Vol. 132, No. 1 Printed in U.S.A.

JOURNAL OF BACTrzIOuLGY, Oct. 1977, p. 233-246 Copyright C 1977 American Society for Microbiology

Deoxyribonucleic Acid Synthesis in Permeabilized Spheroplasts of Saccharomyces cerevisiae WOLFGANG OERTELt AND MEHRAN GOULIAN* Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093 Received for publication 28 March 1977

Osmotically shocked spheroplasts from Saccharomyces cerevisiae incorporated deoxynucleoside triphosphates specifically into double-stranded nuclear and mitochondrial deoxyribonucleic acid (DNA). Results with this in vitro system for cells with and without mitochondrial DNA were compared. Strains lacking mitochondrial DNA were used to study nuclear DNA replication. With a temperature-sensitive mutant defective in DNA replication in vivo, DNA synthesis in vitro was temperature sensitive as well. The product of synthesis with all strains after very short labeling times consisted principally of short fragments that sedimented at approximately 4S in alkali; with longer pulse times or a chase with unlabeled nucleotides, they grew to a more heterogenous size, with an average of 6 to 8S and a maximum of 15S. There was little, if any, integration of these DNA fragments into the high-molecular-weight nuclear DNA. Analysis by CsCl density gradient centrifugation after incorporation of bromodeoxyuridine triphosphate showed that most of the product consisted of chains containing both preexisting and newly synthesized material, but there was also a small fraction (ca. 20%) in which the strands were fully synthesized in vitro. 32P-label transfer ("nearest-neighbor") experiments demonstrated that at least a part of the material synthesized in vitro contained ribonucleic acidDNA junctions. DNA pulse-labeled in vivo in a mutant capable of taking up thymidine 5'-monophosphate, sedimented in alkali at 4S, as in the case of the in vitro experiments. Much of the progress in understanding procaryotic deoxyribonucleic acid (DNA) replication has resulted from the utilization of mutants in specific functions required for replication. The unavailability of mutants in DNA replication functions is a distinct disadvantage with the more complex eucaryotic systems under study; however, some of the simpler eucaryotes allow considerable genetic manipulation. The yeast Saccharomyces cerevisiae has undergone extensive genetic analysis, and several conditional mutants in replicative functions have been partially characterized (24, 25, 26). The relatively small amount of DNA per yeast cell and per yeast chromosome allows certain kinds of characterization of the intact chromosomal DNA not possible with the DNA of the eucaryote cells of higher organisms (10, 41). In addition to nuclear DNA, yeast cells may contain several species of extrachromosomal replication units, including mitochondrial t Present address: Universitat Wurzburg, Institut fur Genetik und Mikrobiologie, Lehrstuhl fur Mikrobiologie, D-8700, Wiirzburg, West Germany.

DNA (5 to 20% of the total cell DNA) (26), small, plasmid-like, circular DNA molecules (7, 8, 21, 26), and the killer factor (4, 6) identified recently as a double-stranded ribonucleic acid (RNA) (54, 55). In contrast to the situation with higher eucaryotes, there are yeast mutants lacking one or all of the extrachromosomal replication units (19, 22), making it possible to investigate nuclear DNA replication independent of the replication of the other nucleic acids. In addition, some of the mutants may permit study of individual proteins and regulatory factors that participate in replication of the different species of extrachromosomal replicons. Certain properties of yeast have a bearing on experimental design for studies on DNA synthesis, including its tough cell wall, lack of thymidine kinase (20), the presence of which is required for specific labeling of DNA in vivo with radioactive thymidine, and its high level of nuclease activities. It may be possible to alter these problem characteristics by using mutants capable of taking up deoxythymidine 5'-monophosphate (dTMP) and dTMP auxo233

