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Replication of Colicin El Plasmid DNA Added to Cell Extracts*

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Replication of Colicin El Plasmid DNA Added to Cell Extracts* Proc. Nat. Acad. &ci. USA Vol. 72, No. 3, pp. 1050-1054, March 1975 Replication of Colicin El Plasmid DNA Added to Cell Extracts* (intermediates/electron microscopy/RNA synthesis/protein synthesis) JUN-ICHI TOMIZAWAt, YOSHIMASA SAKAKIBARAtt, AND TSUYOSHI KAKEFUDA§ t Laboratory of Molecular Biology, National Institute of Arthritis, Metabolism and, Digestive Diseases, and §Chemistry Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014 Communieated by Terrell L. Hill, December 20, 1974 ABSTRACT Closed-circular DNA of colicin El plasmid by precipitation of ammonium magnesium phosphate), 0.5% can undergo a round of semiconservative replication when (w/v) NaCl, 1 mM MgCl2, 20 each of itryptophan added to an extract of Escherichia coli. Extracts of cells gg/ml that do not carry the plasmid are able to perform complete and thymine, 1% (w/v) glucose, 0.16 mM K2HPO4, and replication of the plasmid. Replication requires de novo [32P]H3PO4 (100 mCi/mmol). The bacteria were treated with RNA synthesis but not protein synthesis. chloramphenicol for 2 hr, and a cleared lysate was prepared. Closed-circular DNA was isolated by CsCl/ethidium bromide In a previous paper (1) it has been reported that extracts pre- density gradient centrifugation followed by neutral sucrose pared from Escherichia coli carrying colicin El plasmid (Col gradient centrifugation (1). When preferable, the 82P-labeled El) are able to support a round of semiconservative replica- DNA was diluted with nonlabeled DNA. 3H-Labeled, closed- tion of endogenous Col El DNA. In this paper evidence is circular DNA was similarly prepared from a cleared lysate presented that these extracts, as well as extracts made from of A745 (Col El) thy bacteria grown in a Casamino acids bacteria that do not carry the plasmid, are capable of carrying medium supplemented with 5,ug/ml of thymidine and 20 out a complete round of replication of exogenous, closed- mCi/mmol of [methyl-'H]thymidine and treated with chlor- circular Col El DNA. amphenicol for 2 hr. MATERIALS AND METHODS One microgram of Col El DNA corresponds to approxi- Materials. Most of the materials used have been described mately 1.4 X 1011 molecules or 750 pmol of incorporated in the previous papers (1-4). E. coli YS1 thr leu thi str minA dTMP. end is an end derivative of P678-54, and YS10 is a derivative Treatment of Closed-Circular DNA with Enzymes. 3H- of YS1 carrying Col El (1). Restriction endonuclease EcoRl Labeled, closed-circular DNA (100 gg/ml) in buffer A was and Pronase were obtained from Miles Laboratories and Sigma treated with a mixture of 100 jsg/ml of RNase A (ribonucleate Chemical Co., respectively. Actinomycin D, streptolydigin 3-pyrimidino-oligonucleotidohydrolase; EC 3.1.4.22) and and puromycin, and Pronase were the products of Merck Co., 50 ug/ml of RNase Ti (ribonucleate 3'-guanylo-oligonucleo- Upjohn Co., and Nutritional Biochemical Corp., respectively. tidohydrolase; EC 3.1.4.8) for 1 hr at 37° and then with 200 i [4,5-JH]Leucine was obtained from New England Nuclear. jsg/ml of Pronase for 5 hr at 37°. After the addition of sodium Preparation of Cell Extracts. Extracts were prepared as lauroyl sarkosinate (0.2%), the mixture was shaken with an described from cells incubated with chloramphenicol for 2 hr equal volume of water-saturated phenol. DNA in the aqueous before harvesting (1). phase was centrifuged in a CsCl/ethidium bromide density gradient. More than 90% of DNA was recovered in the heavy Preparation ofVol El DNA. For preparation of nonlabeled, band. closed-circular Col El DNA, A745 (Col El) thy bacteria were grown exponentially in a Casamino acids medium containing Assay of Incorporation of i-[8H]Leucine into Hot Trichloro- 5 Ag/rnl of thymine (1). The culture was treated with chlor- acetic Acid-Insoluble Material. Incorporation of i-[3H]leucine amphenicol for 2 hr, and a cleared lysate was prepared (5). was measured with the standard reaction mixture (1) (300 DNA was purified by two cycles of CsCl/ethidium bromide 1A) containing a YS1 extract that was passed through a density gradient centrifugation (4). Less than 5% of the total Sephadex G-25 column (2), 8 ug/ml of closed-circular DNA, closed-circular molecules contained a ribonucleotide sequence 10 AM i,[3H]leucine (1100 cpm/pmol), 0.1 mM puromycin, (6), as judged by the stability against alkali treatment (1). and with or without 10% glycerol and 2 mM spermidine. The DNA preparation was dialyzed against buffer A [50 mM The reaction mixtures with the same compositions except that K2HPO4, 50 mM NaCl, 1 mM EDTA, and 50 mM Tris - HCl -[1H]leucine was replaced by nonlabeled ileucine and [a- (pH 8.0)]. 