9
1222 BIOCHEMISTRY: HURWITZ ET AL. PROC. N. A. S. ficity of any kind involved in the determination of what kind of DNA binds with what kind of histone is an interesting question but is not considered in this paper. * Report of work supported by the U.S. Public Health Service, grants no. RG-3977, RG-5143, and A-3102. We wish to acknowledge the fine technical assistance of Hedi Imfeld and the counsel and cooperation of F. William Studier. 1 Huang, R. C., N. Maheshwari, and J. Bonner, Biochem. Biophys. Res. Comm., 3, 689 (1960). 2 Bonner, J., R. C. Huang, and N. Maheshwari, these PROCEEDINGS, 47, 1548 (1961). 3 Other enzymes responsible for the DNA-dependent synthesis of RNA have of course also been described. See Weiss, S. B., these PROCEEDINGS, 46, 1020 (1960); Hurwitz, J., A. Bresler, and R. L'irniger, Biochem. Biophys. Res. Comm., 3, 15 (1960); Stevens, A., Biochem. Biophys. Res. Comm., 3, 92 (1960). 4Ts'o, P. 0. P., and C. S. Sato, Exptl. Cell Res., 17, 227 (1959). 'Burton, K., Biochern. J., 62, 315 (1956). 6 Lowry, 0. H., Nina J. Rosebrough, A. Lewis Farr, and Rose J. Randall, J. Biol. Chem., 193, 265 (1931). 7 Marmur, J., J. AMol. Biol., 3, 208 (1961). 8 Chamberlin, WI., and P. Berg, these PROCEEDINUS, 48, 81 (1962). 9 Zubay, G., and P. Doty, J. Mol. Biol., 1, 1 (1959). '0 Stedman, E., and E. Stedman, Nature, 166, No. 4227, 780 (1950). 11 Rasmussen, P. S., K. Murray, and J. M. Luck, Biochem., 1, 79 (1962). 12 Phillips, D., and P. Simson, Biochem. J., 82, 236 (1962). THE ROLE OF DEOXYRIBONUCLEIC ACID IN RIBONUCLEIC ACID SYNTHESIS, III. THE INHIBITION OF THE ENZYMATIC SYNTHESIS OF RIBONUCLEIC ACID AND DEOXYRIBONUCLEIC ACID BY ACTINOMYCIN D AND PROFLAVIN* BY JERARD HuRwITz,t J. J. FURTH,1 M. MALAMY,§ AND M. ALEXANDER DEPARTMENT OF MICROBIOLOGY, NEW YORK UNIVERSITY SCHOOL OF MEDICINE, NEW YORK 16 Communicated by B. L. Horecker, May 22, 1962 Actinomycin D is one of a number of polypeptide antibiotics isolated in Waks- man's laboratory.1' 2 Bacteriostatic effects, particularly on gram positive bacteria, and antitumor activity have been attributed to this compound.2 3 Kirk4 has demonstrated that the addition of actinomycin D (0.2 to 0.5 ,4M) to exponentially growing cultures of Staphylococcus aureus stops RNA synthesis immediately. This effect is rapidly followed by an inhibition of protein synthesis, and later by a partial inhibition of DNA synthesis. The action of this compound is not related directly to energy production since both respiration and glycolysis of inhibited cells are un- affected by concentrations up to 0.1 mM.4 Kirk also demonstrated that the com- bination of DNA and actinomycin D results in a spectral change of the latter com- pound. These observations suggest the formation of a complex between these two compounds since Kawamata and Imanishi5 found no interaction of actinomycin and RNA and the reaction appears to be relatively specific for DNA. Although Rauen et al.6 have reported complex formation between actinomycin and RNA, 100 times more RNA than DNA is required. Two recent reports have suggested that actinomycin acts by inhibiting the syn- thesis of "messenger" RNA. Nakata et al.7 have shown that reproduction of T2 Downloaded by guest on July 9, 2020

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Page 1: BIOCHEMISTRY: Service,VOL. 48, 1962 BIOCHEMISTRY: HURWITZETAL. 1223 phage and phage protein synthesis are markedly inhibitedbyactinomycinSwhile phage DNAsynthesis is unaffected. Reich

1222 BIOCHEMISTRY: HURWITZ ET AL. PROC. N. A. S.

ficity of any kind involved in the determination of what kind of DNA binds withwhat kind of histone is an interesting question but is not considered in this paper.

