JOURNAL OF BACTrzIOuLGY, Oct. 1977, p. 233-246 Copyright C 1977 American Society for Microbiology Vol. 132, No. 1 Printed in U.S.A. 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 incorpo- rated 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 acid- DNA 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 pro- caryotic deoxyribonucleic acid (DNA) replica- tion has resulted from the utilization of mu- tants in specific functions required for replica- tion. The unavailability of mutants in DNA replication functions is a distinct disadvantage with the more complex eucaryotic systems un- der study; however, some of the simpler eucary- otes allow considerable genetic manipulation. The yeast Saccharomyces cerevisiae has under- gone 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) identi- fied recently as a double-stranded ribonucleic acid (RNA) (54, 55). In contrast to the situation with higher eucaryotes, there are yeast mu- tants lacking one or all of the extrachromo- somal 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 replica- tion of the different species of extrachromo- somal 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 auxo- 233 on June 30, 2018 by guest http://jb.asm.org/ Downloaded from
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JOURNAL OF BACTrzIOuLGY, Oct. 1977, p. 233-246Copyright C 1977 American Society for Microbiology
Vol. 132, No. 1Printed in U.S.A.
Deoxyribonucleic Acid Synthesis in PermeabilizedSpheroplasts 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 incorpo-rated deoxynucleoside triphosphates specifically into double-stranded nuclearand mitochondrial deoxyribonucleic acid (DNA). Results with this in vitrosystem for cells with and without mitochondrial DNA were compared. Strainslacking mitochondrial DNA were used to study nuclear DNA replication. Witha temperature-sensitive mutant defective in DNA replication in vivo, DNAsynthesis in vitro was temperature sensitive as well. The product of synthesiswith all strains after very short labeling times consisted principally of shortfragments that sedimented at approximately 4S in alkali; with longer pulsetimes or a chase with unlabeled nucleotides, they grew to a more heterogenoussize, 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 nuclearDNA. Analysis by CsCl density gradient centrifugation after incorporation ofbromodeoxyuridine triphosphate showed that most of the product consisted ofchains containing both preexisting and newly synthesized material, but therewas also a small fraction (ca. 20%) in which the strands were fully synthesizedin vitro. 32P-label transfer ("nearest-neighbor") experiments demonstrated thatat least a part of the material synthesized in vitro contained ribonucleic acid-DNA junctions. DNA pulse-labeled in vivo in a mutant capable of taking upthymidine 5'-monophosphate, sedimented in alkali at 4S, as in the case of thein vitro experiments.
Much of the progress in understanding pro-caryotic deoxyribonucleic acid (DNA) replica-tion has resulted from the utilization of mu-tants in specific functions required for replica-tion. The unavailability of mutants in DNAreplication functions is a distinct disadvantagewith the more complex eucaryotic systems un-der study; however, some of the simpler eucary-otes allow considerable genetic manipulation.The yeast Saccharomyces cerevisiae has under-gone extensive genetic analysis, and severalconditional mutants in replicative functionshave been partially characterized (24, 25, 26).The relatively small amount of DNA per
yeast cell and per yeast chromosome allowscertain kinds of characterization of the intactchromosomal DNA not possible with the DNAof the eucaryote cells of higher organisms (10,41).
In addition to nuclear DNA, yeast cells maycontain several species of extrachromosomalreplication units, including mitochondrial
t Present address: Universitat Wurzburg, Institut furGenetik 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) identi-fied recently as a double-stranded ribonucleicacid (RNA) (54, 55). In contrast to the situationwith higher eucaryotes, there are yeast mu-tants lacking one or all of the extrachromo-somal replication units (19, 22), making itpossible to investigate nuclear DNA replicationindependent of the replication of the othernucleic acids. In addition, some of the mutantsmay permit study of individual proteins andregulatory factors that participate in replica-tion of the different species of extrachromo-somal replicons.