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trophs (12, 17, 57) and by using enzymes to inorganic orthophosphate (32P,) was added to the remove or weaken the cell wall (1, 31, 61). The desired specific activity. Both media were made lOx concentrated and recent isolation of nuclease-deficient mutants diluted in another yeast-like eucaryote (2) encourages sterilized by filtration. Before use, theyorwere YM-P (YMM water of sterile 9 parts with the hope that this will soon be successful in dium) or sterile 1 M sorbitol (YMMS medium)meor yeast as well. with a solution containing (per liter) 6.5 g of sulfaTwo in vitro yeast systems have been de- nilamide, 55 mg of aminopterin, 1 mmol of KH2PO4, scribed, both employing the detergent Brij to and 0.5 mmol of dTMP (YMM-SAT medium). promote uptake of nucleotide precursors of Reagents. Glusulase (snail gut juice) was obDNA (2, 27). Although DNA synthesis in one tained from Endo Laboratories, Garden City, N.Y. of these systems was temperature sensitive in Yeast cell wall degrading enzyme from Arthrobacter mutants defective in replication in vivo, the luteus was isolated by ammonium sulfate precipita(70% saturation) from the medium of a 3-day products, which were only partially character- tion culture in a minimal medium containing yeast cell ized, may have been mostly or entirely mito- walls as its only carbon source (31). Pancreatic chondrial rather than nuclear (2). A recent, deoxyribonuclease (DNase), pancreatic ribonuclease brief description of a system using yeast cells (RNase), and snake venom phosphodiesterase were treated with snail extract and mercaptoethanol purchased from Worthington Biochemicals Corp., deals with the synthesis of nuclear as well as Freehold, N.J., and Pronase was from Calbiochem, mitochondrial DNA (62). In this report we La Jolla, Calif. Unlabeled nucleotides were from Pdescribe another permeable system in S. cere- L Biochemicals; trisodium phosphoenolpyruvate, Nvisiae, which synthesizes both nuclear and mi- ethylmaleimide, dithioerythritol, saponin, D-(+)sorbitol, and spermidine-hydrochloride were from tochondrial DNA in vitro from deoxynucleoside Sigma Chemical Co., St. Louis, Mo. Sarkosyl NL-30 triphosphates (dNTPs). Properties of the sys- and ethidium bromide were gifts from Geigy and tem are characterized, particularly the nature Boots. 3H-labeled deoxythymidine 5'-triphosphate of nuclear DNA synthesis. (dTTP) and deoxycytidine 5'-triphosphate (dCTP) (15 to 20 mCi/,umol) and [3H]uracil (40 to 60 mCi/ MATERIALS AND METHODS ,mmol) were from Schwarz/Mann, Orangeburg, N.Y. Cells. S. cerevisiae A364A p+ (a adel ade2 ural [32P]dTMP (100 to 500 mCi/,umol) was prepared by gall tyrl his7 lys2, Wt p+) and its derivatives 198, the procedure of Okazaki and Kornberg (39) by temperature sensitive in gene cdc8 (Ts cdc8 p+), using thymidine kinase and [y-32P]adenosine 5'and 146-2-3, temperature sensitive in gene cdc2l (Ts triphosphate (ATP) (18). 5'-[32P]deoxyguanosine 5'cdc2l p+), were generously provided by L. Hartwell triphosphate (dGMP) (200 mCi/Amol) was synthe(24, 25). Mutant A364A tup4 p+ (tup p+), capable of sized from 3'-dGMP by using polynucleotide kinase dTMP uptake (tup), was selected from A364A p+ (44) and [_y-32P]adenosine 5'-triphosphate (rATP), cells by a variation of the method of R. B. Wickner (57). From these p+ strains, the mitochondrial DNAless (p°) strains A364A p0-3 (Wt p°); 198 p0-4 (Ts cdc8 p°); 46-2-3 p°-13 (Ts cdc2l po); and A364 tup4 pQ-17 (tup p°) were derived by prolonged treatment with ethidium bromide (19). Absence of mitochondrial DNA was checked by CsCl density gradient centrifugation of the 32p_ or 3H-uracil-labeled DNA (see Fig. 2). A364A p+ and A364 p0°3 contain oDNA (L. M. Hereford, personal communication and W. Oertel, unpublished experiments) and the killer factor (47); the other strains are not characterized in this respect. Media. YMM medium is based on the formula of Hartwell (23) and contains per liter: 20 g of glucose, 10 g of succinic acid, 6 g of NaOH, 6.7 g of yeast nitrogen base (without amino acids, Difco Laboratories, Detroit, Mich.), 0.5 g of yeast extract, 20 mg of adenine, 20 mg of uracil, and 40 mg each of Ltyrosine, L-lysine, L-histidine, L-threonine, and Lmethionine. YM-P medium contains per liter: 20 g of glucose, 10 g of succinic acid, 5 g of NaOH, 1 g of KOH, 2 g of NH4C1, 0.5 g of MgSO4, 0.1 g of CaCl2, 20 mg of adenine, 20 mg of uracil, amino-acids as in YMM medium, trace elements (45), and vitamins (45). KH2PO4 was included at a final concentration of 10-6 to 10-3 M, depending on the experiment; labeled

