Interaction of actinomycin D with the chymotrypsinogen-DNA complex

457

BIOCHIMICA ET BIOPHYSICA ACTA BBA 96033

INTERACTION OF ACTINOMYCIN D WITH THE CHYMOTRYPSINOGENDNA COMPLEX DOLORES BOBB Basic Biochemistry Division, Palo Alto Medical Research Foundation, 860 Bryant Street, Palo Alto, Calif. (U.S.A.) (Received June I9th, 1968)

SUMMARY

I. In slightly alkaline solutions of low ionic strength, actinomycin D, DNA and chymotrypsinogen A have been shown to form a kinetically stable triple complex which can be isolated by sucrose density gradient sedimentation or gel filtration. This triple complex does not appear to be any less stable than the respective actinomycin-DNA and chymotrypsinogen-I)NA complexes. 2. Spectrophotometric data indicated that the binding capacity of I)NA for actinomycin was not significantly affected by chymotrypsinogen-I)NA binding. 3. No evidence was found for binding of actinomycin to the protein. 4. Both the antibiotic and the protein in the triple complex appeared to contribute to the increase in the DN'A melting temperature. 5. The results of sucrose density gradient sedimentation are consistent with the current belief that actinomycin does not intercalate into the DNA helix. 6. If bound actinomycin is situated in the minor groove of DNA, the present results would indicate that chymotrypsinogen binding does not mask the minor groove to any great extent.

INTRODUCTION

The properties of soluble, stoichiometric complexes formed in vitro between certain purified proteins and I)NA, which have been described and studied in these laboratories (e.g., ref. I-6), make them suitable subjects for studying the effects of chemical and physical agents on the bound DNA and bound protein~,s. The DNA complex with chymotrypsinogen offers certain advantages in that soluble complexes are formed over a wide pit range (6.5 to 9.5) in solutions of relatively low ionic strength 5. Such complexes are stable to ultracentrifugationa, dialysis (D. BOBB, unpublished observations), sedimentation on sucrose gradients and chromatography on Sephadex (see RESULTS). On the other hand, the complexes can be readily dissociated by increasing the ionic strength to above o.13 (ref. 7). The Tm (thermal transition temperature) of the DNA is increased in proportion to the protein concentration up to a maximum of about 5.5 ° at the maximum combining ratio v. The native protein cornAbbreviation: SSC buffer, o.15 M N a C l + o . o I 5 M sodium citrate. Biochim. Biophys. Acta, 169 (1968) 457-465

458

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bines maximally with either native or denatured I)NA of calf thymus or Bacillus subtilis at a protein/DNA dry weight ratio of 12 : I (refs. 4, 7). During heating, the protein denaturation curve parallels the protein release curve*, indicating that as the protein molecules denature, they are released from DNA binding (see also ref. 6). Release from I)NA binding appears to be a consequence of the reversal in the sign of the net charge which occurs during chymotrypsinogen denaturation 9. All evidence suggests that the positively-charged protein binds, primarily at least, by electrostatic interaction with the negatively-charged DNA phosphodiester oxygens. Actinomycin D also forms complexes with DNA and inhibits those biological reactions in which DNA participates (see review of GOLDBERG, ref. I0). HAMILTON, FULLER AND REICH11 have proposed that the fused ring portion of actinomycin lies in the minor groove of the DNA helix and bonds to guanine constituents of one strand while the peptide portions bind to the phosphodiester oxygens of the other strand. This model predicts that constituents in the major groove should not interfere with actinomycin binding while those in the minor groove may do so. The natural nucleoproteins are believed to be formed, at least primarily, by electrostatic interaction of the basic groups of the protein with the negatively-charged DNA phosphate groups, the DNA retaining its characteristic double-helical structure 12. It has been thought that the polypeptide chains of nucleohistone may be wound around DNA in the maior groove 13 as opposed to the probable location of nucleoprotamine in the minor groove 14. More recently, reasons have been advanced for believing that histone may be situated in the minor groove ~5,~6. In any event, the suggested modes of binding for the proteins and for the antibiotic indicate that the one may possibly interfere with binding by the other. In fact, chromatin ~%1sand both natural and reconstituted calf-thymus nucleoprotein 19 have been reported to have only 40-5 ° % as many binding sites for actinomycin per mole of DNA-phosphorous as does DNA. The proteins per se associated with the DNA did not appear to provide binding sites for actinomycin ls,19. Actinomycin at low concentrations inhibits the action of RNA-polymerase which suggested that this enzyme may lie in the minor groove of DNA and that actinomycin inhibition is directly due to steric interference ~°. Since high concentrations of the antibiotic are required to inhibit DNA-polymerase, it was postulated that this protein lies in the major groove and that enzyme inhibition by actinomycin is indirect and due to inhibition of strand separation which is normally required for replication of template DNA. More recent reports ~° indicate that actinomycin does not prevent the formation of the complex of RNA-polymerase with DNA but inhibits the operation of the enzyme on DNA. The active complex per se was found to be essential for the enzymic reaction. Actinomycin has also been found to shield I)NA against the action of micrococcal nuclease 21. Native DNA possesses two classes of actinomycin-binding sites z2,23. The strong binding sites, which constitute about IO % of the total, are dependent on the I)NA helical structure, consist of guanine-containing units, and appear to be involved in the biological activity of actinomycin. For calf thymus DNA, there is one actinomycin strong binding site per 14-15 nucleotides 22,24. At high actinomycin concentrations the so-called weak binding sites are involved. Only one class of sites was found for denatured DNA and these had a binding constant of intermediate value 28. On the basis of the above observations, it seemed pertinent to inquire how Biochim. Biophys. At/a, 169 (1968) 457-465

