Inhibition of DNA-dependent RNA polymerase with partially thiolated polynucleotides

Biochimica et Biophysica Acta, 319 (1973) 294-303

(~ Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands BBA 97784

INHIBITION OF DNA-DEPENDENT RNA POLYMERASE WITH PARTIALLY THIOLATED POLYNUCLEOTIDES A. J. MIKULSKI, T. J. BARDOS*, P. CHAKRABARTI, T. I. KALMAN and A. ZSINDELY Department of Medicinal Chemistry and Biochemical Pharmacology, School of Pharmacy, State University of New York at Buffalo, Bt~ff'alo, N.Y. 14214 (U.S.A.)

(Received April 6th, 1973)

SUMMARY Various modified polynucleotides, prepared from polycytidylic and polyuridylic acids as well as from RNA and DNA isolates by partial thiolation in the 5-position of their uracil and/or cytosine bases, strongly inhibited the DNA-dependent RNA polymerase of Micrococcus lysodeikticus. The inhibition was in the presence of mercaptoethanol partially reversible by the DNA template in a concentration-dependent manner, but was irreversible without the addition of mercaptoethanol. Incubation of the enzyme with the template prior to the addition of the inhibitor afforded some protection and decreased the inhibitory effects. The results suggest that the partially thiolated polynucleotides act by competing with the DNA template for the "template site" of the enzyme. In the absence of mercaptoethanol, covalent binding via mixed disulfide linkages may take place between the inhibitor and the enzyme.

INTRODUCTION In a preliminary report 1, we briefly presented the basic concept and initial results of our new approach to the selective inhibition of RNA and DNA biosynthesis, using chemically modified polynucleotides as inhibitory structural analogs of the natural templates. This approach became possible by the development of a thiolation procedure 2, 3 in our laboratory which permits the selective introduction of a "reactive" 5-mercapto group into some of the cytosine and/or uracil bases present in a polynucleotide without causing degradation or any other chemical alterations of the macromolecule. The partially thiolated polynucleotides thus obtained are currently being studied in a variety of test systems. Two recent communications from this and collaborating laboratories describe the inhibition of DNA polymerases flom RNA tumor viruses 4 and from cultured human Burkitt lymphoma cells 5 by certain partially thiolated polyribonucleotides. The present paper describes our studies of the inhibition of a bacterial DNA-dependent RNA polymerase system by a variety of macromolecular inhibitors prepared by partial thiolation of synthetic polynucleotides and of some natural RNA and DNA isolates. * To whom correspondenceshould be directed.

