Vol. 129, No. 2 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Feb. 1977, p. 916-925 Copyright X) 1977 American Society for Microbiology
Spermidine-Deoxyribonucleic Acid Interaction In Vitro and in Escherichia coli ROBERT L. RUBIN Department of Medicine, National Jewish Hospital and Research Center, Denver, Colorado 80206
Received for publication 23 August 1976
The binding of spermidine to deoxyribonucleic acid (DNA) was studied by equilibrium dialysis in a wide range of salt concentrations. The association constants ranged from 6 x 105 M-I in 1 mM sodium cacodylate, pH 7.5, to 3 x 102 M-' in 0.3 M NaCl. MgCl2 reduced spermidine-DNA interaction even more than NaCl so that in moderate-ionic-strength solutions (0.3 M NaCl, 0.002 M MgCl2) there was little detectable binding. Low-ionic-strength media were used to isolate DNA from Escherichia coli by a method shown to minimize loss of spermidine from the DNA. Considerable spermidine was associated with E. coli DNA, but control experiments indicated that complex formation had taken place during or after lysis of the cells. Exogenous DNA or ribonucleic acid added to spheroplasts at the time of their lysis caused most of the cellular spermidine to be scavenged by the extra nucleic acid. The data suggest that spermidine is relatively free in the cell and thereby capable of strong (high-affinity) associations with nucleic acids only after the ionic strength of the cell environment is lowered.
The function of the naturally occurring poly- and their suspected reactants. Interactions of amines continues to be the elusive subject of putrescine, spermidine, and spermine with nuefforts involving a wide range of experimental cleic acids have been demonstrated in vitro (12, systems. One approach to this problem has 36, 39) and have led to numerous proposals been to attempt to isolate native complexes concerning in vivo associations (3, 8, 20, 29, 36). between polyamines and various cellular con- However, inorganic ions compete with polyastituents. A wide range of polyamine-macromo- mines for binding to nucleic acids (4, 17, 29). lecular complexes has been identified (see re- There is less association of polyamines with views by Stevens [37], Cohen [7], and Tabor nucleic acids as the ionic strength increases (17, and Tabor [41].) However, where adequate con- 21, 36, 39), but the extent of this salt-induced trols had been performed, artifactual associa- dissociation has not been quantitated. Analyses tions were readily demonstrated. Thus the poly- of the inorganic-salt concentration within E. amine content of Escherichia coli ribosomes coli have produced estimates of greater than 0.2 was highly dependent on the method of isola- M in monovalent ions and greater than 0.04 M tion (40), exogenous nucleic acids greatly re- in divalent ions (10, 23). It is not known duced the amount of [3H]spermine bound to whether the polyamine-nucleic acid complexes endogenous nucleic acids of an E. coli lysate previously observed in vitro are maintained in (20), and spermidine in E. coli that appeared the relatively high-ionic-strength environment bound to a membrane fraction completely of the cell. The present study focuses on the interaction equilibrated with [14C]spermidine added at the time of lysis (27). Major alterations of the polya- between spermidine and deoxyribonucleic acid mine levels in E. coli and phage T4 (1, 2) were (DNA). The importance of spermidine in biobrought about by modifications in the composi- logical systems is suggested by its ubiquity and tion of the growth medium. It appears that the tight biosynthetic regulation (7, 41). DNA was chemical activity of the polyamines is particu- chosen because it has a more uniform structure larly sensitive to seemingly small alterations in and function than ribonucleic acid (RNA). In environment, and therefore the possibility of addition, evidence that spermidine may interpolyamine exchange in any biological system act with DNA in E. coli has been deduced from needs rigorous assessment. their temporal biosynthetic relationship (18), An alternative approach toward elucidating the apparent reversal by spermidine of the propolyamine function has been to exploit in vitro tective effect of spermine on mutagenesis (3), binding studies involving various polyamines and the presence of spermidine in the DNA916
VOL. 129, 1977
containing, T-even phage (1). In the following work, the magnitude of saltinduced dissociation of spermidine from DNA is defined for the first time. Based on the results obtained, DNA was isolated from E. coli by a method that minimized exchange among macromolecules of polyamines that might be originally bound. It was found that this DNA contained appreciable amounts of polyamines, but control experiments showed that spermidine had associated with DNA after cell lysis. The finding that isolated E. coli DNA lacked natively bound spermidine may be due to a low affinity of spermidine for DNA in the cell. This novel proposal is consistent with the present as well as previous observations (20, 27, 29, 40) on the extensive exchange of polyamines during isolation of cellular constituents, and it is supported by the equilibrium dialysis data presented herein, which indicate negligible binding of spermidine to DNA in solutions of moderate ionic strength. The concept that spermidine may be relatively free in E. coli suggests that models depicting spernidine as a tightly associated, site-bound ligand (3, 8, 20, 21, 29, 36, 43) may not be relevant to the in vivo environment. MATERIALS AND METHODS Binding studies. Spermidine-DNA interaction was quantitated by equilibrium dialysis in various solutions. Dialysis cells were made by placing strips of 0.63-inch (about 1.6-cm)-wide boiled dialysis tubing (single layer) between the compartments of Lucite multicavity dialysis cells (Chemical Rubber Co.). Calf thymus DNA (Worthington) was prepared by exhaustive dialysis against 2 M NaCl followed by equilibration with 1 mM sodium cacodylate, pH 7.5, and diluted to 0.3 to 1.8 mM DNA phosphate. One