ACTINOMYCIN-DNA-cHYMOTRYPSINOGEN COMPLEX

459

chymotrypsinogen-D1VA binding would affect actinomycin binding to DNA. In this respect, the similarities and differences between the properties of this protein-DNA model complex and those of natural nucleoproteins would be of interest. The drug and chymotrypsinogen were found to combine simultaneously with DNA to form a kinetically stable triple complex.

MATERIALS AND METHODS

Highly polymerized calf thymus D1VA and the 3 times recrystallized chymotrypsinogen were products of Worthington Biochem. Corp. Actinomycin D was kindly supplied by Dr. L. H. SARETTof Merck Sharp and Dohme, Rahway, N. J. The DNA, actinomycin and protein concentrations were determined spectrophotometrically (I-cm light path) using a molar extinction coefficient of 5" lO4 at 280 m# for the protein z5 and assuming that I A260 m/, unit -----1.45.I0-4 M with respect to DNA nucleotide. The molar extinction coefficient of actinomycin at 460 robe is 18 ooo (ref. 23). Unless otherwise stated, experiments were carried out in I mM Tris (pH 8) containing a 5o-fold dilution of o.15 M NaCl+o.oI5 M sodium citrate (SSC buffer) the final ionic strength being approx. 0.004. This solution will be referred to as TSC solution. The preparation of the chymotrypsinogen-DNA complexes has been described previouslys. Complexes of actinomycin and DNA were prepared in a similar manner by adding a dilute solution of actinomycin dropwise to a constantly agitated (Vortex mixer used at slow speed) DNA solution. Mixtures of actinomycin, chymotrypsinogen and DNA were prepared in two ways. Either actinomycin was added to the chymotrypsinogen-DNA complex or the protein was added to the complex of actinomycin and DNA. The protein/DNA ratio employed was about 12:1 (w/w), the maximum combining ratio of these two components. At the actinomycin concentrations employed, the strong binding sites would be essentially saturated zz. The density gradient sedimentation system of Buchler Instruments (triple outlet gradient mixer, polystaltic pump and piercing unit) was employed for preparation and collection of the gradients. After the centrifuge tubes were loaded with 30 ml of a linear 15-6o °/o sucrose gradient in TSC solution, 2 ml of sample was layered over the gradient. Centrifugation at 4 ° was for 16 h at 24 ooo rev./min in a swinging bucket rotor (Spinco 25.1) of the Model L ultracentrifuge, i-ml fractions were obtained in calibrated tubes by collecting drops from a hypodermic needle inserted into the bottom of the centrifuge tube. The samples were assayed spectrophotometrically. For gel filtration, a Kontes chomaflex column (9 mm × 25o ram) with an adapter for attaching a 5 cm long 16 guage Teflon needle was employed. Sephadex G-75 (Pharmacia) was packed to a height of 16 cm. Filtration was done at room temperature, the flow rate being about 25 ml/h, using TSC solution for both equilibration and elution. Drops were collected into tubes calibrated for I ml. All absorbance readings were taken with a Gilford Model-2ooo recording spectrophotometer equipped with the Model 209 absorbance meter. The procedure for obtaining melting profiles has been described previously8. The temperature of the cell compartment was increased automatically at a rate of o.5°/min. Biochim. Biophys. •cta, 169 (1968) 4-57-465