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MATERIALS AND METHODS

Enzymes and substrates DNA-dependent RNA polymerase was isolated from spray-dried Micrococcus lysodeikticus cells purchased from Miles Laboratories, according to Procedure A of Nakamoto et aL6; the purified enzyme corresponding to Fraction V was used, having a specific activity of 250-500 units/mg. Labelled ribonucleoside triphosphates, [G-3H]ATP (spec. act. 9.5 Ci/mmole), [8-aH]GTP (spec. act. 5.6 Ci/mmole) and [5-3H]UTP (spec. act. 19.8 Ci/mmole) were purchased from New England Nuclear Corp., unlabelled nucleosides triphosphates from Calbiochem, and spermidine phosphate from Nutritional Biochemicals Corp. Polynucleotides (unmod(fied) Calf thymus DNA (highly polymerized) was purchased from Worthington Biochemicals Corp. Ehrlich ascites DNA was isolated according to the method of Kay et aL 7, from freshly harvested Ehrlich ascites cells (grown in HA/ICR Swiss female mice for 8 days following inoculation). Ribosomal RNA (rRNA) and soluble RNA fraction (tRNA) were isolated from Ehrlich ascites cells, essentially following the procedures o f Kirby 8 and Littauer et aL 9, respectively. Polycytidylic acid and polyuridylic acid were obtained from Miles Laboratories. Thiolated polynucleotides The partially thiolated polynucleotides were prepared by modifications 3 of our previously described general method 2 for the conversion of pyrimidine nucleotides to their 5-mercapto derivatives. The modified procedure, as employed in the present work for the partial thiolation of cytosine and/or uracil containing polynucleotides, consists of the following two reaction steps: (1) treatment of the polynucleotides with methoxybromide 1° in methanol (0 °C, 1.5 h, stirring), followed by evaporation of the reaction mixture in vacuo to dryness; and (2) subsequent reaction with NaSH in dimethylacetamide (0 °C, 1.5 h, stirring, under nitrogen atmosphere), followed by precipitation of the thiolated product with 3 M NaCI. Before treatment with these reagents, the polynucleotides were usually converted to their organic solvent-soluble cetyltrimethylammonium salts I~'12 and, after thiolation, they were reconverted to their corresponding sodium salts by application of the procedure of Jones 12 (which includes careful purification from trace amounts of the quarternary salt). The thiolated products were gel-filtered through Sephadex G-25 and, in some cases, also through Agarose (Bio-Gel A-1.5 m or A-5 m, purchased from Bio-Rad Laboratories) columns, then dialyzed against distilled water, concentrated with Ficoll, or lyophilized and redissolved in water. The thiolated polycytidylate and DNA samples which were subjected to chromatography through Agarose-5 m (with 0.15 M NaCI) gave a single sharp peak fraction at the same positions as the corresponding unmodified polynucleotide starting materials. Since this peak fraction contained 80-100 ~ of the total thiolated product, it appears that the polymeric structure remains essentially intact in the process of chemical modification. (This is also indicated by the initial results of the light-scattering studies currently in progress in Dr Fiel's laboratory which gave a molecular weight value of 5.30 • 1 0 6 for a "2 ~-thiolated" DNA sample, similar to that of the unmodified calf thymus DNAla). In the case of double-stranded DNA,

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we observed a decrease of the Tm value (from 86 to 73 °C) and also of the thermal hyperchromic effect (from 46 to 25 %); this, however, was due to the effect of the organic solvents on the cetylammonium salt of DNA rather than to the thiolation (see below). The extent of thiolation was controlled with the mole-equivalents of the reagents employed, and it was standardized on the basis of the ultraviolet spectra (absorbance at 335 nm in the presence of dithiothreito114), or, on the basis of radioisotope incorporation studies using Na35SH in the thiolation step. For the determination of both the total extent of thiolation and the relative amount of oxidized and reduced thiol groups, the highly sensitive neutron activation analysis method 15 was employed, after reacting the modified polynucleotides with p-hydroxymercurybenzoate before and after reduction with dithiothreitol. Except in the case of double-stranded DNA, the 5-mercapto groups of the partially thiolated polynucleotides were found to undergo very rapid autoxidation to disulfides; the latter, however, were readily reduced again to the free sulfhydryl groups upon addition of dithiothreitol or mercaptoethanol. These reactions of the 5-mercapto group were indicated by the characteristic changes in the ultraviolet spectra (which were identical with those observed in the case of the thoroughly studied autoxidation and reduction of the monomeric 5-mercaptopyrimidine nucleosides 14'16), as well as by the neutron activation analysis results. We tentatively assume that the disulfide linkages are largely intramolecular because (1) the disulfide formation is much slower and less complete in the case of the more rigid D N A duplex than in the case of single-stranded polynucleotides, and (2) addition of dithiothreitol did not change the molecular weights of a 5.6 %-thiolated poly(C) (3.90 • 106) and of a "2 %-thiolated" D N A (5.30 • 1 0 6 ) while there was some change noted in the radii of gyration. However, further physico-chemical studies on a variety of thiolated polynucleotide samples are still in progress, and we cannot exclude the possibility that intermolecular disulfide linkages may also be formed, depending on the concentration of the solution, distribution of the thiolated bases, or other structural properties of the various polynucleotides; the effects of these variables are presently under investigation. The "%-thiolated" data used in this paper for characterization of the various modified polynucleotide preparations studied, refer to the percent of the total cytosine and/or uracil bases of the polymer that were converted to the corresponding 5-mercapto derivatives, regardless whether the latter are in the reduced or oxidized form. For comparison with the thiolated DNA, we subjected a sample of the calf thymus D N A to the procedure of conversion to the cetyltrimethylammonium salt, treatment with methanol and, after evaporation, with dimethylacetamide, followed by reconversion to the sodium salt and subsequent purification steps, under identical conditions as in the thiolation procedure, except for the omission of the methoxybromide and NaSH reagents. The resulting "solvent-treated D N A " sample gave a lowered helixcoil transition c u r v e ( T m = 72 °C, hyperchromic effect : 24.2 %) which was almost superimposable with that of the "2 %-thiolated" DNA sample. Previous investigators have also noted this change in the secondary structure of D N A upon treatment of its cetyltrimethylammonium salt with ethanol and dimethylformamide and, on the basis of light-scattering studies, concluded that it does not involve complete separation of duplex molecules into two strands, as there was no change in the average molecular weight of the D N A after this treatment ~7.

