecular weight = 1.4 x 105, [DNA] = 1.89 x 10m5~, @tNase] = 1.24 x ~O-‘M.

because of a combination of finite errors in multiple parame-

ters, partially oversimplified models, and most particularly

because the protein ligand itself undergoes a nativesdena-

tured conformational transition which is coupled to that of the

DNA lattice. Nevertheless, the system illustrates the effects of

various factors involved in non-cooperative ligand binding on

DNA melting transitions, and shows that such transitions,

even in complex situations, can be used to estimate ratios of

effective ligand binding constants to single- and double-

stranded polynucleotide lattices. (For further details on the

interpretation of such transitions, see McGhee (32).) As is seen

in the following paper (3), melting transitions generated in the

presence of cooperatively binding melting protein (such as gene

32-protein), in which essentially all the ligand is bound to

single-stranded sequences, are intrinsically easier to analyze in

order to extract estimates of binding parameters, since, in

contrast to the ribonuclease-DNA system, relatively few pa-

rameters dominate the behavior of the system in the observa-

ble regions.

The additional feature of a coupled conformational equilib-

rium involving the protein ligand complicates the interpreta-

tion of the higher temperature portions of the transitions.

However, in the sense that a conformational change (denaturation) of ribonuclese results in its conversion from an effective

DNA conformation destabilizer into a stabilizer, this behavior

does illustrate the subtlety and potential for genome control of

such a system. (The conformational transition of the ligand, in

a “real” genome regulating system, is generally induced by a

small change in the concentration of an ion or other physiologi-

cal regulator, rather than by a change in temperature.)

Overall, the DNA-RNase system provides a rather thor-

oughly worked out example of the interaction of a nucleic acid

with a non-cooperatively binding melting protein, which may

thus serve as a convenient model for the behavior of non-coop-

erative DNA binding proteins which are presumably more

physiologically relevant (e.g. the calf thymus DNA unwinding

protein; see Ref. 51). This system also serves as a transition to

the analysis of cooperatively binding melting protein-nucleic acid interactions, such as the T4 phage gene 32-protein-DNA

system to be considered in the papers that follow (3, 8, 9).

Acknowledgments-We are pleased to acknowledge the

advice and assistance of Dr. James McGhee in applying his


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ligand-coupled nucleic acid helixzcoil transition theory to this 21. system. 22.

REFERENCES 23.

1.

2.

Felsenfeld, G., Sandeen, G., and van Hippel, P. H. (1963) Proc.

Natl. Acad. Sci. U. S. A. 50, 644-651 Alberts, B. M., Amodio, F. J., Jenkins, M., Gutmann, E. D., and

Ferris, F. L. (1968) Cold Spring Harbor Symp. Quant. Riol. 33, 289-305

24. 25. 26.

Richardson. J. P. (1966) J. Mol. Riol. 21, 833114 Raju, E. V., and Davidson, N. (1969) Biopolymers 8, 743-755 Hummel, J. P.. and Drever. W. J. (1962) Biochin. Riophys. Acta

63, 530-532 27.

3.

4.

5.

6.

I.

8.

9

10

11

12

13

14

15

Jensen, D. E., Kelly, R. C., and von Hippel, P. H. (1976) J. Biol. Chem. 251,7215-7228

McGhee, J. D., and von Hippel, P. H. (1974) J. Mol. Biol. 86, 469-489

Kornberg, A. (1974) DNA Synthesis, W. H. Freeman and Co., San Francisco, Calif.

von Hippel, P. H., and Wong, K.-Y. (1971) J. Mol. Biol. 61, 587-613

28. 29. 30. 31.

Walz, F. G., Jr. (1971) Biochemistry 10, 2156-2162 Schellman. J. A. (1975) Hioaolvmers 14. 999-1018 Crothers, D. M. (l968) Bioiolimers 6, i75-584 Zazedatelev. A. S.. Gurskii, G. V.. and Volkenshtein. M. V. (1971)

Mol. Biol: 5, 194-198 McGhee, J. D., and von Hippel, P. H. (1975) Biochemistry 14, 32.

1281-1296 33. McGhee, J. D., and von Hippel, P. H. (1975) Biochemistry 14,

129771303

McGhee, J. D. (1976) Biopolymers 15, 1345-1375 Record, Jr., M. T., de Haseth, P., and Lohman, T. M. (1976) J.