460

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RESULTS

Spec[rophotometric evidence ]or binding o[ actinomycin to the chymotrypsinogen-DNA complex When DNA binds to actinomycin, the absorption peak near 44 ° m/~ of the dye shifts to longer wavelengths and there is a decrease in absorption intensity19,26. Actinomycin concentrations are conveniently measured at the isosbestic point which occurs at 460 m# and where DNA has no effect on absorption. The decrease in absorption is maximal at 425 m/~ may be taken as a measure of binding 2~. The maximum increase in absorption is obtained at 475 m/~. No evidence was obtained from spectrophotometric data for binding of actinomycin to chymotrypsinogen. The addition of increasing amounts of chymotrypsinogen to a constant amount of actinomycin produced little or no change in the visible spectrum of actinomycin. The observed absorbance ratios, e.g., A440m~/A4~5mr,, -4425 m#/A46om~ and A4eo mjA4~ 5 m~, for actinomycin and for mixtures of chymotrypsinogen and actinomycin did not appear significantly different. From absorbance readings of mixtures containing actinomycin, I)NA and chymotrypsinogen, it was apparent that light scattering artifacts were present, particularly at higher DNA concentrations. This was shown by increased absorbance (as compared with actinomycin-DNA) at 34 ° m# (where the dye alone absorbs weakly), at the isosbestic point at 460 m#,, and at wavelengths above about 550 m# (where none of the components absorb). On the basis of these data, the scattering intensity could be approximated by the extrapolation method (see ENGLANDERANDEPSTEIN27) and was found to be proportional to about ~-3.3 which was the value previously found for chymotrypsinogen-DNA complexes 8. If the spectrum of a mixture of chymotrypsinogen, DNA and actinomycin was corrected for light scattering, the corrected spectrum did not appear significantly different from that of the respective actinomycinDNA mixture. As shown in Fig. I, the addition of increasing amounts of DNA or of the 12 : I chymotrypsinogen-DNA complex, where DNA is fully complexed with protein, to a fixed quantity of actinomycin resulted in almost identical decreases in the A4~5 m/,/ A4s0 m~ ratios. It was concluded, therefore, that the bound protein did not significantly interfere with subsequent binding of actinomycin to the nucleic acid.

Sucrose density gradient sedimentation o/ complexes Isolation of chymotrypsinogen-DNA-actinomycin complexes was attempted in order to determine (I) the relative stability of such complexes, (2) the effects of binding on sedimentation rates and (3) whether or not the order of mixing of the components might have an effect on binding. It was known (D. BOBB, unpublished observations) that after sedimentation in a suitable sucrose density gradient, the relative position in the centrifuge tube of a chymotrypsinogen-DNA complex was a function of the protein/DNA ratio, i.e., the amount of protein bound. As shown in Fig. 2C the complex sediments much faster than the DNA. The protein and the actinomycin, either individually or in admixture, remain at the top of the gradient (not shown). It is clear from the absorbance at 460 m# that actinomycin sediments along with DNA both in the presence (Fig. 2B) and in the absence (Fig. 2A) of protein. From the relative positions of the complexes in the gradient, it is apparent that Biochim. Biophys. Acta, 169 (I968) 457-465