compartment of each cavity was filled with 0.9 ml of the DNA solution; to the other compartment was added 50 ,ul of a NaCl or MgCl2 solution in 1 mM sodium cacodylate, pH 7.5, increasing volumes of tetramethylene-[1,4-"4C]spermidine (1 x 104 to 2 x 104 cpm/jAmol; New England Nuclear) in 1 mM sodium cacodylate, pH 7.5, and 1 mM sodium cacodylate to make the final volume 0.9 ml. Equilibrium was reached after gently shaking the cells for 17 to 20 h at 23 to 26°C. No net change in the solution volume within the compartments was detectable. Spermidine concentration was determined by counting a 0.5-ml sample from each compartment in 5 ml of 3a70 scintillation fluor (Research Products International) using a Packard model 3003 liquid scintillation spectrometer. DNA coxrcentration was determined by absorbancy at 260 nm (A260) of a diluted sample removed from the dialysis cell using El260 g = 21.4. Recovery of spermidine was essentially complete in all experiments, indicating the absence of significant spermidine binding to the dialysis membrane. Data were analyzed by the method of Scatchard
SPERMIDINE-DNA INTERACTION
917
(34) according to the following relation: nK - rK = rlc, where r is the number of spermidine molecules bound per DNA phosphate and c is the concentration of free spermidine. A plot of rlc versus r can be drawn, allowing the DNA-spermidine association constant (K) to be calculated from the negative of the slope and the total number of spermidine binding sites per DNA phosphate (n) to be estimated by extrapolating the line to the x-axis. The amount of spermidine on the DNA side of the membrane is due not only to the intrinsic affinity of spermidine for DNA but also to the necessity for maintaining electrical neutrality. This latter factor is particularly important at low ionic strength and can be estimated by calculating the equilibrium concentration of ions on each side of the membrane according to the Donnan expression (44), [Af]l[Am] = [Aj]2/(Z . [M] + 2- [A,]), where [Af]/[Am] is the ratio of the diffusible-anion activity on the free side of the membrane to that on the macromolecular side at equilibrium, [Ai] is the initial activity of diffusible anions, [M] is the molarity of DNA phosphates, and Z is the effective negative charge per DNA phosphate. The values for Z were obtained by plotting the data of Ross (31) against [NaClI and extrapolating to the equivalent average [NaClI present in each dialysis cell. Z ranged from 0.21 in 1 mM sodium cacodylate to 0.34 in NaCl concentrations of 50 mM or higher. For the Scatchard plots, each value of r was first corrected for the Donnan effect by dividing by [Afll[AmI. These correction factors are shown in Table 1. Because of the difficulty in determining the chemical activity of all diffusible ions, the uncertainty in the values for the Donnan factor is approximately + 10%. Scatchard plots for noninteracting ligands that bind nonspecifically to more than one lattice residue should be convex downward at high values of r (24) (see Discussion). In the present study, all plots of r/c versus r were drawn by least-squares fit to a linear regression curve because the extent of scatter in the data did not permit distinguishing between a plot that was linear and one that curved slightly at high values of r. Calculations were done with the aid of a Wang 2200 computer. DNA isolation. E. coli strain 15 TAU was grown to late log phase in 100 ml of synthetic medium (28) containing 20 ,uCi of [methyl-3H]thymine (13 ,uCi/ ,umol; New England Nuclear). Approximately 1.3 x 1011 cells were harvested and washed in 70 ml of 0.05 M tris(hydroxymethyl)aminomethane (pH 7.8) by centrifugation at 10,000 rpm for 15 min at 25°C. The resuspended pellet was incubated in 10 ml of a solution containing 0.05 M tris(hydroxymethyl)aminomethane, 20% sucrose, and 100 ,ug of lysozyme (Worthington) per ml for 20 min at 25°C (pH 7.8). The cells were washed twice in 70 ml of 20% sucrose-1 mM ethylenediaminetetraacetic acid (EDTA) as before. The spheroplasts were suspended in 1.2 ml of the sucrose-EDTA solution and lysed by gentle mixing with 23 ml of 1 mM EDTA, 0°C, containing 0.25 tg of ribonuclease (RNase) B (Sigma, preheated, 80°C, 15 min) per ml. The lysate was immediately layered on a sucrose step gradient consisting of 2 ml
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of 40% sucrose and 5 ml of 30% sucrose, both in 2 M urea (Schwarz/Mann, ultrapure)-l mM EDTA (pH 7.5) and centrifuged in the SW25.1 rotor (Beckman) at 22,000 rpm for 18 h at 00C. The bottom 3 ml was dispersed by three strokes in a rotating Potter-Elvehjem homogenizer and dialyzed in a 0.25-inch (about 0.6-cm)-diameter, preboiled dialysis bag against 1 mM cacodylate-0.1 mM EDTA (pH 7.5) for 2 h at 0°C. The preparation was then incubated with 10 jig of RNase (Worthington, RAF) per ml and 1 U of RNase T2 (Sigma) per ml for 45 min at 250C and layered on a sucrose gradient consisting of 1 ml of 70% sucrose and 2 ml of 60% sucrose overlayed with 22 ml of a 10 to 30% linear sucrose gradient, all containing 1 mM cacodylate-0.1 mM EDTA. After centrifugation as before, 1.2-ml fractions were collected from the bottom through an 18-gauge needle. Analytical determinations. DNA was quantitated by the diphenylamine method of Burton (6), using phenol-purified E. coli 15 TAU DNA as the standard. Bovine serum albumin was the standard for protein determination (22). RNA was analyzed by base lability of perchloric acid-precipitable A260 material (9). Polyamines were extracted from 0.2-ml aliquots of samples into 0.2 N perchloric acid and quantitated by a dansylation procedure (8), modified for dilute solutions. Putrescine and spermidine standards (Calbiochem) were dissolved in the same solvent as present in the samples. Radioactivity was measured as described in the binding studies. For double-label experiments, the windows of the spectrometer were set such that 16.7% of the 14C counts overlapped into the 3H channel and less than 0.01% of the 3H counts were detectable in the 14C channel.