Certain properties of yeast have a bearingon experimental design for studies on DNAsynthesis, including its tough cell wall, lack ofthymidine kinase (20), the presence of which isrequired for specific labeling of DNA in vivowith radioactive thymidine, and its high levelof nuclease activities. It may be possible toalter these problem characteristics by usingmutants capable of taking up deoxythymidine5'-monophosphate (dTMP) and dTMP auxo-
trophs (12, 17, 57) and by using enzymes toremove or weaken the cell wall (1, 31, 61). Therecent isolation of nuclease-deficient mutantsin another yeast-like eucaryote (2) encouragesthe hope that this will soon be successful inyeast as well.Two in vitro yeast systems have been de-
scribed, both employing the detergent Brij topromote uptake of nucleotide precursors ofDNA (2, 27). Although DNA synthesis in oneof these systems was temperature sensitive inmutants defective in replication in vivo, theproducts, which were only partially character-ized, may have been mostly or entirely mito-chondrial rather than nuclear (2). A recent,brief description of a system using yeast cellstreated with snail extract and mercaptoethanoldeals with the synthesis of nuclear as well asmitochondrial DNA (62). In this report wedescribe another permeable system in S. cere-visiae, which synthesizes both nuclear and mi-tochondrial DNA in vitro from deoxynucleosidetriphosphates (dNTPs). Properties of the sys-tem are characterized, particularly the natureof nuclear DNA synthesis.
MATERIALS AND METHODSCells. S. cerevisiae A364A p+ (a adel ade2 ural
gall tyrl his7 lys2, Wt p+) and its derivatives 198,temperature sensitive in gene cdc8 (Ts cdc8 p+),and 146-2-3, temperature sensitive in gene cdc2l (Tscdc2l p+), were generously provided by L. Hartwell(24, 25). Mutant A364A tup4 p+ (tup p+), capable ofdTMP uptake (tup), was selected from A364A p+cells by a variation of the method of R. B. Wickner(57). From these p+ strains, the mitochondrial DNA-less (p°) strains A364A p0-3 (Wt p°); 198 p0-4 (Tscdc8 p°); 46-2-3 p°-13 (Ts cdc2l po); and A364 tup4pQ-17 (tup p°) were derived by prolonged treatmentwith ethidium bromide (19). Absence of mitochon-drial DNA was checked by CsCl density gradientcentrifugation 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 killerfactor (47); the other strains are not characterizedin this respect.
Media. YMM medium is based on the formula ofHartwell (23) and contains per liter: 20 g of glucose,10 g of succinic acid, 6 g of NaOH, 6.7 g of yeastnitrogen base (without amino acids, Difco Labora-tories, Detroit, Mich.), 0.5 g of yeast extract, 20 mgof adenine, 20 mg of uracil, and 40 mg each of L-tyrosine, L-lysine, L-histidine, L-threonine, and L-methionine.YM-P medium contains per liter: 20 g of glucose,
10 g of succinic acid, 5 g of NaOH, 1 g of KOH, 2 gof NH4C1, 0.5 g of MgSO4, 0.1 g of CaCl2, 20 mg ofadenine, 20 mg of uracil, amino-acids as in YMMmedium, trace elements (45), and vitamins (45).KH2PO4 was included at a final concentration of10-6 to 10-3 M, depending on the experiment; labeled
inorganic orthophosphate (32P,) was added to thedesired specific activity.
Both media were made lOx concentrated andsterilized by filtration. Before use, they were dilutedwith 9 parts of sterile water (YMM or YM-P me-dium) or sterile 1 M sorbitol (YMMS medium) orwith a solution containing (per liter) 6.5 g of sulfa-nilamide, 55 mg of aminopterin, 1 mmol of KH2PO4,and 0.5 mmol of dTMP (YMM-SAT medium).