* Report of work supported by the U.S. Public Health Service, grants no. RG-3977, RG-5143,and A-3102. We wish to acknowledge the fine technical assistance of Hedi Imfeld and the counseland cooperation of F. William Studier.

1 Huang, R. C., N. Maheshwari, and J. Bonner, Biochem. Biophys. Res. Comm., 3, 689 (1960).2 Bonner, J., R. C. Huang, and N. Maheshwari, these PROCEEDINGS, 47, 1548 (1961).3 Other enzymes responsible for the DNA-dependent synthesis of RNA have of course also

been described. See Weiss, S. B., these PROCEEDINGS, 46, 1020 (1960); Hurwitz, J., A. Bresler,and R. L'irniger, Biochem. Biophys. Res. Comm., 3, 15 (1960); Stevens, A., Biochem. Biophys.Res. Comm., 3, 92 (1960).

4Ts'o, P. 0. P., and C. S. Sato, Exptl. Cell Res., 17, 227 (1959).'Burton, K., Biochern. J., 62, 315 (1956).6 Lowry, 0. H., Nina J. Rosebrough, A. Lewis Farr, and Rose J. Randall, J. Biol. Chem., 193,

265 (1931).7 Marmur, J., J. AMol. Biol., 3, 208 (1961).8 Chamberlin, WI., and P. Berg, these PROCEEDINUS, 48, 81 (1962).9 Zubay, G., and P. Doty, J. Mol. Biol., 1, 1 (1959).

'0 Stedman, E., and E. Stedman, Nature, 166, No. 4227, 780 (1950).11 Rasmussen, P. S., K. Murray, and J. M. Luck, Biochem., 1, 79 (1962).12 Phillips, D., and P. Simson, Biochem. J., 82, 236 (1962).

THE ROLE OF DEOXYRIBONUCLEIC ACID IN RIBONUCLEICACID SYNTHESIS, III. THE INHIBITION OF THE ENZYMATICSYNTHESIS OF RIBONUCLEIC ACID AND DEOXYRIBONUCLEIC

ACID BY ACTINOMYCIN D AND PROFLAVIN*

BY JERARD HuRwITz,t J. J. FURTH,1 M. MALAMY,§ AND M. ALEXANDERDEPARTMENT OF MICROBIOLOGY, NEW YORK UNIVERSITY SCHOOL OF MEDICINE, NEW YORK 16

Communicated by B. L. Horecker, May 22, 1962

Actinomycin D is one of a number of polypeptide antibiotics isolated in Waks-man's laboratory.1' 2 Bacteriostatic effects, particularly on gram positive bacteria,and antitumor activity have been attributed to this compound.2 3 Kirk4 hasdemonstrated that the addition of actinomycin D (0.2 to 0.5 ,4M) to exponentiallygrowing cultures of Staphylococcus aureus stops RNA synthesis immediately. Thiseffect is rapidly followed by an inhibition of protein synthesis, and later by a partialinhibition of DNA synthesis. The action of this compound is not related directlyto energy production since both respiration and glycolysis of inhibited cells are un-affected by concentrations up to 0.1 mM.4 Kirk also demonstrated that the com-bination of DNA and actinomycin D results in a spectral change of the latter com-pound. These observations suggest the formation of a complex between these twocompounds since Kawamata and Imanishi5 found no interaction of actinomycinand RNA and the reaction appears to be relatively specific for DNA. AlthoughRauen et al.6 have reported complex formation between actinomycin and RNA,100 times more RNA than DNA is required.Two recent reports have suggested that actinomycin acts by inhibiting the syn-

thesis of "messenger" RNA. Nakata et al.7 have shown that reproduction of T2

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VOL. 48, 1962 BIOCHEMISTRY: HURWITZ ET AL. 1223