RESULTS Equilibrium dialysis. It is generaly assumed that putrescine, spermidine, and spermine bind to DNA principally by bonds between polyamine amino and imino groups and DNA phosphates (15, 17, 35) (see Discussion). Consequently, inorganic salts would be expected to dissociate polyamine-DNA complexes, and this effect could be quantitated by equilibrium dialysis in various solvents. Such analyses were performed for spermidine-DNA interactions in concentrations of NaCl and MgCl2 applicable to the isolation of a spermidine-DNA complex from E. coli. Figure 1 shows typical Scatchard plots for spermidine binding to DNA as measured by equilibrium dialysis in increasing concentrations of MgCl2. Each data point was first corrected for Donnan effects as described in Materials and Methods. It can be seen that low [MgCl2] caused a marked decrease in the slope (which is the negative of the association constant, K) and a small decrease in the x-intercept (the total number of sites on DNA, n, available for spermidine binding). The results of a more extensive study of both MgCl2 and NaCl effects onK and n are shown in Table 1.
The sensitivity of the spermidine-DNA binding constants to salt concentration is clearly seen in Fig. 2. K changed little below 10 mM NaCl but decreased 2,000-fold as the [NaClI was increased to 0.3 M. MgCl2 had an even greater ability to reduce the interaction between spermidine and DNA. Just 10 mM MgCl2 decreasedK to 1,000 M-', a value only observed in [NaClI greater than 0.1 M. In the presence of both NaCl and MgCl2, spermidine binding to DNA was even less than the binding in the presence of either salt alone. This effect is shown in Fig. 3 as the moles of spermidine bound per DNA phosphate (r) as a function of increasing [NaCl] in the absence or presence of 2 mM MgCl2. It appears, therefore, that inorganic salts such as NaCl and MgCl2 effectively compete with spermidine for binding to DNA phosphates and, when simultaneously present in moderate concentrations, essentially eliminate measurable associations between spermidine and DNA. Isolation of a spermidine-DNA complex from E. coli. The binding data shown in Fig. 2 indicate that a spermidine-DNA complex would have maximum stability in solvents containing less than 2 mM NaCl and 0.2 mM MgCl2. Therefore, if spermidine is bound to DNA in the cell, these low-ionic-strength solvents would tend to "freeze" the complex, permitting its isolation from other components of the cell. However, if there is any spermidine not bound to 20,
150
K
0
FIG. 1. Scatchard plots of spermidine-DNA interaction based on equilibrium dialysis data. Solvents contained 1 mM sodium cacodylate (a) in addition to 01 mM MgC12 (U), 0.5 mM MgCl2 (A), and 2 mM
MgC12 (0).
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TABLE 1. Total numbers of binding sites, n (moles of spermidine/mole of DNA phosphate), and association constants, K, obtained by equilibrium dialysis in various solutionsa Solution
NaCl (mM) 0
Donnan correction
MgCl2 (mM)
K + SE (M-1)
n (± 10%)
0 1.03-1.11 6.47 ± 0.25 x 105 0.36 2.0 0 1.01-1.07 4.18 ± 0.11 x 105 0.31 10.0 0 1.01-1.02 2.51 ± 0.09 x 105 0.29 40.0 0 1.01 2.05 ± 0.06 x 104 0.21 100 0 1.00 2.48 ± 0.23 x 103 0.20 200 0 1.00 4.35 ± 0.19 x 102 0.19 300 0 1.00 3.19 ± 0.20 x 102 0.20 0 0.1 1.09-1.19 4.77 ± 0.09 x 105 0.25 0 0.5 1.07-1.10 8.12 ± 0.23 x 104 0.24 0 2.0 1.03-1.04 1.41 ± 0.22 x 104 0.19 0 4.0 1.02-1.03 4.38 + 0.23 x 103 0.23 0 6.0 1.02 2.14 ± 0.35 x 103 0.21 0 10.0 1.01 9.99 ± 0.12 x 102 0.21 a Each solution contained 1 mM sodium cacodylate in addition to the amount of NaCl or MgCl2 shown. The Donnan correction is the range of values calculated for each solution. SE is the standard error based on least-squares fit.
r .15-
.10\
0
.05C -7~ 1
0
1
2
3
log mMolarity -1
0 log
1
2
3
mMolarity
FIG. 2. Effect of[NaCI] (-) or [MgCIJ (U) on the spermidine-DNA association constant (K) in the presence of 1 mM cacodylate. The open circle represents the value of K in 1 mM cacodylate.