followed by treatment with 3'-nucleotidase (3) (R. Fox, J. Mynderse, and M. Goulian, unpublished data). [a-32P]dTTP and [a-32P]deoxyguanosine 5'triphosphate (dGTP) were prepared from [nP]deoxynucleoside monophosphates (dNMPs) by enzymatic phosphorylation (39). a-32P-labeled dATP, dGTP, dCTP, and dTTP (100 mCi/iLmol) were purchased from New England Nuclear Corp., Boston, Mass. Bromodeoxyuridine 5'-triphosphate (BrdUTP) was prepared by the procedure of Bessman et al. (5), and arabinosylcytosine triphosphate (araCTP) was prepared by the methods of Yoshikawa et al. (63) and Sowa et al. (T. Sowa, T. Kusaki, K. Sato, H. Osawa, and S. Ouchi, Ger. Offen. 2,014,440. 26 November 1970; Japan, Appl. 17 May 1969). 32P-labeled marker DNA. (i) Nuclear DNA. Wt p0 cells were grown in 100 ml of YMM medium at 30°C to a density of 3 x 107/ml, harvested by centrifugation, and suspended in 100 ml of YM-P medium (0.1 mM KH232P04; 0.5 mCi/,umol). After 6 h of growth at 30°C, the cells were recovered by centrifugation, washed once with 1 M sorbitol, and incubated (5 min, 37°C) in 5 ml of a solution containing 1 M sorbitol, 50 mM tris(hydroxymethyl)aminomethane (Tris)-hydrochloride (pH 7.5), 10 mM ethylenediaminetetraacetic acid (EDTA), 50 mM mercaptoethylamine, and an amount of A. luteus enzyme sufficient to result in immediate lysis when

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treated as follows. The mixture was centrifuged, and the cell pellet was lysed by suspending it in 5 ml of a solution containing 0.1 M Tris-hydrochloride (pH 8.2), 0.1 M EDTA, 3% Sarkosyl, and 1 mg of Pronase per ml. After 3 h at 600C, the mixture was extracted three times with phenol, and insoluble material was removed by centrifugation; the nucleic acids were recovered by ethanol precipitation and incubated (1 h, 37°C) in 3 ml of 50 mM Tris-hydrochloride (pH 7.5), 10 mM EDTA, containing 100 ,ug of pancreatic RNase (heated before use for 10 min, 800C) per ml. After phenol extraction and removal of the phenol with ether, the DNA was reprecipitated with ethanol and further purified by CsCl density gradient centrifugation (10 g of CsCl plus 8 ml of DNA solution [p = 1.68 g/cm3]) for 60 h at 32,000 rpm and 200C in a Beckman type 65 angle rotor (Beckman Instruments, Inc., Fullerton, Calif.). (ii) Mitochondrial DNA. Wt p+ cells were grown under vigorous aeration in 100 ml of YMM medium at 300C to a density of 4 x 107/ml, 20 mg of cycloheximide was added, and the mixture was incubated for 30 min. The cells were harvested and suspended in 100 ml of YM-P medium (0.1 mM KH232P04; 0.5 mCi/,umol) with 20 mg of cycloheximide per 100 ml. After 6 h of incubation under vigorous aeration at 30°C, the labeled cells were harvested and the DNA isolated and purified as described for nuclear DNA. Preparation of permeable spheroplasts. Cells were grown in 300 ml of YMM medium (30°C for Wt, 23°C for Ts) to a density of about 3 x 107/ml and collected by centrifugation. The cells were washed once with 30 ml of 1 M sorbitol and suspended in 30 ml of 1 M sorbitol containing 1/100 volume of Glusulase. The suspension was gently agitated on a shaker at room temperature for 20 to 40 min, depending on the strain, until conversion to spheroplasts was complete as judged by their susceptibility to lysis with 3% Sarkosyl and appearance under the phase microscope. The spheroplasts were collected by centrifugation, gently resuspended, and incubated in 150 ml of YMMS medium at 23°C, with gentle agitation, for 2.5 to 5 h (30). When the nucleic acids in the cells were to be prelabeled in vivo, the unlabeled uracil in the YMMS medium was replaced by [3H]uracil (0.01 mM; 0.5 mCi/,umol). The spheroplasts were collected by centrifugation and suspended without delay in 10 ml of buffer containing 75 mM Tris-hydrochloride (pH 7.5), 15 mM MgCl2, 1.5 mM CaCl2, 7.5 mM spermidinehydrochloride, 1.5 mM dithioerythritol at 0°C, after which 5 ml of 3 M sorbitol and 0.75 ml of 2 M KCl were added. The resulting osmotically shocked spheroplasts were separated from any contaminating cell lysate by centrifugation and resuspension in fresh buffer (0WC; 2 x 109 cells per ml); these "permeable spheroplasts" were always used within 15 min after preparation. Incorporation of dNTP's into permeable spheroplast DNA and sample processing. A 0.9 volume of the suspension of permeable spheroplasts was brought to the desired incubation temperature (23°C, unless stated otherwise), and the reaction was begun by adding 0.1 volume of a mixture containing 1 mM each of dATP, dCTP, dGTP, UTP,