phage and phage protein synthesis are markedly inhibited by actinomycin S whilephage DNA synthesis is unaffected. Reich and co-workers8 have demonstratedthat actinomycin D at concentrations of 0.1 ,uM inhibits the synthesis of RNA inL-cells in tissue culture and decreases the yield of the DNA containing vacciniavirus but does not inhibit cellular DNA synthesis or the multiplication of the RNAcontaining Mengo virus.The present report is concerned with the effects of actinomycin on RNA polymer-

ase and DNA polymerase, both of which require DNA. Both syntheses are in-hibited by actinomycin, the RNA polymerase reaction being somewhat moresensitive.Another compound which binds to DNA is proflavin.9 This dye has been shown

by a number of investigators to be mutagenic. 10 Proflavin exerts an action similarto that of actinomycin D and inhibits both enzymatic reactions leading to RNA andDNA synthesis. In this case, however, DN A synthesis is more sensitive than RNAformation.

Materials and Methods.-Actinomycin D was a gift from Merck Sharp and DohmeResearch Laboratories, West Point, Pennsylvania, and for this gift we are indebtedto E. Alpert. Proflavin sulfate was a commercial preparation of the Mann Re-search Laboratories. Mitomycin C was a gift of Y. Takagi, Department of Bio-chemistry, Kanazawa, Ishikawa, Japan.RNA polymerase and DNA polymerase were purified and assayed as previously

described.", 12 The nucleoside triphosphates were obtained as described in a pre-vious publication.'1Most of the DNA preparations tested were prepared by published procedures"3

or were obtained as previously described.Results.-Effects of actinomycin D on RNA polymerase and DNA polymerase:

It was suggested to us by R. M. Franklin of the Rockefeller Institute that the effectsof actinomycin D might be due to the inhibition of RNA synthesis by the DNA-linked RNA polymerase. As shown in Figure 1, actinomycin D is indeed a potentinhibitor of RNA polymerase. The extent of inhibition can be reduced by increas-ing the DNA concentration but is unaltered by the addition of the other com-ponents required for the synthesis of RNA.A double reciprocal plot'4 of the velocity versus the substrate concentration

(DNA) in the presence of a constant amount of actinomycin D is shown in the insetto Figure 1. Inhibition by actinomycin D appears to be competitive with DNA.From this plot, K1 for actinomycin D was calculated to be 2.76 X 10-8M. TheK1 value reported here is not a measure of the association of the enzyme and theinhibitor but a measure of the association between the enzyme and the inhibitorcomplexed with DNA.Actinomycin D also inhibits the enzymatic synthesis of DNA catalyzed by DNA

polymerase (Fig. 2). This inhibition is also reduced by increasing the DNA con-centration. K1 for actinomycin D in the DNA polymerase reaction is 2.7 X 10 7M.It appears that the synthesis of DNA is approximately 10-fold less sensitive to ac-tinomycin D than the corresponding synthesis of RNA.

The effects of actinomycin D with various DNA primers: As shown in Table 1,the inhibitory effect of actinomycin D is not limited to calf thymus DNA. RNApolymerase-catalyzed reactions which are primed with human bone marrow DNA

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1224 BIOCHEMISTRY: HURWITZ ET AL. PROC. N. A. S.

o ~ I I

22.5 6.0<

5.0-o X2.0 ~~~~~~~~~4.0-

0

- 0.5 02.00~~~~~~~~~~~~~~~~~.D 1.0. .02.0m4.0c6 .08 .10 .12 .14

02 0.5

0.2 0.4 0.6 0.8p.0 2.0

ACTINOMYCIN (puM)FIG. 1.-The inhibition of RNA polymerase by actinomycin D. The reaction mixture

(0.5 ml) contained: 80.MM each of ATP, GTP, and CTP, 80 MM a-P32 UTP containing1.42 X 106 cpm per M~mole, 4 mM MnCl2, 8 mM Mg9l2, 2 mM mercaptoethanol, 50 mMTris buffer, pH 7.5, calf thymus DNA (38 miumoles as deoxynucleotide), 1.84 Ag of RNApolymerase (ammonium sulfate IIB), and varying amounts of actinomycin D as indicated.This was incubated for 20 min at 380 after which time the activity was measured as pre-viously described."'The determination of the KI of actinomycin D was carried out with 0.1 MAM actinomycin

D and varying amounts of DNA. The reciprocal velocity versus the reciprocal substrateconcentration plot is presented in the inset figure.