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Enzyme assay The DNA-directed RNA polymerase assay system of Nakamoto et aL 6, was employed as previously described 18 but using tritiated ATP (GTP or UTP) as the labelled substrate (see Fig. 1 legend). For inhibitory activity, the polynucleotides were tested in the presence of standard amount of calf thymus DNA template. In the "reversal" studies, the template DNA (either calf thymus or Ehrlich ascites DNA) was applied at increasing concentrations over the practically feasible range (limited by solubility) while keeping the inhibitor concentration constant at each given level through a series of 4-5 assay tubes. RESULTS Fig. 1 shows the inhibitory activity of a 5.1 ~-thiolated polycytidylate (i.e. a polymer consisting of 5.1 ~ 5-mercaptocytidylate and 94.9 ~ cytidylate units), compared to that of the corresponding unmodified polycytidylate in the DNA-dependent RNA polymerase system. Using the standard concentration (200 pg/ml) of calf thymus DNA as the template, the thiolated polynucleotide gave 50 ~ inhibition at a concentration of 20 pg/ml, and essentially complete inhibition at 80 pg/ml, while the unmodified polycytidylate (which was reported to inhibit this enzyme in a competitive manner 19) showed even at 240/tg/ml only about 25 ~ inhibition under the same assay conditions. In either case, the incorporation values were not significantly different whether [aH]ATP, [aH]GTP or [aH]UTP was used as the labelled substrate. Somewhat less difference was found between the inhibitory activities of modified and unmodified polyuridylic acids (Fig. 2). Poly(U) itself showed significant inhibition in this assay. Although the thiolated poly(U) preparations were much more active than poly(U) at low concentrations, they failed to inhibit the enzyme completely; thus, at high concentration, the inhibition obtained with poly(U) almost reached the maximum inhibition (80-85 ~ ) attained with its thiolated analogs. It can be seen from Fig. 2, that the extent of thiolation (2.0, 4.7, and 8.5 ~, respectively) made relatively little difference in the activities of the modified polynucleotides in this assay. The unmodified rRNA and tRNA isolated from Ehrlich ascites cell were similar to poly(U) in their inhibitory activities2°; however, the corresponding thiolated preparations, particularly in the case of tRNA, were considerably more active. The "2 ~o-thiolated" tRNA was even more active than the 5.1 ~-thiolated poly(C) (Fig.