cell constituents, it might become associated with DNA in the complex-favoring environment of low-ionic-strength solvents. It appeared desirable, therefore, to lyse cells into a
FIG. 3. Effect of 2 mM MgCl2 on r (moles of spermidine bound/mol of DNA phosphate) in the presence of increasing [NaCI]. Each point was obtained by equilibrium dialysis of 0.40 umol of spermidine with 121 pmol of DNA phosphate in 1 mM sodium cacodylate and increasing [NaCI] in the presence (-) or absence (0) of 2 mM MgCl2. All values were corrected for the Donnan effect. The open symbols represent r in the absence of NaCl.
low-ionic-strength medium to maintain any spermidine-DNA complexes that may preexist in the cells and also to bring about a larger dilution of the cytoplasm to minimize artifac-. tual associations.
920
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J . BACTERIOL .
Lysozyme-treated E. coli strain 15 TAU was osmotically lysed into 1 mM EDTA, resulting in an approximately 200-fold dilution in the concentration of the cytoplasm (see Materials and Methods). One millimolar EDTA was intended to inhibit deoxyribonuclease activity (11) while still maintaining a solvent for which equilibrium dialysis data indicated a spermidine-DNA association constant equal to that of 4 mM NaCl (data not shown). The lysate was centrifuged through a sucrose step gradient as described in Materials and Methods. The DNAcontaining pellet was then banded on a second sucrose gradient (Fig. 4). Fractions corresponding to each of the two main peaks shown in Fig. 4 (fractions 1 through 7 and 8 through 16) were pooled and chemically analyzed as described in Materials and Methods and summarized in Table 2. A total recovery of 77% of the DNA present in the original lysate was obtained in the two peaks. The bottom peak (fractions 1 through 7) contained a large amount of protein and had a high turbidity (320/260 ratio) and buoyant density characteristic of bacterial DNA in association with cell membrane and wall fragments (14, 26). Ten
FR ACTION
FIG. 4. Sucrose gradient centrifugation profile of DNA-containing extract obtained from E. coli grown in the presence of [3H]thymine as described in Materials and Methods. Fractions were analyzed for absorbance at 260 nm and radioactivity of a 0.1-ml aliquot. Fraction 23 was from the top of the gradient. There was no detectable pelleted material.
percent of the nucleic acid in the bottom peak RNA. Approximately half the cellular DNA was present in fractions 8 through 16 along with considerable protein but no detectable RNA. The two DNA peaks contained a total of 3.8% of the putrescine and 10.6% of the spermidine found in the original lysate. This material, therefore, may be a polyamine-DNA complex isolated from E. coli, but modifications may have been introduced during the isolation. To estimate the extent to which spermidine might have been lost from DNA during the isolation, an artificial complex of DNA and [14C]spermidine (1 spermidine/15 DNA P042-) was subjected to the isolation procedure exactly as performed on the native DNA. The specific was
radioactivity ([14C]spermidine cpm/DNA P042-) of this complex decreased by only 13% after banding in the second sucrose gradient. The presence of a spheroplast lysate sonicated to minimize sedimentation of cellular DNA resulted in an additional 11% decrease in specific radioactivity of the artificial DNA-spermidine complex (Table 3). Although this is a relatively small loss (76% recovery of spermidine originally complexed to DNA), it suggests that the spermidine content of the DNA peaks (Table 2) may be an underestimation. Taken together with the 77% value for the recovery of DNA (Table 2), it can be estimated that 18% of the cellular spermidine may have been complexed to the cellular DNA in the original lysate. To estimate the extent of artifactual uptake of spermidine during the isolation of DNA, a sonicated lysate prepared from cells grown in [14C]spermidine was mixed with washed spheroplasts at the time of lysis in such a way (Table 4) that any 14C present in the DNA peaks shown in Fig. 4 could only arise if free spermidine or a spermidine-containing molecule associated with DNA during its isolation. There were significant levels of ['4C]spermidine in the DNA peaks (Table 4). The amount of spermidine taken up from the sonicated lysate by the DNA isolated from unlabeled cells was calculated from the specific radioactivity of the original mixture of lysates of unlabeled plus spermidine-labeled cells by assuming equilibration of spermidine between both lysates. Comparison of these levels (last column of Table 4) with the amount of spermidine obtained by direct chemical analysis (last column of Table 2) reveals no difference within experimental error. This finding indicates that all the spermidine on the isolated DNA could be accounted for by associations taking place during or after lysis of the cells. It appears, therefore, that the observed polyamine composition of the isolated DNA was not due to a native interaction between spermi-
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SPERMIDINE-DNA INTERACTION
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TABLE 2. Composition of DNA-containing peaksa Pooled frac- ['Hithymine % of cpm tions (cpm x 10-3) in lysate
ug of pro-
DNA
tein/ DNAg of
(Lg)
RNA/ DNA DN
AJ20IA
Putrescine (nmol)
Spermidine (nmol)
17.0 ± 1.1 25.8 + 1.2 0.11 0.240 1-7 5.56 331 108 26 0.005 13.7 ± 0.9 14.5 + 0.7 657 1.96
JOURNAL OF BACTERIOLOGY, Feb. 1977, p. 916-925 Copyright X) 1977 American Society for Microbiology
Spermidine-Deoxyribonucleic Acid Interaction In Vitro and in Escherichia coli ROBERT L. RUBIN Department of Medicine, National Jewish Hospital and Research Center, Denver, Colorado 80206
Received for publication 23 August 1976
The binding of spermidine to deoxyribonucleic acid (DNA) was studied by equilibrium dialysis in a wide range of salt concentrations. The association constants ranged from 6 x 105 M-I in 1 mM sodium cacodylate, pH 7.5, to 3 x 102 M-' in 0.3 M NaCl. MgCl2 reduced spermidine-DNA interaction even more than NaCl so that in moderate-ionic-strength solutions (0.3 M NaCl, 0.002 M MgCl2) there was little detectable binding. Low-ionic-strength media were used to isolate DNA from Escherichia coli by a method shown to minimize loss of spermidine from the DNA. Considerable spermidine was associated with E. coli DNA, but control experiments indicated that complex formation had taken place during or after lysis of the cells. Exogenous DNA or ribonucleic acid added to spheroplasts at the time of their lysis caused most of the cellular spermidine to be scavenged by the extra nucleic acid. The data suggest that spermidine is relatively free in the cell and thereby capable of strong (high-affinity) associations with nucleic acids only after the ionic strength of the cell environment is lowered.