C]~~~ ~~~1.8IO 1.8

1.616o 12

Ir 1.4 v0z 1.2 4

~-1.0 2 4 6 10

FG 0.- DNAaD.0/mi)

U0.60

0.4

0.2

2 4 6 8 10 12 14 16 1 8 20

ACTINOMYCIN (juM)

FIG. 2.-The inhibition of DNA polymerase by actinomycin D. The reaction midxture(0.5 ml) contained: 14 MM (each) dATP, dGTP, and dTTP, 8MuM a-P32 dCTP containing0.87 X 106 cpm per jMmole, 4 mM MgCl2, 2 mM mercaptoethanol, 50 mM Tris buffer, pH8.0, calf thymus DNA (24 mjumoles as deoxynucleotide), 0.14 jg of DNA polymerase (am-monium sulfate 50%-40% fraction), and actinomycin D as indicated. The reaction mix-ture was incubated and assayed as described in Figure L.'The reciprocal velocity versus reciprocal substrate concentration plot was carried out with

8 MM actinomycin D.

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VOL. 48, 1962 BIOCHEMISTRY: HURWITZ ET AL. 1225

TABLE 1EFFECTS OF ACTINOMYCIN D ON THE RNA POLYMERASE REACTION PRIMED BY DIFFERENT DNA

PREPARATIONS-Nucleotide Incorporation-

Addition of AMP UMP CMPSource of DNA actinomycin D (mar moles in 20 minutes)

1. Human bone marrow - 2.41 2.47"t it it + 0.34 0.31 -

2. M. lysodeikticus - 0.52 1.30It it + 0.04 0.05

3. bX 174 - 0.70it it + 0.07

4. Thymus (heated at 100° for 4 nin) - 1.14itccitit it it it + ~~~~~~~0.16

5. Polydeoxythymidylate - 1.46i' + 1.51

The activity of RNA polymerase was measured as described in Figure 1. The amount of DNA added was50 mimoles of human bone marrow DNA, 100 m;smoles of polydeoxythymidylate, 38 mjsmoles of calf thy-mus DNA, 25 mgsmoles of M. lysodeikticus DNA, or 5.5 mismoles of OX 174 DNA. One /AM actinomycin Dwas added where indicated. The enzyme was ammonium sulfate ITB (2.04 ,ug of protein).

or Micrococcus lysodeikticus DNA are also inhibited. The degree of inhibition isindependent of the nature of the ribonucleotide which is used to measure the reaction.The degree of inhibition is also similar with OX 174 DNA, suggesting that double-

stranded DNA, as such, is not required for the observed effects. Reaction mixturescontaining heat-denatured DNA as primer are also inhibited by actinomycin D.On the other hand, reaction mixtures primed with polydeoxythymidylate are

not affected by actinomycin D nor is the DNA-dependent production of polyribo-adenylate or polyribouridylate sensitive to actinomycin D. Recently, Chamber-lin and Berg"5 have reported the formation of polyriboadenylate (poly A) by RNApolymerase preparations. The production of poly A has already been noted by us'6as well as by Stevens.'7 In agreement with the results obtained by Chamberlinand Berg, the reaction leading to poly A is dependent on DNA, Mn++, and ATP.However, in contrast to their results, our partially purified enzyme fractions cata-lyze a very limited poly A production. The ratio of AMP incorporation with ATPas the only nucleoside triphosphate to AMP incorporation in the presence of theother three ribonucleoside triphosphates has varied in our hands from 0.07 to 0.3.This is in contrast to the value of 10 reported by Chamberlin and Berg'5 for theirpurified enzyme preparations. The marked differences between our results andthose of Chamberlin and Berg appear to be related to different assay conditionsused to measure the above reaction. In addition to the formation of poly A, asmaller amount of polyuridylate is also produced. This DNA-dependent poly Aand polyuridylate synthesis is also insensitive to actinomycin.