3). The most active inhibitor among the polynucleotides so far tested in this assay system was a modified calf thymus DNA (see Fig. 4) in which only 2.5 ~ of the cytosine bases were thiolated (i.e. it contained one 5-mercaptocytidylate per approx. 200 nucleotide units); this gave 75 ~ inhibition of the enzyme at 10 pg/ml concentration. (The "2 ~-thiolated" DNA, referred to under Materials and Methods, showed essentially the same activity). In comparison, unmodified calf thymus DNA, added to the assay system in addition to the amount already present as template, caused no change of the rate of RNA synthesis. As a control, the "solvent-treated DNA" sample (see Materials and Methods) was also included in this assay; this was much less inhibitory than the thiolated sample. A 2.1 ~o-thiolated Ehrlich ascites D N A sample

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Fig. 1. Inhibition of DNA-dependent R N A polymerase by poly(C) and thiolated poly(C). T h e reaction mixture, 0.50 ml, contained 100/~g of calf thymus DNA; 30-40 units of enzyme; 0.4/=mole each of ATP, GTP, CTP and UTP (one of them aI-I-iabelled, 4.4" ]05 cpm/0.4/=mole); 0.8/=mole of sperm|dine phosphate; 1.25/~moles of MnC12; 50/~moles of Tris-HCl, pH 7.5; and the indicated concentration of the inhibitors: modified (5.1% -thiolated) poly(C) (solid lines) or unmodified poly(C) (broken lines). R N A polymerase activity was assayed by measuring the formation of radioactive RNA from [aH]ATP (circles) or [aH]GTP (squares) during 30 rain incubation at 30 °C, as described (ref. 18). Ordinate: Radioactivity of R N A formed per 0.1 ml of the reaction mixture (i.e. ] • 103 cpm corresponds to 4.55 mmoles of the radioactive precursor incorporated Lnto RNA in the total 0.5 ml of the reaction mixture). Abscissa: polynucleotide concentration (/=g/ml); 100 mg/ml corresponds to approx. 0.28/=mole/ml monomer unit concentration. Fig. 2. Inhibition of RNA polymeras¢ by poly(U) and thiolated poly(U). Enzyme activity was

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presence of indicated concentrations of polynucleotide inhibitors. O - - - O , unmodified poly(U); & - A , 2.0; B-B, 4.7; and 0 - 0 , 8.5 ~/o-thiolated poly(U). The reaction conditions and radioactivity i n c o r p o r a t i o n m e a s u r e m e n t s were the same as in Fig. 1.

is shown to be somewhat less effective than the thiolated calf thymus DNA as inhibitor of this enzyme in the presence of the calf thymus DNA template (Fig. 4). It should be pointed out that the thiolated DNA samples showed no template activity in this assay system. The inhibitory effect of the thiolated polynucleotides could not be reversed by increasing the concentration of the DNA template, unless mercaptoethanol was added to the incubation mixture; in the latter case, the inhibition was partially reversible with the template (see Fig. 5). However, even in the absence of mercaptoethanol, the enzyme could be to some extent protected from the inhibitory action of the thiolated calf thymus DNA by incubation with the template DNA for 10 rain prior to the addition of the inhibitor (see Fig. 6). When the thiolated poly(C) was employed as the inhibitor, the degree of "protection" afforded by the DNA template under the same conditions was much more significant, particularly against low concentration of the inhibitor (see Fig. 7).

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Fig. 3. I n h i b i t i o n o f R N A p o l y m e r a s e b y u n m o d i f i e d a n d t h i o l a t e d E h r l i c h a s c i t e s t R N A a n d r R N A . Enzyme activity was assayed as in Fig. 2 in the presence of the indicated amounts of Ehrlich ascites R N A preparations. O - - - O , t R N A ; 0 - 0 , 2.0 ~othiolated tRNA; 71- - -[], r R N A ; I1-11, 2,1 ~ thiolated rRNA. Fig. 4. Inhibition of R N A polymerase by thiolated DNA. Enzyme activity was assayed as in Fig. 2, in the presence of varying concentrations of 2.5 ~-thiolated calf thymus D N A ( O - O ) , 2.1 ~-thiolated Ehrlich ascites D N A ( O - O ) native calf thymus D N A ( [ ] - [ ] ) ; or "solvent treated" calf thymus D N A ( • - A ) , as indicated (in addition to the 200/~g/ml calf thymus D N A template present in the reaction mixture). DISCUSSION