Reagents. Glusulase (snail gut juice) was ob-tained from Endo Laboratories, Garden City, N.Y.Yeast cell wall degrading enzyme from Arthrobacterluteus was isolated by ammonium sulfate precipita-tion (70% saturation) from the medium of a 3-dayculture in a minimal medium containing yeast cellwalls as its only carbon source (31). Pancreaticdeoxyribonuclease (DNase), pancreatic ribonuclease(RNase), and snake venom phosphodiesterase werepurchased from Worthington Biochemicals Corp.,Freehold, N.J., and Pronase was from Calbiochem,La Jolla, Calif. Unlabeled nucleotides were from P-L Biochemicals; trisodium phosphoenolpyruvate, N-ethylmaleimide, dithioerythritol, saponin, D-(+)-sorbitol, and spermidine-hydrochloride were fromSigma Chemical Co., St. Louis, Mo. Sarkosyl NL-30and ethidium bromide were gifts from Geigy andBoots. 3H-labeled deoxythymidine 5'-triphosphate(dTTP) and deoxycytidine 5'-triphosphate (dCTP)(15 to 20 mCi/,umol) and [3H]uracil (40 to 60 mCi/,mmol) were from Schwarz/Mann, Orangeburg, N.Y.[32P]dTMP (100 to 500 mCi/,umol) was prepared bythe procedure of Okazaki and Kornberg (39) byusing thymidine kinase and [y-32P]adenosine 5'-triphosphate (ATP) (18). 5'-[32P]deoxyguanosine 5'-triphosphate (dGMP) (200 mCi/Amol) was synthe-sized from 3'-dGMP by using polynucleotide kinase(44) and [_y-32P]adenosine 5'-triphosphate (rATP),followed by treatment with 3'-nucleotidase (3) (R.Fox, J. Mynderse, and M. Goulian, unpublisheddata). [a-32P]dTTP and [a-32P]deoxyguanosine 5'-triphosphate (dGTP) were prepared from [nP]deoxy-nucleoside monophosphates (dNMPs) by enzymaticphosphorylation (39). a-32P-labeled dATP, dGTP,dCTP, and dTTP (100 mCi/iLmol) were purchasedfrom New England Nuclear Corp., Boston, Mass.Bromodeoxyuridine 5'-triphosphate (BrdUTP) wasprepared by the procedure of Bessman et al. (5),and arabinosylcytosine triphosphate (araCTP) wasprepared 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. 26November 1970; Japan, Appl. 17 May 1969).
32P-labeled marker DNA. (i) Nuclear DNA. Wtp0 cells were grown in 100 ml of YMM medium at30°C to a density of 3 x 107/ml, harvested by centrif-ugation, and suspended in 100 ml of YM-P medium(0.1 mM KH232P04; 0.5 mCi/,umol). After 6 h ofgrowth at 30°C, the cells were recovered by centrif-ugation, washed once with 1 M sorbitol, and incu-bated (5 min, 37°C) in 5 ml of a solution containing1 M sorbitol, 50 mM tris(hydroxymethyl)amino-methane (Tris)-hydrochloride (pH 7.5), 10 mM eth-ylenediaminetetraacetic acid (EDTA), 50 mM mer-captoethylamine, and an amount of A. luteus en-zyme sufficient to result in immediate lysis when
treated as follows. The mixture was centrifuged,and the cell pellet was lysed by suspending it in 5ml of a solution containing 0.1 M Tris-hydrochloride(pH 8.2), 0.1 M EDTA, 3% Sarkosyl, and 1 mg ofPronase per ml. After 3 h at 600C, the mixture wasextracted three times with phenol, and insolublematerial was removed by centrifugation; the nucleicacids were recovered by ethanol precipitation andincubated (1 h, 37°C) in 3 ml of 50 mM Tris-hydro-chloride (pH 7.5), 10 mM EDTA, containing 100 ,ugof pancreatic RNase (heated before use for 10 min,800C) per ml. After phenol extraction and removalof the phenol with ether, the DNA was reprecipi-tated with ethanol and further purified by CsCldensity gradient centrifugation (10 g of CsCl plus 8ml of DNA solution [p = 1.68 g/cm3]) for 60 h at32,000 rpm and 200C in a Beckman type 65 anglerotor (Beckman Instruments, Inc., Fullerton, Calif.).
(ii) Mitochondrial DNA. Wt p+ cells were grownunder vigorous aeration in 100 ml of YMM mediumat 300C to a density of 4 x 107/ml, 20 mg of cyclohex-imide was added, and the mixture was incubatedfor 30 min. The cells were harvested and suspendedin 100 ml of YM-P medium (0.1 mM KH232P04; 0.5mCi/,umol) with 20 mg of cycloheximide per 100 ml.After 6 h of incubation under vigorous aeration at30°C, the labeled cells were harvested and the DNAisolated and purified as described for nuclear DNA.