The function of the naturally occurring poly- and their suspected reactants. Interactions of amines continues to be the elusive subject of putrescine, spermidine, and spermine with nuefforts involving a wide range of experimental cleic acids have been demonstrated in vitro (12, systems. One approach to this problem has 36, 39) and have led to numerous proposals been to attempt to isolate native complexes concerning in vivo associations (3, 8, 20, 29, 36). between polyamines and various cellular con- However, inorganic ions compete with polyastituents. A wide range of polyamine-macromo- mines for binding to nucleic acids (4, 17, 29). lecular complexes has been identified (see re- There is less association of polyamines with views by Stevens [37], Cohen [7], and Tabor nucleic acids as the ionic strength increases (17, and Tabor [41].) However, where adequate con- 21, 36, 39), but the extent of this salt-induced trols had been performed, artifactual associa- dissociation has not been quantitated. Analyses tions were readily demonstrated. Thus the poly- of the inorganic-salt concentration within E. amine content of Escherichia coli ribosomes coli have produced estimates of greater than 0.2 was highly dependent on the method of isola- M in monovalent ions and greater than 0.04 M tion (40), exogenous nucleic acids greatly re- in divalent ions (10, 23). It is not known duced the amount of [3H]spermine bound to whether the polyamine-nucleic acid complexes endogenous nucleic acids of an E. coli lysate previously observed in vitro are maintained in (20), and spermidine in E. coli that appeared the relatively high-ionic-strength environment bound to a membrane fraction completely of the cell. The present study focuses on the interaction equilibrated with [14C]spermidine added at the time of lysis (27). Major alterations of the polya- between spermidine and deoxyribonucleic acid mine levels in E. coli and phage T4 (1, 2) were (DNA). The importance of spermidine in biobrought about by modifications in the composi- logical systems is suggested by its ubiquity and tion of the growth medium. It appears that the tight biosynthetic regulation (7, 41). DNA was chemical activity of the polyamines is particu- chosen because it has a more uniform structure larly sensitive to seemingly small alterations in and function than ribonucleic acid (RNA). In environment, and therefore the possibility of addition, evidence that spermidine may interpolyamine exchange in any biological system act with DNA in E. coli has been deduced from needs rigorous assessment. their temporal biosynthetic relationship (18), An alternative approach toward elucidating the apparent reversal by spermidine of the propolyamine function has been to exploit in vitro tective effect of spermine on mutagenesis (3), binding studies involving various polyamines and the presence of spermidine in the DNA916
VOL. 129, 1977
containing, T-even phage (1). In the following work, the magnitude of saltinduced dissociation of spermidine from DNA is defined for the first time. Based on the results obtained, DNA was isolated from E. coli by a method that minimized exchange among macromolecules of polyamines that might be originally bound. It was found that this DNA contained appreciable amounts of polyamines, but control experiments showed that spermidine had associated with DNA after cell lysis. The finding that isolated E. coli DNA lacked natively bound spermidine may be due to a low affinity of spermidine for DNA in the cell. This novel proposal is consistent with the present as well as previous observations (20, 27, 29, 40) on the extensive exchange of polyamines during isolation of cellular constituents, and it is supported by the equilibrium dialysis data presented herein, which indicate negligible binding of spermidine to DNA in solutions of moderate ionic strength. The concept that spermidine may be relatively free in E. coli suggests that models depicting spernidine as a tightly associated, site-bound ligand (3, 8, 20, 21, 29, 36, 43) may not be relevant to the in vivo environment. MATERIALS AND METHODS Binding studies. Spermidine-DNA interaction was quantitated by equilibrium dialysis in various solutions. Dialysis cells were made by placing strips of 0.63-inch (about 1.6-cm)-wide boiled dialysis tubing (single layer) between the compartments of Lucite multicavity dialysis cells (Chemical Rubber Co.). Calf thymus DNA (Worthington) was prepared by exhaustive dialysis against 2 M NaCl followed by equilibration with 1 mM sodium cacodylate, pH 7.5, and diluted to 0.3 to 1.8 mM DNA phosphate. One compartment of each cavity was filled with 0.9 ml of the DNA solution; to the other compartment was added 50 ,ul of a NaCl or MgCl2 solution in 1 mM sodium cacodylate, pH 7.5, increasing volumes of tetramethylene-[1,4-"4C]spermidine (1 x 104 to 2 x 104 cpm/jAmol; New England Nuclear) in 1 mM sodium cacodylate, pH 7.5, and 1 mM sodium cacodylate to make the final volume 0.9 ml. Equilibrium was reached after gently shaking the cells for 17 to 20 h at 23 to 26°C. No net change in the solution volume within the compartments was detectable. Spermidine concentration was determined by counting a 0.5-ml sample from each compartment in 5 ml of 3a70 scintillation fluor (Research Products International) using a Packard model 3003 liquid scintillation spectrometer. DNA coxrcentration was determined by absorbancy at 260 nm (A260) of a diluted sample removed from the dialysis cell using El260 g = 21.4. Recovery of spermidine was essentially complete in all experiments, indicating the absence of significant spermidine binding to the dialysis membrane. Data were analyzed by the method of Scatchard