Previous work in this laboratory indicated that the most sensitive biochemical parameter correlating with the cytotoxic activity of various alkylating agents is their ability to decrease the template activity of DNA for RNA polymerase18; this has been confirmed by other investigators21'22. Various antibiotics have also been shown to inhibit RNA polymerase by interacting with the DNA and abolishing its template activity2a, 24. These agents are generally non-selective in their inhibition of RNA polymerase from various sources, but their activity is at least to some extent dependent on the base composition of the DNA template. On the other hand, rifamycin and some of its derivatives (also the chemically related streptovaricin25) which specifically inhibit bacterial but not mammalian RNA polymerases, were shown to interact with the enzyme and not with the template 2~'27. The action of these compounds was found to be independent of the base composition of the template and was not influenced by the formation of the DNA-enzyme complex 26. It appears that the binding site of these inhibitors on the enzyme is distinctly different from that of the template 27. In contrast to the aforementioned inhibitors of DNA-dependent RNA polymerase, the partially thiolated polynucleotides described in this paper were designed to act as macromolecular "analog-inhibitors" (antimetabolites) which would, pre-

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Fig. 5. The effect of mercaptoethanol on the reversal by D N A of the enzyme inhibition produced by thiolated D N A . Enzyme activity was assayed as in Fig. 4, but in the presence of varying concentrations of the 2.5 %-thiolated calf thymus D N A , in absence (solid lines) and presence (broken lines) of 1 mM mercaptoethanol. The concentrations of the thiolated D N A were; ( I and F1), 100/~g/ml; ( A and m), 25/~g/ml; (O and O), none (control). Fig. 6. Protection of R N A polymerase by D N A against inhibition by thiolated D N A . In all tubes the radioactive substrate, [3H]ATP, was added after 15 • i n incubation of the enzyme with the indicated amounts of calf thymus D N A at 30 °C, and the reaction was stopped after additional 30 • i n incubation. (Solid lines), the inhibitor was added simultaneously with the D N A ; (broken lines), the inhibitor was added 10 • i n later. The concentrations of 2.5 %-thiolated calf thymus D N A were: • and [], 100/~g/ml; • and A, 25 # g / • l ; O - O , none (control).