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Fig. I. Actinomycin binding to DNA and to the chymotrypsinogen-DBlA complex. Decrease in the absorbmlce ratio (A425m~/ A45om~) of an actinomycin solution upon adding increasing a~ounts of D•A (O) or of a I2:I (w/w) chymotrypsinogen-DBlA complex (&). The final concentrationof actinomycinwas 6.5/ZM and the buffer (I mM Tris (pH8)) contained a 2o-fold dilution of SSC (/Z ~ o.oi). Fig. 2. Sucrose density gradient sedimentation of complexes of actinomycin and chymotrypsinogenwith DNA. The molar actinomycin/DNA nucleotide ratio was i:18, the protein/DNA ratio (w/w) was 13 : I, and the DNA concentration was 28/zg/ml. A. Mixture of actinomycin and DNA. B. Mixture of actinomycin, DNA and chymotrypsinogen. C. Individual runs for DNA (peak fraction 12) and for the chymotrypsinogen-DNA mixture (peak fraction 6). Solid lines connect absorbance readings at 260 m/z; dashed lines connect absorbance readings at 280 m/z; shaded areas are defined by absorbance readings at 45o m/z.

actinomycin-binding did not noticeably affect the sedimentation rate of either DNA (c/. Figs. 2A and C) or the protein-DNA complex (c/. Figs. 2B and C). If significantly less protein were bound to DNA in the presence of actinomycin, the triple complex (Fig. 2B) would band nearer to the top of the tube than the respective protein-DNA control (Fig. 2C). The complex shown (Fig. 2B) was formed b y adding the protein to a DNA complex with actinomycin. Virtually identical profiles were obtained (not shown) if the dye was added to the DNA complex with chymotrypsinogen. These results imply that approximately the same amount of protein is bound to DNA whether or not actinomycin is present and that the DNA binding sites for the drug and for the protein must be relatively independent. Under like conditions, the acridine orange-DNA complex was found (D. BOBB, unpublished observations) to sediment considerably slower than the DNA control presumably due to intercalation of the dye into the helix which would decrease the DNA mass per unit length and lower the sedimentation rate ~s. The present results then are compatible with the idea that actinolnycin does not intercalate 2~,~3,~9,3°. The isolation of a triple complex (Fig. 2B) after 16 hours centrifugation on the gradient attests to its kinetic stability. In the presence of the protein, an apparent lesser amount of dye remains bound to DNA and free dye is found throughout the upper portions of the gradient. Qualitatively these results would suggest that the rate of dye dissociation for the triple complex is higher than for the d y e - D N A complex. However, in the latter case dissociation might not be detected since the distance travelled is much less and the dye-DNA complex and free dye exist in closer proximity to each other during sedimentation on the gradient. Indeed, the results of rechromatography on Sephadex (see below) indicate that the dye dissociation rates for the two complexes are not significantly different.

Biochim. Biophys. Acta, 169 (I968) 457-465

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Isolation o/ complexes by gel filtration L1ERSCHAND ~ARTMAN 26 demonstrated that actinomycin-DNA complexes were sufficiently stable to be isolated by gel filtration on Sephadex. In gel filtration, DNA (Fig. 3A) and its protein complex (Fig. 3B) rapidly pass through the column and elute in the void volume while free chymotrypsinogen is retarded (Fig. 3D). It is apparent that the bulk of the protein, which elutes as a peak in fraction 7 (Fig. 3D), when mixed with DNA elutes as a unit with the nucleic acid (Fig. 3B). The calculated elution pattern (sums of absorbances of Figs. 3A and D) if DNA and protein were present together but not complexed is shown in Fig. 3C. When a mixture (Fig. 3 F) of actinomycin and the protein were applied to the column, two absorption peaks appeared in the same relative positions as when each component was applied separately to the column (Fig. 3D and E). Thus, at least within the limits of the procedure, no interaction between protein and dye was detectable.