Effect of actinomycin D on the pyrophosphate exchange reaction catalyzed by RNApolymerase: RNA polymerase catalyzes an exchange reaction between PP1 andribonucleoside triphosphates."1 This reaction is completely dependent on the pres-ence of DNA but does not require the presence of all four ribonucleoside triphos-phates. The exchange reaction is inhibited by DNase but is insensitive to RNase.In view of the different requirements for the exchange reaction compared with syn-thesis of RNA, it was of interest to determine the effect of actinomycin D on thisreaction. As shown in Table 2, actinomycin D inhibits PP1 incorporation onlyslightly. Concentrations which markedly inhibit RNA synthesis produce onlyabout 20 per cent inhibition of the exchange reaction.

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1226 BIOCHEMISTRY: HURWITZ ET AL. PROC. N. A. S.

TABLE 2

THE EFFECT OF ACTINOMYCIN D ON THE PYROPHOSPHATE EXCHANGE REACTION CATALYZED BYRNA POLYMERASE

PP, Incorporated Per centAdditions (msmoles) inhibition

1. None 5.252. 0.2 ,M actinomycin D 4.34 173. 0.5 " " 4.20 204. 1.0 " " 3.92 255. 5.0 " " 3.36 36The reaction mixture (0.5 ml) contained 80 MM each of the 4 ribonucleoside triphos-

phates, 8 mM MgCl2, 50 mM Tris buffer, pH 7.5, 4 mM mercaptoethanol, 1 MM Ppi32containing 2.22 X 106 cpm per pmole, 38 msmoles of calf thymus DNA, and 2.4 Mig ofRNA polymerase (ASIIB) This mixture was incubated for 30 min at 380, after whichthe extent of PPi32 incorporation was measured as previously described."'

RNA polymerase preparations also catalyze a slow pyrophosphorolysis of theRNAproduced.1' This pyrophosphorolytic reaction is completely insensitive to actino-mycin D.

Effect of proflavin on RNA polymerase and DNA polymerase: We have examinedthe effect of proflavin on DNA and RNA synthesis and have found this compoundto inhibit both reactions (Figs. 3 and 4). Proflavin appears to inhibit RNA and

l

2.4I .6O ~~~~~~~~2.0

Ld0._

'101o 1.2-2a- 1.20

o21.000.2 0O4 0.6 0.6 .10 .12 .14

CL0.8~ ~ ~ ROA I/S(DN OD/ml)

a- 0.8

E 0.2

20 40 60 80 100 120 140 160

PROFLAVIN (0i)FIG. 3.-The inhibition of RNA polymerase by proflavin. The additions were the same

as those described in Figure 1 with the exception that proflavin replaced actinomycin D.

DNA synthesis in a manner analogous to that observed with actinomycin D. Italso acts as a competitive inhibitor, and the degree of inhibition is dependent on theDNA concentration. K1 of proflavin in the RNA polymerase reaction is 4.05 X10-5M; in the DNA polymerase reaction, the inhibition is more pronounced andthe KI is 2.05 X 10-6M.

The effect of actinomycin in vivo: In view of the selective effect of actinomycinD on the RNA polymerase system in vitro, it was of interest to determine the effectof this drug in intact bacteria. Initially, the effect of actinomycin D on Escherichiacoli was examined but it was found not to affect either whole cells or spheroplasts.

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VOL. 48, 1962 BIOCHEMISTRY: HURWITZ ET AL. 1227

1 1 1 1O 1.8w

< 1.6 \-

A~~~ ~~~tIavI<W 1.4o 6oz 1.2

Q. 1.0 IV

E 0.8 2

WL 0.60

E 0.2

6 12 18 24 30 36 42 48 54 60

PROFLAVIN %(pM)FIG. 4.-The inhibition of DNA polymerase by proflavin. The additions were as de-

scribed in Figure 2 with the exception that proflavin was added as indicated in place of ac-tinomycin D.