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RNA POLYMERASEINHIBITION BY POLYNUCLEOTIDES

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sumably, compete with the DNA template for the specific "template site" of the enzyme. Such inhibitors may show structural selectivities in their competition with different templates, as well as in their binding to different polymerases4'5. The procedure used for the preparation of the thiolated polynucleotides permits a wide range of reproducible structural variations, based on choice of the polynucleotide to be thiolated and on the extent of thiolation. The assumption that the thiolated polynucleotides would act as inhibitors rather than templates was based on our previous studies with 5-mercaptopyrimidine nucleosides2s'29, which revealed the unusual reactivity and "binding" capacity of the 5-mercapto group. The results obtained with the M . lysodeikticus enzyme, which are presented in this paper, appear to be consistent with our assumptions concerning the inhibitory activity and mode of action of the thiolated polynucleotides. It is clear that even a small extent of thiolation greatly enhances the relatively low inhibitory activities of the polyribonucleotides19 in this enzyme system (Figs 1-3), and that this modification also converts the template DNA into an effective inhibitor (Fig. 4), while abolishing its template activity. The partial reversibility of the inhibition with increasing concentration of the template DNA in the presence of mercaptoethanol (Fig. 5), as well as the partial "protection" of the enzyme by prior incubation with the DNA template (Figs 6 and 7, further discussed below), suggest that the thiolated polynucleotides bind to the "template site" of the enzyme. The fact that the "reversal" was only partial through a 3-4-fold range of template concentration (limited by the solubility of the DNA), is consistent with the behavior of a competitive inhibitor which is much more strongly bound to the enzyme than is the template. In addition, the complete irreversibility of the inhibition in the absence of mercaptoethanol suggests that covalent binding via mixed disulfide linkage(s) may take place between the thiolated polynucleotides and RNA polymerase; thus, these polynucleotides could be perhaps considered as a special kind of "active-site-directed irreversible inhibitors" (ref. 30). The results provide some indications only of the potential template specificity of the inhibitory effects that can be achieved with this approach. Although all thiolated polynucleotide samples tested were inhibitory in this enzyme system, there were some quantitative differences in their activities, and it may be significant that the thiolated calf thymus DNA sample, i.e. the closest structural analog of the template, was the most potent inhibitor among all those tested. The greater effectiveness of this analog, e.g. in comparison with thiolated poly(C), was particularly apparent in the preincubation study in which the enzyme was partially protected by prior addition of the template (cf~ Figs 6 and 7, respectively). It may be noted that Fox et al. 2° observed complete protection of the enzyme activity from the inhibitory effects of unmodified polyribonucleotides by prior addition of the DNA template; however, the "protection" appears to be a time-dependent phenomenon which is due to the slow rate of reversal of the binding of RNA polymerase to DNA (ref. 3 I). We did not study this phenomenon in detail in the present work but used arbitrarily selected experimental conditions, to show both the site and the relative strength of the binding of the thiolated polynucleotides to the RNA polymerase. Detailed studies of the effects of these inhibitors (in relation to their diverse structures) on the initiation and elongation steps of RNA synthesis are in progress. Further studies, using different DNA templates and different thiolated polynucleotide inhibitors may provide more information concerning the degree of struc-