32P]dTTP (160 cpm/pmol) were simultaneously incubated. For preparation of J2P-labeled, closed-circular Col El DNA, After incubation for 60 min, hot trichloroacetic acid-insoluble A745 (Col El) thy bacteria were grown in a medium contain- 3H radioactivity and cold trichloroacetic acid-insoluble 82P ing 1% Casamino acids (phosphate concentration was reduced radioactivity were measured. Abbreviation: Col El, colicin El plasmid. Other Aethods. Assay of DNA synthesis, density gradient and * This paper is the fifth of a series. The fourth is ref. 4. centrifugation analysis, electron microscopic examination, t On leave from the National Institute of Health, Tokyo, Japan preparation of the reference [3H]DNA were performed as (present address). described (1, 3). 1050 Proc. Nat. Acad. Sci. USA 72 (1976) Replication of Exogenous Col El DNA 1051 I c -1L0 'V I x 0 II ILa. II - II 5 6. I, 0-, ,I I \ -" III11 .- I------ .-Jol 20 40 60 20 40 60 20 40 60 FRACTION NUMBER FIG. 1. Sedimentation analysis of 32P-labeled, closed-circular DNA incubated with a YS10 extract. 2P-Labeled, closed-circular DNA (0.01 Mg; 5 X 104 cpm) was incubated at 300 with a YS10 extract in the standard reaction mixture (600 M1) with 10% glycerol and 2 mM spermiidine and with (c) or without (a and b) rifampicin (10 Mg/ml, 13fiM). DNA was extracted after incubation -for 75 min and centrifuged in neutral sucrose gradients at 45,000 rpm for 150 min at 10' in a Beckman SW50.1 rotor. In (b); DNA was heated at 900 for 90 sec before centrifugation. (@--*) 32P-labeled DNA; -- -) H-labeled reference closed-circular and open-circular Col El DNA. RESULTS instead of dTTP, the density of the labeled exogenous DNA centrifugation was in- Col El DNA Added to Extracts of Cells That measured by CsCl density gradient Replication of one Of the of the El. Col El DNA in YSId(Col E1)+ extracts is creased by roughly tenth difference light Carry Col not shown). The density converted to early replicative intermediates that sediment at density and the hybrid density (data of these molecules corresponds to that of the molecules con- approximately 26 S by incubation in the presence of 10% These results show glycerol and 2 mM spermidine (2). The intermediates contain taining BrdUMP labeled 6S DNA (1, 2). is dissociated that exogenous DNA was efficiently converted to the early a newly synthesized DNA fragment(s) that in the presence of from parental DNA by heating at 900 for 90 sec (2). Closed- replicative intermediates in the extracts El added to a reaction mixture containing 10% glycerol and 2 mM spermidine. circular Col DNA, of a round of semiconservative a YS10 extract, glycerol, and spermidine, was also converted Evidence of the completion replication of exogenous Col El DNA in a YS10 extract is to molecules sedimenting at 26 S (Fig. 1). More than 80% closed- of the labeled exogenous DNA was converted to 26S molecules presented in the following experiments. 3H-Labeled, After heating at 90° for circular molecules were incubated for 90 min and 180 min in a by incubation for 75 min (Fig. la). YSiO extract, BrdUTP, 90 sec,the labeled product sedimented with the closed-circular reaction mixture that contained a The formation of 26S molecules and [a-_2P]dATP in the absence of glycerol and spermidine. reference DNA (Fig. lb). at was inhibited by rifampicin (Fig. lc) as similar conversion of The 32P-labeled, newly synthesized DNA formed a band endogenous DNA in YS10 extracts (2). Rifampicin is known the half-heavy position in CsCl density gradients, and the El DNA replication (2). When a 3H-labeled, exogenous DNA formed a band at the light posi- to block initiation of Col side 2a similar experiment was carried out in the presence of BrdUTP tion with a considerable skew toward the heavy (Fig. and b). Recentrifugation9 of the DNA in the half-heavyy frac- '7 mixture (1.6 ml) contained 50,MM each of dATP, dGTP, dCTP, C~~~~~~~~~ 2 b1 II and BrdUTP, 200 MM each of the four ribonucl6oside triphos- a 0 4- phates, 2 mM NAD, 7.5mM MkC4, 25 mM potassium phosphate 10 buffer (pH 7.4), 50 mM KCl, 20 Ci/mmol of [a..32P]dATP, 3H- 2- labeled, closed-circular DNA (0.2 Mug; 1.4 X -105 cpm), and 0.8 ml 5 of an extract. DNA was extracted from half' f the reattion mix- ture after incubation for 90 min. To the remaining half, an equal same reaction mixture without 3H-labeled DNA 40 50 60 70 volume of the was added, and incubation was continued for. tn additional 90 C~~~~~~~~~~C .-~~~~~~~~~~~~~~~I1 min before extraction of DNA. The DNA samples were centri- fuged in CsCl density gradients at 36,000 rpm for 60 hr at 15° 15- 8 4.5 ml of 4 in a SW50.1 rotor.
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