It was evident that permeability barriers may prevent access of the drug to its sitesof action. In contrast to E. coli, Bacillus subtilis is extremely sensitive to actino-mycin and 0.2 1AM actinomycin D completely blocks growth. With lower concen-trations, there is observed a marked decrease in growth (as measured by opticaldensity) and the formation of long, snake-like forms (Fig. 5). These observationswere similar to those found with an inhibitor such as mitomycin C,18 or by other

FIG. 5. The effect of actinomycin D on intact Bacillus subtilzs. Cells of B. subtilis weregrown overmight on glucose-semi-synthetic media without (A) and with (B), 0.1 M'M acti-nomycin D. Phase contrast microscopy, the magmification was identical for both fields asindicated by the reference line.

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1228 BIOCHEMISTRY: HURWITZ ET AL. PROC. N. A. S.

conditions causing unbalanced growth,19 and prompted us to examine the effect ofactinomycin D on the synthesis of RNA, DNA, and protein by B. sub-tilis.

In agreement with the results of Kirk with S. aureus,4 RNA synthesis is markedlyinhibited (90-95%), protein synthesis is also inhibited but to a lesser degree (50-75%), while DNA synthesis is least affected (25%) at a level of actinomycin D equalto 0.2 ,gM. The small amount of RNA synthesis that still occurs (2-8% of the nor-mal) in the case of actinomycin D-intoxicated cells has been examined after pulse-labeling (30 see) such cells with P,12. The distribution of p32 of the RNA, meas-ured after alkaline hydrolysis, in the 2'(3')-mononucleotides does not resemble thebase composition found either for the RNA or for the DNA of B. subtilis.We have also measured the effect of actinomycin D on a number of other en-

zymatic reactions involved in RNA metabolism. None of these enzymatic reac-tions are inhibited at concentrations which affect RNA polymerase and DNA poly-merase. The systems examined included the RNA-dependent production ofpolyriboadenylate,20 an RNA-dependent polymerase obtained from E. coli andthe enzyme which adds CMP and AMP to the terminal end of soluble RNA.21 22These reactions, as well as both RNA and DNA polymerases, are not affected bymitomycin.Both actinomycin D and proflavin inhibit the stimulation of amino acid incor-

poration into protein produced by the RNA polymerase system.23' 24 These stud-ies were carried out with the E. coli cell free preparation;25 the observed effects willbe described in greater detail elsewhere.Discussion.-The inhibitory effects of actinomycin D and proflavin appear to be

related to their ability to bind to DNA. The DNA complex formed inhibits RNAsynthesis and DNA synthesis in a qualitatively similar manner, but with significantquantitative differences. These quantitative differences are in accord with thein vivo action of these inhibitors: namely, there is very little effect of actinomycinD on DNA synthesis, while RNA synthesis is markedly inhibited. The DNA-dependent synthesis of RNA is exceedingly sensitive to this inhibitor. At 0.5,gM actinomycin D, RNA polymerase reaction is inhibited nearly 80 per cent: atthis concentration, there is virtually no effect on the DNA polymerase system.

Proflavin has been shown to alter phage replication in E. coli.9 When added tobacteria infected with T2 or T4,9 lysis occurs but the yield of progeny liberated islimited to those phage particles already present at the time of proflavin addition.If the inhibitor is added early, during the latent period, no viable phage are producedbut cell lysis can occur. The material lysed as above or prematurely with cyanide27contains incomplete phage particles which have little or no nucleic acid but appre-ciable amounts of sulfur. Such observations suggest that "messenger" RNA28production occurs to some extent while DNA synthesis has been blocked. Thein vitro effects of proflavin are in accord with these observations. Proflavin con-centrations (30 ,uM) which inhibit DNA synthesis 85 per cent inhibit RNA poly-merase by only 30 per cent.The observation that RNA synthesis can be inhibited in vivo with only partial

inhibition of protein synthesis is of particular interest. This raises some questionsconcerning the nature of "messenger" RNA, which, according to Jacob and Mo-nod,28 should possess a short half-life. A possible explanation is that not all "mes-