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Fraction Number Fig. 3. Column chromatography on Seph&dex G-75 of DNA, &ctinomycin and chymotrypsinogen solutions. The following samples were applied to the column: A, 2.9 m! of D N A (28/~g/m|); ]3, 2.9 ml of a solution of chymotrypsinogen and D N A (35 o and 28 #g/ml, respectively); D, 2.9 m! of chymotrypsinogen (35o #g/ml); E, 2.5 ml &ctinomycin (25/~g/m]); F, 2.5 m! of a solution of actinomycin and chymotrypsinogen (25 &nd 325 #g/ml, respectively), C represents the calculated sum of the absorbances shown for DNA (A) and chymotrypsinogen (D). The designations for the absorbance readings are the same as in Fig. 2. (Note change in scale for bottom row.)

The elution patterns for the actinomycin-DNA and actinomycin-DNA-protein complexes after one and two passages through the column are compared in Fig. 4Approximately the same amount of dye (A4~oms) appeared to be bound to DNA in the absence (Fig. 4A) as in the presence (Fig. 4]3) of bound protein. This was also true after rechromatography (Figs. 4 C and D) of the peak fractions. The order ot mixing of the components of the triple mixture had no significant effect (not shown) on the elution profiles depicted in Figs. 4 B and D. In comparison with the protein-DNA complex itself (Fig. 3B), absorbance values indicated that at least as much protein is complexed to actinomycin-bound DNA (Fig. 4B). After the second passage there was a continued partial release of protein (Fig. 4D, second peak at fraction I I where A~80ms > A~e0ms) and a slight decrease in the actinomycin/nucleotide ratio. However, the reisolation of the proBiochim. Biophys. Acta, 169 (1968) 457-465

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Fig. 4. Elution patterns for actinomycin-DNA (A) and for actinomycin-DNA-chymotrypsinogen (B) complexes obtained after column chromatography on Sephadex. The applied samples of 3.0 ml contained 6, 28 and 35 °/~g/ml of actinomycin, DNA and ckymotrypsinogen, respectively. Tke elution patterns in C and D were obtained after the three peak fractions from A and t3, respectively, were combined and z. 7 ml of each pool were passed through the column. The designations for the absorbance readings are tke same as in Fig. 2.

tein-DNA~clye complex reaffirms the kinetic stability both of chymotrypsinogenDNA and actinomycin-DNA binding. Individual melting profiles on the combined peak fractions (4 and 5 of Figs. 4C and D) showed a Tm of 68.0 ° for the actinomycin-DNA mixture as compared with a Tm of 72.o ° for the actinomycin-DNA-protein mixture. Since each mixture contained essentially equal amounts of the dye (Aan0m~),the similar hyperchromicities found upon heating (o.165 for actinomycin-DNA and o.16o for dye-DNAprotein) showed that like amounts of DKA were also present. Therefore, the increased Tm of the protein-containing mixture presumably indicates the contribution of chymotrypsinogen to DNA stabilization. The apparent effect of the protein was to delay the onset of DNA denaturation as was shown previously to be the case in the absence of the dye~,8. The behaviour of the triple complex upon heating will be the subject of a separate communication. DISCU SSION

The spectrophotometric data (Fig. I) indicate that in the presence of bound chymotrypsinogen, there is little if any apparent change in the binding capacity of DNA towards actinomycin, That is, the drug is still able to bind maximally to DNA and to both classes of sites~" even when the DI~A has combined maximally with the protein. These results indicate that the DNA-binding sites for actinomycin and for the protein are, at least to a major extent, independent. This is in contrast to the case with certain natural nucleoprotein preparations where actinomycin binding to DNA is restricted by tile presence of bound protein x~-19. As in the case of these natural nucleoproteins, no evidence was found in the present experiments for binding of actinomycin to the protein (chymotrypsinogen) itself. Simultaneous binding of actinomycin and chymotrypsinogen to DNA was demBiochim. Biophys. Acta, 169 (1968) 457-465