Mol. Riol., in press 34.

Kelly, R. C., and von Hippel, P. H. (1976) J. Biol. Chem. 251, 7229-7239

McConnell, B., and von Hippel, P. H. (1970) J. Mol. Biol. 50, 317-332

Kelly, R. C., Jensen, D. E., and von Hippel, P. H. (1976) J. Biol. Chem. 251, 7240-7250

35. 36. 37.

Kosaganov, Yu. N., Lazurkin, Yu. S., and Sidorenko, N. V. (1967) Mol. Biol. 1, 301-309

Jensen, D. E. (1974) Thesis, University of Oregon, Eugene, Oregon Gennis, R. B., and Cantor, C. R. (1972) J. Mol. Biol. 65, 381-399 Rosenberg, A. H., and Studier, F. W. (1969) Biopolymers 7,

765-774

Lacombe, C., Cattan, D., and Laigle, A. (1974) Biochimie 56, 649-657

Tanford, C., and Hauenstein, J. D. (1956) J. Am. Chem. Sot. 78, 5287-5291

38. 39. 40. 41.

Triebel, H., and Reinert, K. E. (1971) Riopolymers 10, 827-837 Scruggs. R. L., and Ross, P. D. (1964) Riopolymers 2, 593-609 Hearst, J. E. (1972) J. Mol. Riol. 4, 415-417 Ulrich, D. V., Kupke, D. W., and Beams, J. W. (1964) Proc. Natl.

Acad. Sci. U. S. A. 52, 349-356 Lazurkin, Yu. S., Frank-Kamenetskii, M. D., and Trifonov, E. N

(1970) Biopolymers 9, 125331306 Sekine, H., Nakano, E., and Sakaguchi, K. (1969) Biochim.

Biophys. Acta 174,202-210

42. 43.

Foss, J. G. (1961) Biochim. Riophys. Actn 47, 569-579 van Hippel, P. H., Revzin, A., Gross, C. A., and Wang, A. C. (1974)

Proc. N&l. Acad. Sci. U. S. A. 71, 4808-4812 44

Richards, F. M., and Wyckoff, H. W. (1971) in The Enzymes (Boyer, P. D., ed) 3rd Ed, Vol. 4, pp. 647-896, Academic Press, New York

Wingert, L., and von Hippel, P. H. (1968) Biochim. Biophys. Acta 157, 114-126

45 46

16. 3909-3923

von Hippel, P. H., and Wong, K.-Y (1965) J. Biol. Chem. 240,

I-man, R. B., and Baldwin, R. L. (1962) J. Mol. Biol. 5, 172-184 Jensen. D. E.. and von HiDDd. P. H. (1976) Anal. Biochem., in

17 18

press

47. Ukita, T., Waku, K., Irie, M., and Hoshino, 0. (1961) J. Biochem.

Marmur, J., and Doty, P. (1962) J. Mol. Biol. 5, 1099118 Felsenfeld, G., and Hirschman, S. Z. (1965) J. Mol. Sol. 13,

(Tokyo) 50, 405-415

407-427

48. Hummel. J. P.. Ver Ploea. D. A., and Nelson, C. A. (1961) J. BiOl. I .

Chem.‘236, 3168-3172” 19 Olins, D. E., Olins, A. L., and von Hippel, P. H. (1967) J. Mol. 49. Irie, M., and Sawada, F. (1967) J. Riochem. (Tokyo) 62, 282-284

Eigner, J., and Doty, P. (1965) J. Mol. Biol. 12, 549-580 Riggs, A. D., Suzuki, H., and Bourgeois, S. (1970) J. Mol. Biol. 48,

67-83 Hinkle, D. C., and Chamberlin, M. J. (1972) J. Mol. Biol. 70,

1577185

Biol. 24, 157-176 50. Irie, M. (1968) J. Biochem. (Tokyo) 64, 3477353 20. Hirschman, S. Z., and Felsenfeld, G. (1966) J. Mol. Riol. 16, 51. Herrick, G., and Alberts, B. (1976) J. Biol. Chem. 251, 2133m

347-358 2141


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D E Jensen and P H von Hippelthe conformation and stability of DNA.

DNA "melting" proteins. I. Effects of bovine pancreatic ribonuclease binding on

1976, 251:7198-7214.J. Biol. Chem.

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dna “melting” proteins · manifested by a shift of the thermally induced helix-coil transition...

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