SPERMIDINE-DNA INTERACTION
917
(34) according to the following relation: nK - rK = rlc, where r is the number of spermidine molecules bound per DNA phosphate and c is the concentration of free spermidine. A plot of rlc versus r can be drawn, allowing the DNA-spermidine association constant (K) to be calculated from the negative of the slope and the total number of spermidine binding sites per DNA phosphate (n) to be estimated by extrapolating the line to the x-axis. The amount of spermidine on the DNA side of the membrane is due not only to the intrinsic affinity of spermidine for DNA but also to the necessity for maintaining electrical neutrality. This latter factor is particularly important at low ionic strength and can be estimated by calculating the equilibrium concentration of ions on each side of the membrane according to the Donnan expression (44), [Af]l[Am] = [Aj]2/(Z . [M] + 2- [A,]), where [Af]/[Am] is the ratio of the diffusible-anion activity on the free side of the membrane to that on the macromolecular side at equilibrium, [Ai] is the initial activity of diffusible anions, [M] is the molarity of DNA phosphates, and Z is the effective negative charge per DNA phosphate. The values for Z were obtained by plotting the data of Ross (31) against [NaClI and extrapolating to the equivalent average [NaClI present in each dialysis cell. Z ranged from 0.21 in 1 mM sodium cacodylate to 0.34 in NaCl concentrations of 50 mM or higher. For the Scatchard plots, each value of r was first corrected for the Donnan effect by dividing by [Afll[AmI. These correction factors are shown in Table 1. Because of the difficulty in determining the chemical activity of all diffusible ions, the uncertainty in the values for the Donnan factor is approximately + 10%. Scatchard plots for noninteracting ligands that bind nonspecifically to more than one lattice residue should be convex downward at high values of r (24) (see Discussion). In the present study, all plots of r/c versus r were drawn by least-squares fit to a linear regression curve because the extent of scatter in the data did not permit distinguishing between a plot that was linear and one that curved slightly at high values of r. Calculations were done with the aid of a Wang 2200 computer. DNA isolation. E. coli strain 15 TAU was grown to late log phase in 100 ml of synthetic medium (28) containing 20 ,uCi of [methyl-3H]thymine (13 ,uCi/ ,umol; New England Nuclear). Approximately 1.3 x 1011 cells were harvested and washed in 70 ml of 0.05 M tris(hydroxymethyl)aminomethane (pH 7.8) by centrifugation at 10,000 rpm for 15 min at 25°C. The resuspended pellet was incubated in 10 ml of a solution containing 0.05 M tris(hydroxymethyl)aminomethane, 20% sucrose, and 100 ,ug of lysozyme (Worthington) per ml for 20 min at 25°C (pH 7.8). The cells were washed twice in 70 ml of 20% sucrose-1 mM ethylenediaminetetraacetic acid (EDTA) as before. The spheroplasts were suspended in 1.2 ml of the sucrose-EDTA solution and lysed by gentle mixing with 23 ml of 1 mM EDTA, 0°C, containing 0.25 tg of ribonuclease (RNase) B (Sigma, preheated, 80°C, 15 min) per ml. The lysate was immediately layered on a sucrose step gradient consisting of 2 ml
918
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of 40% sucrose and 5 ml of 30% sucrose, both in 2 M urea (Schwarz/Mann, ultrapure)-l mM EDTA (pH 7.5) and centrifuged in the SW25.1 rotor (Beckman) at 22,000 rpm for 18 h at 00C. The bottom 3 ml was dispersed by three strokes in a rotating Potter-Elvehjem homogenizer and dialyzed in a 0.25-inch (about 0.6-cm)-diameter, preboiled dialysis bag against 1 mM cacodylate-0.1 mM EDTA (pH 7.5) for 2 h at 0°C. The preparation was then incubated with 10 jig of RNase (Worthington, RAF) per ml and 1 U of RNase T2 (Sigma) per ml for 45 min at 250C and layered on a sucrose gradient consisting of 1 ml of 70% sucrose and 2 ml of 60% sucrose overlayed with 22 ml of a 10 to 30% linear sucrose gradient, all containing 1 mM cacodylate-0.1 mM EDTA. After centrifugation as before, 1.2-ml fractions were collected from the bottom through an 18-gauge needle. Analytical determinations. DNA was quantitated by the diphenylamine method of Burton (6), using phenol-purified E. coli 15 TAU DNA as the standard. Bovine serum albumin was the standard for protein determination (22). RNA was analyzed by base lability of perchloric acid-precipitable A260 material (9). Polyamines were extracted from 0.2-ml aliquots of samples into 0.2 N perchloric acid and quantitated by a dansylation procedure (8), modified for dilute solutions. Putrescine and spermidine standards (Calbiochem) were dissolved in the same solvent as present in the samples. Radioactivity was measured as described in the binding studies. For double-label experiments, the windows of the spectrometer were set such that 16.7% of the 14C counts overlapped into the 3H channel and less than 0.01% of the 3H counts were detectable in the 14C channel.