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VOL. 48, 1962 BIOCHEMISTRY: HURWITZ ET AL. 1229

senger" RNA molecules are short-lived but that some may act catalytically fora long time after synthesis of new messenger is interrupted.The finding that virtually all synthesis of RNA is blocked by actinomycin D sug-

gests that the DNA-directed synthesis of RNA may be involved in the formation ofribosomal RNA and soluble-RNA. Gros and coworkers have shown that the com-ponents of "messenger RNA" ultimately appear in ribosomal RNA.29 The preciserole of RNA produced by the RNA polymerase in the synthesis of the other RNAspecies remains to be elucidated.Summary.-The inhibition of the enzymatic synthesis of DNA and RNA by

actinomycin D and by proflavin has been reported. The inhibition is competitivein nature and can be reversed by increasing concentrations of DNA. It has beendemonstrated that the DNA-dependent synthesis of RNA is more sensitive to thisdrug than DNA synthesis.The inhibition of RNA polymerase by actinomycin D may depend on the type of

DNA used to prime the reaction. The synthesis of RNA is sensitive to this in-hibition when primers such as human marrow DNA, heated thymus DNA, OX174 DNA, and M. lysodeikticus DNA are employed. Reaction mixtures primedwith the synthetic oligonucleotide, polydeoxythymidylate, are not inhibited byactinomycin D.By contrast, the DNA-dependent exchange reaction between inorganic pyrophos-

phate and the ribonucleoside triphosphates is only poorly inhibited, although thesame enzyme appears to be involved. Concentrations of actinomycin D which com-pletely inhibit the synthesis of RNA inhibit the exchange reaction only about 20 percent.The addition of actinomycin D to Bacillus subtilis results in the production of

long "snake-like"cells. This appears to be related to unbalanced growth resultingfrom the preferential inhibition of RNA synthesis. The role of DNA-like RNA asan intermediate in protein and RNA synthesis has been discussed.

* These experiments were supported by grants from the National Institutes of Health and theHealth Research Council of the City of New York.

t Senior Postdoctoral Fellow of the National Institutes of Health.t Postdoctoral Fellow of the National Institutes of Health.§ National Science Foundation Cooperative Fellow.1 Vining, L. C., and S. A. Waksman, Science, 120, 389 (1954).2 Pugh, L. H., E. Katz, and S. A. Waksman, J. Bacteriol., 72, 660 (1956).3 Farber, S., in Amino Acids and Peptides with Antimetabolic Activity, Ciba Foundation Sym-

posia (general series), ed. G. E. W. Wolstenholme and C. M. O'Connor (Boston: Little, Brown andCompany, 1958), p. 140.

4Kirk, J. M., Biochim. Biophys. Acta., 42, 167 (1960).5 Kawamata, J., and M. Imanishi, Biken J., 4, 13 (1961).6 Rauen, H. M., H. Kernsten, and W. Kernsten, Z. Physiol. Chem., 321, 139 (1960).7Nakata, A., M. Sekiguchi, and J. Kawamata, Nature, 189, 246 (1961).8 Reich, E., R. M. Franklin, A. J. Shatkin, and E. L. Tatum, Science, 134, 556 (1961).9 DeMars, R. I., S. E. Luria, H. Fisher, and C. Levinthal, Ann. Inst. Pasteur, 84, 113 (1953).

10 Orgel, A., and S. Brenner, J. Mol. Biol., 3, 762 (1961).11 Furth, J. J., J. Hurwitz, and M. Anders, J. Biol. Chem. (in press).12 Hurwitz, J., J. J. Furth, and M. Anders, J. Biol. Chem. (in press).13 Marmur, J., J. Mol. Biol., 3, 208 (1961).14 Lineweaver, H., and D. Burk, J. Am. Chem. Soc., 56, 658 (1934).16 Chamberlin, M., and P. Berg, these PROCEEDINGS, 48, 81 (1962).