464

D. BOBB

onstrated directly by means of sucrose gradient sedimentation and gel filtration. The isolation of the triple complex, even after rechromatography on Sephadex (Fig. 4C), attests to its kinetic stability. From the almost identical dye content of the DNA zones after recbromatography (Figs. 4C and D), it can be inferred that the rate of dissociation of the drug from DNA is not measurably influenced by the binding of the protein to the nucleic acid. Conversely, the rate of dissociation of the protein from DNA does not appear to be influenced appreciably, if at all, by DNA binding to actinomycin. This is shown by the same relative positions of the triple complex and of the protein-DNA complex after sedimentation on the sucrose gradient (Figs. 2B and C) and by the relative absorbance at 280 m# of the DNA fractions (Figs. 3B and 4 B) after gel filtration, taking into account the absorbance of the dye at this wavelength. If, as proposed by HAMILTON, FULLER AND R E I C H 11, actinomycin lies in the minor groove of DNA, then chymotrypsinogen binding does not appear to mask this groove to any great extent. The number of strong binding sites for actinomycin on native calf thymus DNA is about one per 14-15 nucleotides or about one per seven base pairs 2~,~. In the proposed modeP 1, the peptide portions of the molecule are estimated to fill the minor groove over a distance corresponding to about three base pairs. Chymotrypsinogen is bound at a ratio of about one protein molecule for every 6- 7 nucleotides (or one per 3 base pairs). Therefore, it is less than likely that, if actinomycin does extend into the minor groove, both the protein and the drug could simultaneously occupy the groove without the one interfering with binding of the other. Either the protein would extend into the major groove or be oriented along the backbone in such a way that neither groove would be obstructed. The DNA binding ratio for chymotrypsinogen (and for chymotrypsin*) and the large size (tool. wt. 25 ooo) of the essentiallly globular molecule indicate that the protein must be closely packed along the DNA sugar-phosphate chain. If only a very small region of the protein chain at one extreme of the molecule were bound to DNA, with the bulk of the protein extending beyond the double helix, a chymotrypsinogenDN'A structure which would allow the approach of a molecule such as actinomycin into the minor groove could be more readily visualized. In this respect, it is of particular interest that ill the model of ~ATTHEWS et al. 3~ for the conformation of ~-chymotrypsin, two areas of concentrated cationic charge distribution apparently are found in relatively close proximity to each other at one extreme of the molecule. (The same situation appears to hold for the zymogen as well.) The methionine loop of the C-chain contains four lysine residues while the neighboring loop of the B-chain contains six. One or both of these regions would then represent the most logical site(s) for DN'A binding assuming that the protein combines, at least primarily, by electrostatic forces with the DNA-phosphate groups. Lysines 84, 87 and 9 ° (B-chain) are of particular interest since they are separated from each other b y two non-polar residues which could form loops as postulated for protamine-DNA complexes 1~ and, thereby, might allow the three lysines to bind to an array of three phosphate groups on DNA. * C h y m o t r y p s i l i also f o r m s a soluble s t o i c h i o m e t r i c c o m p l e x w i t h n a t i v e DNA3, 4 a n d c o m bines a t a ratio of a b o u t one proteili molecule p e r four IIucleotides. E l e c t r o n - m i c r o g r a p h m e a s u r e m e n t s on s u c h a c o m p l e x (usilig t h e d i i s o p r o p y l p h o s p h o r y l d e r i v a t i v e ~of ¢¢-chymotrypsin) s u g g e s t e d t h a t t h e D N A double helix w a s covered b y a moliomoleculax l a y e r of protein. A similar s i t u a t i o n c a n be p r e s u m e d to e x i s t for c h y m o t r y p s i n o g e n .

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465

NOTE ADDED IN PROOF (Received October 24th, 1968 ). In a recent paper 32 a new molecular model for the actinomycin-DNA complex is proposed in which the actinomycin chromophore is actually intercalated into the DNA helix adjacent to a GC base pair. However, as in the previous model 11, the peptide rings lie in the minor groove which is the point pertinent to the present DISCUSSION.

ACKNOWLEDGEMENTS

The author is indebted to Dr. B. H. J. HOFSTEE for his helpful suggestions during preparation of the manuscript and to EDITH EDLIN for excellent technical assistance. This investigation was primarily supported by Public Health Service Research Grants GM 11832 and FR 05513 and aided by C-2289.

REFERENCES i 2 3 4 5 6 7 8 9 io II 12 13 14 15 16 17 18 19 2o 2i 22 23 24 25 26 27 28 29 30 31 32

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Biochim. Biophys. Acta, 169 (1968) 457-465