RESULTS Equilibrium dialysis. It is generaly assumed that putrescine, spermidine, and spermine bind to DNA principally by bonds between polyamine amino and imino groups and DNA phosphates (15, 17, 35) (see Discussion). Consequently, inorganic salts would be expected to dissociate polyamine-DNA complexes, and this effect could be quantitated by equilibrium dialysis in various solvents. Such analyses were performed for spermidine-DNA interactions in concentrations of NaCl and MgCl2 applicable to the isolation of a spermidine-DNA complex from E. coli. Figure 1 shows typical Scatchard plots for spermidine binding to DNA as measured by equilibrium dialysis in increasing concentrations of MgCl2. Each data point was first corrected for Donnan effects as described in Materials and Methods. It can be seen that low [MgCl2] caused a marked decrease in the slope (which is the negative of the association constant, K) and a small decrease in the x-intercept (the total number of sites on DNA, n, available for spermidine binding). The results of a more extensive study of both MgCl2 and NaCl effects onK and n are shown in Table 1.
The sensitivity of the spermidine-DNA binding constants to salt concentration is clearly seen in Fig. 2. K changed little below 10 mM NaCl but decreased 2,000-fold as the [NaClI was increased to 0.3 M. MgCl2 had an even greater ability to reduce the interaction between spermidine and DNA. Just 10 mM MgCl2 decreasedK to 1,000 M-', a value only observed in [NaClI greater than 0.1 M. In the presence of both NaCl and MgCl2, spermidine binding to DNA was even less than the binding in the presence of either salt alone. This effect is shown in Fig. 3 as the moles of spermidine bound per DNA phosphate (r) as a function of increasing [NaCl] in the absence or presence of 2 mM MgCl2. It appears, therefore, that inorganic salts such as NaCl and MgCl2 effectively compete with spermidine for binding to DNA phosphates and, when simultaneously present in moderate concentrations, essentially eliminate measurable associations between spermidine and DNA. Isolation of a spermidine-DNA complex from E. coli. The binding data shown in Fig. 2 indicate that a spermidine-DNA complex would have maximum stability in solvents containing less than 2 mM NaCl and 0.2 mM MgCl2. Therefore, if spermidine is bound to DNA in the cell, these low-ionic-strength solvents would tend to "freeze" the complex, permitting its isolation from other components of the cell. However, if there is any spermidine not bound to 20,
150
K
0
FIG. 1. Scatchard plots of spermidine-DNA interaction based on equilibrium dialysis data. Solvents contained 1 mM sodium cacodylate (a) in addition to 01 mM MgC12 (U), 0.5 mM MgCl2 (A), and 2 mM
MgC12 (0).
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TABLE 1. Total numbers of binding sites, n (moles of spermidine/mole of DNA phosphate), and association constants, K, obtained by equilibrium dialysis in various solutionsa Solution
NaCl (mM) 0
Donnan correction
MgCl2 (mM)
K + SE (M-1)
n (± 10%)
0 1.03-1.11 6.47 ± 0.25 x 105 0.36 2.0 0 1.01-1.07 4.18 ± 0.11 x 105 0.31 10.0 0 1.01-1.02 2.51 ± 0.09 x 105 0.29 40.0 0 1.01 2.05 ± 0.06 x 104 0.21 100 0 1.00 2.48 ± 0.23 x 103 0.20 200 0 1.00 4.35 ± 0.19 x 102 0.19 300 0 1.00 3.19 ± 0.20 x 102 0.20 0 0.1 1.09-1.19 4.77 ± 0.09 x 105 0.25 0 0.5 1.07-1.10 8.12 ± 0.23 x 104 0.24 0 2.0 1.03-1.04 1.41 ± 0.22 x 104 0.19 0 4.0 1.02-1.03 4.38 + 0.23 x 103 0.23 0 6.0 1.02 2.14 ± 0.35 x 103 0.21 0 10.0 1.01 9.99 ± 0.12 x 102 0.21 a Each solution contained 1 mM sodium cacodylate in addition to the amount of NaCl or MgCl2 shown. The Donnan correction is the range of values calculated for each solution. SE is the standard error based on least-squares fit.
r .15-
.10\
0
.05C -7~ 1
0
1
2
3
log mMolarity -1
0 log
1
2
3
mMolarity
FIG. 2. Effect of[NaCI] (-) or [MgCIJ (U) on the spermidine-DNA association constant (K) in the presence of 1 mM cacodylate. The open circle represents the value of K in 1 mM cacodylate.