CTP and GTP, 10 mM ATP, 0.1 mM dTTP labeled with 3H (1.8 mCi/,umol) or 32p (10 to 100 mCi/,umol) and 100 mM phosphoenolpyruvate. To stop the reaction, the whole sample or a portion was diluted 10 times with a solution containing 50 mM Trishydrochloride (pH 8.2), 50 mM EDTA, 50 mM K4P207, and 1 M sorbitol at 00C and centrifuged at 2,800 x g for 5 min at 0°C; the supernatant was discarded. To measure alkali-stable, acid-insoluble radioactivity, the cell pellet was taken up in 1 ml of a solution containing 0.5 M NaOH, 10 mM K4P207, 10 mM EDTA, and 50 ,ug of herring sperm DNA per ml and heated (100°C) for 15 min. It was then chilled in ice, and 4 ml of 10% trichloroacetic acid was added and the precipitate collected on a glassfiber filter (Whatman GF/C). The filter was thoroughly washed with 0.01 M HCl and then with acetone and dried, and the radioactivity was measured in a toluene scintillation fluid. To analyze the undenatured nucleic acids, pellets of spheroplasts (ca. 2 x 109 cells) were lysed by treatment with 2 ml of a solution containing 0.1 M Tris-hydrochloride (pH 8.2), 0.1 M EDTA, 3% Sarkosyl, and 1 mg of Pronase per ml at 600C for 10 min. After further incubation at 400C for 10 h, insoluble material was removed by centrifugation (15,000 x g for 10 min at 00C) and the mixture extracted 3 times with phenol and 3 times with ether, after which it was precipitated with ethanol. To analyze DNA in alkaline sucrose gradients, the cell pellet was lysed with 0.5 ml of 0.3 M KOH and left for 30 min at room temperature. Insoluble material was removed by centrifugation, and the clear solution, which contained all acid-insoluble radioactivity, was applied directly to the gradient. For density gradient analysis of denatured DNA, the alkaline lysate was incubated for 15 h at room temperature and then neutralized with 0.1 ml of 1 M Tris-hydrochloride (pH 7.5) and 0.5 ml of 0.3 M HCI. In experiments in which intact cells (labeled in vivo with [32P]dTMP or [3H]uracil) were analyzed, the cells were treated with A. luteus enzyme before lysis in alkali as described above (32P-labeled marker DNA). Sample processing for 32p transfer experiments. Permeable spheroplasts were labeled in vitro with each of the [a-32P]dNTPs in separate incubations (4.5 x 109 cells in 1.5 ml) by using standard conditions, except that the [a-32P]dNTP was at 10 ,uM instead of at 100 ,uM. After incubation (15 min, 2300), the spheroplasts were lysed under neutral conditions and deproteinized with Sarkosyl, Pronase, and phenol as described above. The nucleic acids were precipitated with ethanol 3 times and then passed through a column of Sephadex G-100 (prewashed with saturated diethylpyrocarbonate) in 10 mM Tris-hydrochloride (pH 7.5) and 1 mM EDTA (autoclaved). The combined fractions containing acid-insoluble radioactivity (immediately after the void volume) were precipitated with ethanol; the pellet was washed with 95% ethanol, air dried, and dissolved in 0.3 ml of 0.3 M KOH. The mixture was incubated at 370C for 15 h, after which 0.3 ml of 1 M HC104 was added, and, after a

