Hagen, U.

Kroeger, H.

Petersen, E.

Schuchmann, L.

English

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Juli 1967Institut für StrahlenbiologieBiochemisches Institut der Universität, Freiburg/Br.Studies on-Homopolymer Synthesis with Irradiated DNAin the RNA Polymerase SystemU. Hagen, H. Kröger, L. Schuchmann, E. PetersenKFK 651'Reprinted fromBWcIWnica cl Biophysi,;a ActaEbevier Publishing CompanyAmoterdamPrinted in The NetherlandsBBA 93236SHORT COMMUNICATIONSStudies on homopolymer synthesis with irradiated DNAin the RNA polymerase systemPreparations of RNA polymerase (RNA nucleotidyltransferase, EC 2.7.7.6),isolated from Escherichia coli according to the method of CHAMBERLIN AND BERG!,catalyse, apart from the synthesis of RNA, the synthesis of poly (A) and poly (U) inthe presence of primer DNA, ATP and UTP, respectively. In conneetion with earlierinvestigations into the priming ability of irradiated DNA2-S, its aetivity was studiedwith respeet to homopolymer synthesis. Calf-thymus DNA was irradiated either with60CO y-rays (30000 R, 5oo,ug/ml, 0.01 M NaCl) or with ultraviolet light (4.42' IOserg/mI, measured with the aid of uranyl acetate6). These DNA sampIes will beabbreviated to "y-DNA" or "UV-DNA", respectively. Further experimental detailshave been described previously3,~.The effeet of various doses of y-irradiation on native DNA is shown in Table 1.Small doses lead to an increase of poly A synthesis, high doses to a decrease. This is inTABLE IEFFECT OF y-IRRADIATION ON THE PRIMING ABILITY OF DNA FOR POLY (A) SYNTHESISThe incubation mixture (0.25 ml) contained in Jlmoles: Tris buffer (pH 7.9), 10; MnCl., 1.0;MgCl., 5.0; ß-mercaptoaethanol, 0.2; [8-14CJATP, O. I, specific activity: 258 countsImin permJlrnole; 10 Jlg DNA; 100 Jlg of protein. Incubation time 20 min at 37°.mJlmoles [8- 14CJAMP incorporatedDose(krad)o2461030Native DNA4.05. 1 55.66·55.834.42DNA denaturedafter irradiation25·927.821.221.925·419.6contrast to RNA synthesis, where the dose--effeet curve shows a continuous decreaseof the priming ability3. If the DNA is denatured after irradiation, the initial increaseis very small.The priming ability of DNA in the synthesis of poly (A) is dependent on thetemperature of incubation (Table 1I). The increase after y-irradiation is more pro-nounced at low temperatures(200). A similar effect is observed for poly (U) synthesis,but not for RNA synthesis. The inhibition of poly (A) synthesis on UV-DNA, how-ever, is independent of the incubation temperature. These results are in agreementwith similar experiments by MEHROTHRA AND KHORANA7 on thymidine polynucleo-tides.Abbreviations: poly (A), polyadenylic acid; poly (U), polyuridylic acid.Biochim. Biophys. Acta, 142 (1967) 542-544SHORT COMMUNICATIONSTABLE U543TEMPERATURE DEPENDENCE OF THE SYNTHESIS OF POLY (A), POLY (U), AND RNA IN THE PRESENCEOF UNTREATED, y-IRRADIATED AND ULTRAVIOLET IRRADIATED DNAThe incubation mixture was the same as in Table I, except for 30-min incubation, 20 pg DNAand temperature as indicated. For poly (A) synthesis specific activity of [8-14CJATP: 324 counts/min per mpmole, for poly (U) synthesis specific activity of [2-14CJUTP: 90 counts/min per mpmole,for RNA synthesis specific activity of [8-14C]ATP: 305 counts/min per mpmole.mpmoles nucleotide incorporatedISO 20° 25°Poly (A)synthesis Untreated DNA 1.5 2.6 4·9y-DNA 3·7 9·2 10·5UV-DNA 0·7 LI 1.5Poly (U)synthesis Untreated DNA 0·5 0·9y-DNA 1.3 1.5RNA synthesis Untreated DNA 3.6 4·7 5.6y-DNA 0·4 0.6 0·7UV-DNA <0.1 <0.1 <0.1TABLE UI8.211.42.07·30·7<0.110.111.92·52.01.37·30·70.113. 112.02.67. 