cell constituents, it might become associated with DNA in the complex-favoring environment of low-ionic-strength solvents. It appeared desirable, therefore, to lyse cells into a
FIG. 3. Effect of 2 mM MgCl2 on r (moles of spermidine bound/mol of DNA phosphate) in the presence of increasing [NaCI]. Each point was obtained by equilibrium dialysis of 0.40 umol of spermidine with 121 pmol of DNA phosphate in 1 mM sodium cacodylate and increasing [NaCI] in the presence (-) or absence (0) of 2 mM MgCl2. All values were corrected for the Donnan effect. The open symbols represent r in the absence of NaCl.
low-ionic-strength medium to maintain any spermidine-DNA complexes that may preexist in the cells and also to bring about a larger dilution of the cytoplasm to minimize artifac-. tual associations.
920
RUBIN
J . BACTERIOL .
Lysozyme-treated E. coli strain 15 TAU was osmotically lysed into 1 mM EDTA, resulting in an approximately 200-fold dilution in the concentration of the cytoplasm (see Materials and Methods). One millimolar EDTA was intended to inhibit deoxyribonuclease activity (11) while still maintaining a solvent for which equilibrium dialysis data indicated a spermidine-DNA association constant equal to that of 4 mM NaCl (data not shown). The lysate was centrifuged through a sucrose step gradient as described in Materials and Methods. The DNAcontaining pellet was then banded on a second sucrose gradient (Fig. 4). Fractions corresponding to each of the two main peaks shown in Fig. 4 (fractions 1 through 7 and 8 through 16) were pooled and chemically analyzed as described in Materials and Methods and summarized in Table 2. A total recovery of 77% of the DNA present in the original lysate was obtained in the two peaks. The bottom peak (fractions 1 through 7) contained a large amount of protein and had a high turbidity (320/260 ratio) and buoyant density characteristic of bacterial DNA in association with cell membrane and wall fragments (14, 26). Ten
FR ACTION
FIG. 4. Sucrose gradient centrifugation profile of DNA-containing extract obtained from E. coli grown in the presence of [3H]thymine as described in Materials and Methods. Fractions were analyzed for absorbance at 260 nm and radioactivity of a 0.1-ml aliquot. Fraction 23 was from the top of the gradient. There was no detectable pelleted material.
percent of the nucleic acid in the bottom peak RNA. Approximately half the cellular DNA was present in fractions 8 through 16 along with considerable protein but no detectable RNA. The two DNA peaks contained a total of 3.8% of the putrescine and 10.6% of the spermidine found in the original lysate. This material, therefore, may be a polyamine-DNA complex isolated from E. coli, but modifications may have been introduced during the isolation. To estimate the extent to which spermidine might have been lost from DNA during the isolation, an artificial complex of DNA and [14C]spermidine (1 spermidine/15 DNA P042-) was subjected to the isolation procedure exactly as performed on the native DNA. The specific was
radioactivity ([14C]spermidine cpm/DNA P042-) of this complex decreased by only 13% after banding in the second sucrose gradient. The presence of a spheroplast lysate sonicated to minimize sedimentation of cellular DNA resulted in an additional 11% decrease in specific radioactivity of the artificial DNA-spermidine complex (Table 3). Although this is a relatively small loss (76% recovery of spermidine originally complexed to DNA), it suggests that the spermidine content of the DNA peaks (Table 2) may be an underestimation. Taken together with the 77% value for the recovery of DNA (Table 2), it can be estimated that 18% of the cellular spermidine may have been complexed to the cellular DNA in the original lysate. To estimate the extent of artifactual uptake of spermidine during the isolation of DNA, a sonicated lysate prepared from cells grown in [14C]spermidine was mixed with washed spheroplasts at the time of lysis in such a way (Table 4) that any 14C present in the DNA peaks shown in Fig. 4 could only arise if free spermidine or a spermidine-containing molecule associated with DNA during its isolation. There were significant levels of ['4C]spermidine in the DNA peaks (Table 4). The amount of spermidine taken up from the sonicated lysate by the DNA isolated from unlabeled cells was calculated from the specific radioactivity of the original mixture of lysates of unlabeled plus spermidine-labeled cells by assuming equilibration of spermidine between both lysates. Comparison of these levels (last column of Table 4) with the amount of spermidine obtained by direct chemical analysis (last column of Table 2) reveals no difference within experimental error. This finding indicates that all the spermidine on the isolated DNA could be accounted for by associations taking place during or after lysis of the cells. It appears, therefore, that the observed polyamine composition of the isolated DNA was not due to a native interaction between spermi-
VOL. 129, 1977
SPERMIDINE-DNA INTERACTION
921
TABLE 2. Composition of DNA-containing peaksa Pooled frac- ['Hithymine % of cpm tions (cpm x 10-3) in lysate
ug of pro-
DNA
tein/ DNAg of
(Lg)
RNA/ DNA DN
AJ20IA
Putrescine (nmol)
Spermidine (nmol)
17.0 ± 1.1 25.8 + 1.2 0.11 0.240 1-7 5.56 331 108 26 0.005 13.7 ± 0.9 14.5 + 0.7 657 1.96