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minimum of 15 min at 00C, the precipitate containing DNA and KC104 was removed by centrifugation. The relative proportions of acid-insoluble and -soluble radioactivity were determined by measuring Cerenkov radiation of the HCIO4 precipitate and supernatant. The ClO4- ions were removed from the supernatant fraction by neutralizing with KOH, chilling, and centrifuging. The supernatant was concentrated by a stream of air and analyzed by descending chromatography on Whatman 3MM paper in isobutyric acid-water-concentrated ammonium hydroxide (66:33:1, vol/vol/vol), together with markers of the 2'-, 3'-, and 5'-rNMPs and the 3'and 5'-dNMP's. Radioactivity in the segments in and between the nucleotide spots (detected by ultraviolet light) and the oligonucleotide spot at the origin was measured in toluene scintillation fluid. The scintillation fluid was then removed with ethanol, and the labeled nucleotides were eluted with 3 M NH4OH and reanalyzed by paper chromatography in isopropanol-concentrated HCl-water (65:17:18, vol/vol/vol). With the combined use of both chromatographic steps, the 2'(3')-rNMPs can be separated from the 5'-rNMP's and the 3'and 5'dNMP's. The percentage of transfer to 2'(3')-rNMPs was expressed relative to total incorporation into DNA, determined from HCIO4 precipitate and the oligonucleotide spot on chromatography. In experiments that included a KI density gradient step, pooled fractions were desalted on Sephadex G-100 and precipitated with ethanol (with 100 ,ug of yeast carrier RNA) before alkaline hydrolysis, as described above. Subsequent procedure was as described except for omission of the last (isopropanol-water-HCl) chromatographic step. Ultracentrifugation. Velocity sedimentation was carried out in a Beckman SW40 rotor. Alkaline sucrose gradients were 5 to 20% (wt/vol) sucrose, 0.3 M KOH, 0.7 M KCI, and 1 mM EDTA. Neutral sucrose gradients were 5 to 20% (wt/vol) sucrose in 1 M KCI, 10 mM Tris-hydrochloride (pH 7.5), and 1 mM EDTA. All gradients were layered over a 1-ml cushion of 60% sucrose. The molecular weights were estimated from S values by the empirical formulas of Studier (48). Equilibrium density gradient centrifugation was carried out in a Beckman type 65 angle rotor. For separation of nuclear and mitochondrial DNA, samples were centrifuged to equilibrium in a mixture of 10 g of CsoCl plus an 8-ml sample in 10 mM Trishydrochloride (pH 7.5)-l mM EDTA (p = 1.68 g/ cm3); for DNA containing BrdUMP, samples were centrifuged to equilibrium in a mixture of 11 g of CsCl plus 8 ml of solution (p = 1.74 g/cm3). KI solutions were prepared by dissolving 8.40 g of powdered KI in 8 ml of the same buffer (p = 1.58 g/ cm3) containing, in addition, NaHSO3 (10 mM). All fractions were collected from below. Unless stated otherwise, alkali-stable, acid-insoluble radioactivity was determined ior each fraction of the gradients as described above. Of the radioactivities, 64 to 78% were recovered from the CsCl gradients, and >80% was recovered from the alkaline sucrose gradients. Further details of the procedure are given in the figure legends.

Incorporation of [32P]dTMP into DNA in vivo. tup po cells were grown at 23°C in 300 ml of YMM medium (1 mM KH2PO4) to a density of about 3 x 107/ml. The cells were recovered by centrifugation, suspended in 250 ml of YMM-SAT medium, and incubated with aeration for 4 h to allow adaptation to the sulfanilamide, aminopterin, and dTMP in that medium. The cells were collected again (without cooling) by centrifugation (23°C) and suspended in 3 ml of fresh YMM-SAT medium at 23°C. Incorporation was begun by adding 3 mCi of [a-32P]dTMP (150 mCi/ymol) in 0.2 ml of YMM-SAT medium. The reaction was stopped by pipetting portions of 0.75 ml into 10-ml amounts of a mixture of EDTA (50 mM)-acetone-ether (4:5:1, vol/vol/vol) at 00C, which were underlayered with 2 ml of a solution containing 50 mM Tris-hydrochloride (pH 8.2), 50 mM EDTA, 1 M sorbitol, and was centrifuged (6,300 x g for 5 min at 0°C). The cells were washed once with Tris-EDTA-sorbitol, treated with A. luteus enzyme, and processed for alkali-stable, acid-insoluble radioactivity and alkaline sucrose gradient centrifugation, as described above. Other procedures. Cells were counted with a microscope with a counting chamber. Unless specified, all centrifugations of whole yeast cells were at 3,500 x g for 10 min at 20°C and at 1,200 x g for 5 min at 00C for spheroplasts or cells treated with A. luteus enzyme. Ethanol precipitation of nucleic acid was carried out by making the solution 1 M in LiCl followed by 2.5 volumes of 95% ethanol. After 18 h at -20°C, the precipitate was collected by centrifugation (27,000 x g for 30 min at 00C), washed once with 95% ethanol, and redissolved in 10 mM Tris-hydrochloride (pH 7.5) and 1 mM EDTA. For analysis of nucleotides in the incubation mixture, polyethyleneimine chromatography was carried out on thin-layer plates of polyethyleneimine cellulose (Brinkmann Instruments Inc., Westburg, N.Y.) with 1 M LiCl as solvent.