20·90·312.012·92.80·90·75·50·70.2EFFECT OF ULTRAVIOLET lRRADlATED DNA ON THE POLY (A) SYNTHESIS ON UNTREATED DNAThe incubation mixture was the same as in Table I except for 10 pg irradiated and untreatedDNA each.DNA sampIes in tke incubation mix/ureUntreated DNA aloneUV-DNA aloneUntreated DNA, UV-DNA simultaneouslyUntreated DNA, UV-DNA after 40 secUntreated DNA, UV-DNA after 120 secUntreated DNA, UV-DNA after 210 secUntreated DNA, UV-DNA after 300 secmpmoles [8~14CJAMPincorporated10·43.15.67·98.610·510.6UV-DNA, as weIl as y-DNA, is able to inhibit RNA synthesis on untreatedDNAS,4. The same phenomenon was found for poly (A) synthesis (Table III). Simul-taneous addition of IO p,g UV~DNA to the incubation mixture with IO p,g untreatedDNA lowers the [8-14C]AMP incorporation by half. When the UV-DNA was addedafter the synthesis had started, this inhibitory effect was reduced. Addition after2IO sec no longer had any effect. This shows that the enzyme is bound to the untreatedDNA within 3.5 min and cannot be removed by the UV-DNA. Corresponding resultswere found in the case of RNA synthesis3,4.The kinetics of poly (A) synthesis under certain conditions is shown in Fig. I-On UV-DNA, the rate of synthesisis low; addition of untreated DNA 2 ruin after thesynthesis has started leads to a small increase in the synthesis (Fig. IA). UV-DNA,added 2 min after the start of the synthesis with untreated DNA, reduces the syn-thetic rate. This shows that there is a competition for the polymerase between thetwo DNA sampIes. A corresponding observation was made on y-DNA (Fig. IB).Here, irradiated DNA has the higher priming ability.Biockim. BioPkys. Acta, 142 (1967) 542-5445441l 1OJJ...ogaoL-aIJc.- 6.00-:I< 4.0eilIläE2.0::I..EA10 15lOJJ20TimeB5 10 15SHORT COMMUNICATIONS20Fig. I. Kinetics of poly (A) synthesis in the presence of untreated and irradiated DNA. A. Ultra-violet irradiation. B. y-Irradiation. • , untreated DNA; A, irradiated DNA; 0, untreated DNA,irradiated DNA after 2 min; !:,., irradiated DNA, untreated DNA after 2 min. Addition of thesecond DNA sampIe to the incubation mixture is indicated by arrow. The incubation mixture(1.50 ml) contained in,umoles:Tris buffer (ph, 7.9), 60; MgCl., 30; MnCl., 6; ß-mercaptoethanol, 1.2;[8-14C]ATP, 0.6, specific activity: 197 countsJmin per m,umole; 60,ug of each DNA sampIe;600 ug protein. At the times indicated, 0.25 ml of the incubation mixture were removed and theincorporation of [8-14C]AMP determined in the usual way.The increase of poly (A) synthesis after y-irradiation may be explained by thefact that ionizing radiation denatures the DNA molecules apart from its effect on thephosphodiester bonds or on the base moieties8,9. As only the denatured regions of theDNA molecule act as primer for homopolymer synthesis10, the priming ability in-creases in a manner corresponding to the radiation-induced denaturation. Thedecrease at higher doses may be due to destmction of the nucleotide bases.The work was supported by the Deutsche Forschungsgemeinschaft.Biochemisches Institutder Universität,FreiburgIBr., (Germany)I nstit-ut tür Strahlenbiologie,Kernjorschungszentrum Karlsruhe,Karlsruhe (Germany)HANS KRÖGERLOTHAR SCHUCHMANNEIKO PETERSENULRICH HAGENI M. CHAMBERLIN AND P. BERG, Proe. Natl. Aead. Sei. U.S., 48 (1962) 81.2 F. ZIMMERMANN, H, KRÖGER, U. HAGEN AND K. KECK, Bioehim. Biophys. Acta, 87 (1964) 160.3 U. HAGEN, K. KECK, H. KRÖGER, F. ZIMMERMANN AND TH. LÜCKING, Bioehim. Biophys.Acta, 95 (1964) 418. .4 F. ZIMMERMANN, H. KRÖGER AND TH. LÜCKING, Bioehem. Z., 342 (1965) II5.5 H. KRÖGER AND L. SCHVCHMANN, Bioehem. Z., 346 (1966) 191.6 E. 1. BOWEN, The Chemieal Aspects 0/ Light, Oxford University Press, New York, I946,P' 283.7 B. D. MEHROTHRA AND H. G. KHORANA, J. Biol. Chem., 240 (1965) 1750.8 G. SCHOLES, J. F. WARD AND J. WEISS, J. Mol. Biol., 2 (1960) 379.9 U. HAGEN AND R. WILD, Strahlentherapie, 124 (1964) 275.10 M. CHAMBERLIN AND P. BERG, J. Mol. Biol., 8 (1964) 708.Received Febmary 15th, 1967Bioehim. Biophys. Acta, 142 (1967) 542-544

Studies on homopolymer synthesis with irradiated DNA in the RNA polymerase system

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Studies on homopolymer synthesis with irradiated DNA in the RNA polymerase system

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