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tural selectivity o f these inhibitors with respect to various templates. (However, the M . l y s o d e i k t i c u s enzyme, by itself m a y not be the m o s t suitable system for such studies as it is k n o w n to show relatively little d i s c r i m i n a t i o n t o w a r d D N A templates f r o m different sources 19). O n the o t h e r h a n d , it will be interesting to see whether the selectivity o f these i n h i b i t o r s with respect to R N A p o l y m e r a s e s f r o m different sources c o u l d be achieved, o r e n h a n c e d , by varying the extent o f thiolation. F u t h e r m o r e , in view o f the very s t r o n g b i n d i n g o f the t h i o l a t e d p o l y n u c l e o t i d e s to the R N A polymerase, a n d the a p p a r e n t dissociability o f the e n z y m e - i n h i b i t o r complexes after the a d d i t i o n o f m e r c a p t o e t h a n o l , these c o m p o u n d s m a y be useful in the i s o l a t i o n o f this e n z y m e as well as in future s t u d i e s o f its structure a n d m o d e o f o p e r a t i o n . ACKNOWLEDGEMENTS W e express o u r t h a n k s to D r R. J. Fiel (Springville L a b o r a t o r i e s , Roswell P a r k M e m o r i a l I n s t i t u t e ) for the light-scattering data, a n d to M r s I l o n a C s a b a f o r h e r a s s i s t a n c e in the p r e p a r a t i o n o f the enzyme. This investigation was s u p p o r t e d by a g r a n t f r o m the A m e r i c a n C a n c e r Society (IC-27) a n d b y U . S . P . H . S . R e s e a r c h G r a n t No. CA-06695-10 f r o m the N a t i o n a l C a n c e r Institute. REFERENCES 1 Bardos, T. J., Baranski, K., Chakrabarti, P., Kalman, T. I. and Mikulski A. J. (1972) Proc. Am. Assoc. Cancer Res. 13, 359 2 Szabo, L., Kalman, T. I. and Bardos, T. J. (1970) J. Org. Chem. 35, 1934-1937 3 Bardos, T. J., Kalman, T. I., Mikulski, A. J. and Novak, L. (1972) Proc. 8th Int. Syrup. Chem. Nat. Prod. New Delhi, pp. 329-330 4 Chandra, P. and Bardos, T. J. (1972) Res. Commun. Chem. Pathol. PharmacoL 4, 615-622 5 Srivastava, B. I. S. and Bardos, T. J. (1973) Life Sci. 13, 37-44 6 Nakamoto, T., Fox, C. F. and Weiss, S. B. (1964) J. Biol. Chem. 239, 167-174 7 Kay, E. R. M., Simmons, N. S. and Dounce, A. L. (1952) J. Am. Chem. Sac. 74, 1724-1726 8 Kirby, K. S. (1965) Biochem. J. 96, 266--269 9 Littauer, U, Z., Yankofsky, A. S., Novogrodsky, A., Borsztyn, H., Galenter, Y. and Katcbalski, E. (1969) Biochim. Biophys. Acta 195, 29-49 10 Duschinsky, R., Gabriel, T., Tanty, W., Nussbaum, A., Hoffer, M., Grunberg, E., Burchanal, J. H. and Fox, J. J. (1967) J. Med. Chem. 10, 47-58 11 Aubel-Sadron, G., Beck, G. and Ebel, J. P. (1961) Biochim. Biophys. Acta 53, 11-18 12 Jones, A. S. (1963) Nature 199, 280-282 13 Fiel, R. J., Bardos, T. J., Chmielewicz, Z. F. and Ambrus, J. L. (1965) Cancer Res. 25, 1244-1253 14 Bardos, T. J. and Kalman, T. I. (1966) J. Pharm. Sci. 55, 606-610 15 Bruce, A. K., Mahoney, G. F. and Thomas, Jr, C. C. (1969) Radiat. Res. 40, 298-309 16 Kalman, T. I. and Bardos, T. J. (1967) J. Am. Chem. Soc. 89, 1171-1175 17 Aubel-Sadron, G., Beck, G. and Ebel, J. P. (1964) Biochim. Biophys. Acta 80, 448-455 18 Chmielewicz, Z. F., Fiel, R. J., Bardos, T. J. and Ambrus, J. L. (1967) Cancer Res. 27, 1248-1257 19 Fox, C. F. and Weiss, S. B. (1964) J. Biol. Chem. 239, 175-185 20 Fox, C. F., Gumport, R. I. and Weiss, S. B. (1965) J. Biol. Chem. 240, 2101-2109 21 Ruddon, R. W. and Johnson, J. M. (1968) Mol. Pharmacol. 4, 258-273 22 Puschendorf, B., Wolf, H. and Grunicke, H. (1971) Biochem. PharmacoL 20, 3039-3050 23 Goldberg, I. H. and Friedman, P. A. (1971) Annu. Rev. Biochem. 40, 775-805 24 Chandra, P., Gotz, A., Wacker, A., Verini, M. A., Casazza, A. M., Fioretti, A., Arcamone, F. and Ghione, M. (1971) FEBS Lett. 16, 249-252 25 Mizuno, S., Yamazaki, H., Nitta, K. and Umezawa, H. (1968) Biochim. Biophys. Acta 157, 322332

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26 Sippel, A. and Hartmann, G. (1968) Biochim. Biophys. Acta 157, 218-219 27 Wehrli, W., Knusel, F., Schmid, K. and Staehelin, M. (1968) Proc. Natl. Acad. Sci. U.S. 61, 667-673 28 Kalman, T. I. and Bardos, T. J. (1970) Mol. Pharmacol. 6, 621-630 29 Schwartz, S. H., Bardos, T. J., Burgess, G, H. and Klein, E. (1970) J. Med. 1, 174-179 30 Baker, B. R. (1967) in Design o f Active-Site-Directed Irreversible Enzyme lnhibitors, Wiley, J. and Sons Inc., New York 31 Richardson, J. P. (1966) J. Mol. Biol. 21, 83-114