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1230 BIOCHEMISTRY: LEVINTHAL ET AL. PROC. N. A. S.

16Furth, J. J., J. Hurwitz, and M. Goldmann, Biochem. Biophys. Res. Comm., 4, 431 (1961).17 Stevens, A., in Cellular Regulatory Mechanisms, Cold Spring Harbor Symposia on Quantita-

tive Biology, vol. 26 (1961), p. 100.18 Reich, E., A. J. Shatkin, and E. L. Tatum, Biochim. Biophys. Acta, 53, 132 (1961).19 Cohen, S. S., and H. Barner, these PROCEEDINGS, 40, 885 (1954).20 Hurwitz, J., J. J. Furth, M. Anders, P. 0. Ortiz, and J. T. August, in Cellular Regulatory

Mechanisms, Cold Spring Harbor Symposia on Quantitative Biology, vol. 26 (1961), p. 91.21 Furth, J. J., J. Hurwitz, R. Krug, and M. Alexander, J. Biol. Chem., 236, 3317 (1961).22 Preiss, K., M. Dieckmann, and P. Berg, J. Biol. Chem., 236, 1748 (1961).23 Wood, W. B., and P. Berg, these PROCEEDINGS, 48, 94 (1962).24 Ning, C., A. Stevens, J. C. Loper, Biochem. Biophys. Res. Comm. 7, 30 (1962).25 Tissieres, A., D. Schlessinger, and F. Gros, these PROCEEDINGS, 46, 1450 (1960).26 After this paper had been written, Goldberg and Rabinowitz3n reported that actinomycin D

inhibits DNA-linked RNA synthesis in HeLa cells.27 Maaloe, O., and N. Symonds, J. Bact., 65, 177 (1953).28 Jacob, F., and J. Monod, J. Mol. Biol., 3, 318 (1961).29Gros, F., W. Gilbert, H. H. Hiatt, G. Attardi, P. F. Spahr, and J. D. Watson, in Cellular

Regulatory Mechanisms, Cold Spring Harbor Symposia on Quantitative Biology, vol. 26 (1961),p. 111.

30 Goldberg, I. H., and M. Rabinowitz, Science, 136, 315 (1962).

REACTIVATION AND HYBRIDIZATION OF REDUCED ALKALINEPHOSPHA TASE*

BY CYRUS LEVINTHAL, ETHAN R. SIGNER, t AND KATHLEEN FETHEROLF

DEPARTMENT OF BIOLOGY, MASSACHUSETTS INSTITUTE OF TECHNOLOGY

Communicated by S. E. Luria, May 28, 1962

There is an abundance of evidence that the structural gene for a protein controlsits primary amino acid sequence," 2 and there is strong evidence that the foldingof the polypeptide chain of ribonuclease is largely determined by the primary struc-ture.' However, for relatively large proteins, especially those composed of morethan one polypeptide chain, it is not clear whether folding and polymerization areentirely determined by primary structure or require additional information avail-able within the cell. It has been suggested, for example, that both a chains ofhemoglobin are made on the same ribosome, are released as a dimer, and combinein the cytoplasm with a similarly made f-chain dimer to form the complete hemo-globin molecule.4As part of a study of the synthesis and polymerization of the separate peptides

of a protein molecule, experiments have been carried out on the dimerization andconcurrent restoration of enzymatic activity of alkaline phosphatase inactivated byreduction of disulfide bridges. This enzyme,5 purified from Escherichia coli, hasa molecular weight of 80,000 and in its native state appears to be a tightly foldedglobular molecule. It is resistant to proteolysis by trypsin and chymotrypsinand is stable at 850C for at least 30 min in the presence of 10-2 M Mg++. Enzy-matic activity persists even after incubation in 6M urea, but a combined treatmentwith urea and thioglycollic acid leads to reduction of disulfide bonds and makes theresultant sulfhydryl groups on the molecule accessible to alkylation by iodoacetic

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