RESULTS

Properties of yeast spheroplasts. Cells of S. cerevisiae, harvested in mid-log phase and converted to spheroplasts by treatment with snail gut juice (30), enter into a resting phase, probably due to starvation. When spheroplasts are returned to medium with sorbitol for osmotic support, the rate of DNA synthesis resumes and reaches a maximum of 50% that of untreated cells in the same medium or about 10% that of untreated cells in the medium without sorbitol (30; W. Oertel, unpublished data). The reason for diminished incorporation of [3Hluracil by intact cells in the presence of sorbitol is not known, but it may result from interference of uracil uptake by sorbitol. DNA synthesis in the spheroplasts continues for at least 18 h, and under these conditions more than doubles their DNA content (30). The DNA product in p0 spheroplasts is indistinguishable from nu-

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clear DNA synthesized in intact cells, by sedimentation analysis and CsCl density gradient centrifugation (data not shown). Requirements for incorporation of nucleotides into permeabilized spheroplasts. The in vitro system utilizes spheroplasts made permeable ito nucleotides by brief exposure to a hypotonic buffer. p0 mutants were used for examining nuclear DNA synthesis without concurrent mitochondrial DNA synthesis. Results with p0 and p+ strains are compared below. Optimal DNA synthesis in permeable spheroplasts prepared from p0 strains of yeast required all four dNTPs, rATP, phosphoenolpyruvate, and Mg2+ (Table 1) and a pH between 7.0 and 8.0. rATP can be replaced partially by another rNTP, e.g., rUTP. In the absence of phosphoenolypyruvate, rATP and the other NTPs were degraded very rapidly, as determined by chromatography (polyethyleneimine) of the incubation mixture after various incubation times (data not shown). Inclusion of the other three rNTPs (rUTP, rGTP, and rCTP) did not stimulate the system to a significant extent when ATP was present at a high concentration (1 mM). Spermidine and CaCl2 were included to stabilize the cytoplasmic membranes and help prevent lysis of the permeable spheroplasts, and sorbitol provided a nonfermentable osmotic support. In vitro DNA synthesis is inhibited by araCTP, a klnown inhibitor of DNA replication but not DNA repair in E. coli and mammalian cells (9, 34). The in vitro system described here requires relatively high ratios of araCTP/dCTP for inhibition, resembling, in this respect, the yeast polymerase B (60). In the presence of DNase, the product is greatly reduced; a similar effect of DNase has been observed with toluenized E. coli (36). The sensitivity to ethidium bromide contrasts the resistance of nuclear DNA replication to the drug in vivo, where it only affects replication of mitochondrial DNA (19). The system is also sensitive to the sulfhydryl inhibitor, N-ethylmaleimide. Kinetics of incorporation into DNA. In vitro synthesis of DNA in permeable spheroplasts of the Wt p0 strain proceeded at 23°C in approximate linear fashion for the first 5 to 10 min and then at a slowly decreasing rate for another 40 to 50 min (Fig. 1A). At 38°C the initial rate of DNA synthesis with the Wt p0 used in these studies (A364A p0-3) was 1.5 times as high as at 23°C. With some other p0 derivatives of otherwise normal wild-type strains, the initial rate of DNA synthesis at 38°C was more than twice the rate at 230C. With Wt p+ cells containing intact mitochondria, the rate and extent of

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TABLE 1. Requirements and inhibitors of the in vitro systema Conditions Activity (%) 100 Complete ........................... -rATP, -PEP ...................... 22 -PEP ............................. 85 -rATP ............................. 59 -rATP, +rUTP (1 mM), -PEP ...... 55 -rGTP, -rCTP, -rUTP ....... ..... 95 -dATP, -dCTP, -dGTP ....... ..... 15 +araCTP (0.25 mM ......... ........ 50 +araCTP (0.8 mM) .......... ........ 23