Preparation of permeable spheroplasts. Cellswere grown in 300 ml of YMM medium (30°C forWt, 23°C for Ts) to a density of about 3 x 107/mland collected by centrifugation. The cells werewashed once with 30 ml of 1 M sorbitol and sus-pended in 30 ml of 1 M sorbitol containing 1/100volume of Glusulase. The suspension was gentlyagitated on a shaker at room temperature for 20 to40 min, depending on the strain, until conversion tospheroplasts was complete as judged by their sus-ceptibility to lysis with 3% Sarkosyl and appearanceunder the phase microscope. The spheroplasts werecollected by centrifugation, gently resuspended, andincubated in 150 ml of YMMS medium at 23°C,with gentle agitation, for 2.5 to 5 h (30). When thenucleic acids in the cells were to be prelabeled invivo, the unlabeled uracil in the YMMS mediumwas replaced by [3H]uracil (0.01 mM; 0.5 mCi/,umol).The spheroplasts were collected by centrifugationand suspended without delay in 10 ml of buffercontaining 75 mM Tris-hydrochloride (pH 7.5), 15mM MgCl2, 1.5 mM CaCl2, 7.5 mM spermidine-hydrochloride, 1.5 mM dithioerythritol at 0°C, afterwhich 5 ml of 3 M sorbitol and 0.75 ml of 2 M KClwere added. The resulting osmotically shockedspheroplasts were separated from any contaminat-ing cell lysate by centrifugation and resuspensionin fresh buffer (0WC; 2 x 109 cells per ml); these"permeable spheroplasts" were always used within15 min after preparation.
Incorporation of dNTP's into permeable sphero-plast DNA and sample processing. A 0.9 volume ofthe suspension of permeable spheroplasts wasbrought to the desired incubation temperature(23°C, unless stated otherwise), and the reactionwas begun by adding 0.1 volume of a mixturecontaining 1 mM each of dATP, dCTP, dGTP, UTP,
DNA SYNTHESIS IN S. CEREVISIAE 235
CTP and GTP, 10 mM ATP, 0.1 mM dTTP labeledwith 3H (1.8 mCi/,umol) or 32p (10 to 100 mCi/,umol)and 100 mM phosphoenolpyruvate. To stop the re-action, the whole sample or a portion was diluted10 times with a solution containing 50 mM Tris-hydrochloride (pH 8.2), 50 mM EDTA, 50 mMK4P207, and 1 M sorbitol at 00C and centrifuged at2,800 x g for 5 min at 0°C; the supernatant wasdiscarded. To measure alkali-stable, acid-insolubleradioactivity, the cell pellet was taken up in 1 ml ofa solution containing 0.5 M NaOH, 10 mM K4P207,10 mM EDTA, and 50 ,ug of herring sperm DNA perml and heated (100°C) for 15 min. It was thenchilled in ice, and 4 ml of 10% trichloroacetic acidwas added and the precipitate collected on a glass-fiber filter (Whatman GF/C). The filter was thor-oughly washed with 0.01 M HCl and then withacetone and dried, and the radioactivity was mea-sured in a toluene scintillation fluid.
To analyze the undenatured nucleic acids, pelletsof spheroplasts (ca. 2 x 109 cells) were lysed bytreatment with 2 ml of a solution containing 0.1 MTris-hydrochloride (pH 8.2), 0.1 M EDTA, 3% Sar-kosyl, and 1 mg of Pronase per ml at 600C for 10min. 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 mixtureextracted 3 times with phenol and 3 times withether, 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 KOHand left for 30 min at room temperature. Insolublematerial was removed by centrifugation, and theclear solution, which contained all acid-insolubleradioactivity, was applied directly to the gradient.
For density gradient analysis of denatured DNA,the alkaline lysate was incubated for 15 h at roomtemperature and then neutralized with 0.1 ml of 1M Tris-hydrochloride (pH 7.5) and 0.5 ml of 0.3 MHCI.
In experiments in which intact cells (labeled invivo with [32P]dTMP or [3H]uracil) were analyzed,the cells were treated with A. luteus enzyme beforelysis in alkali as described above (32P-labeledmarker DNA).Sample processing for 32p transfer experiments.
Permeable spheroplasts were labeled in vitro witheach of the [a-32P]dNTPs in separate incubations(4.5 x 109 cells in 1.5 ml) by using standard condi-tions, except that the [a-32P]dNTP was at 10 ,uMinstead of at 100 ,uM. After incubation (15 min,2300), the spheroplasts were lysed under neutralconditions and deproteinized with Sarkosyl, Pro-nase, and phenol as described above. The nucleicacids were precipitated with ethanol 3 times andthen passed through a column of Sephadex G-100(prewashed with saturated diethylpyrocarbonate)in 10 mM Tris-hydrochloride (pH 7.5) and 1 mMEDTA (autoclaved). The combined fractions con-taining acid-insoluble radioactivity (immediatelyafter the void volume) were precipitated withethanol; 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, afterwhich 0.3 ml of 1 M HC104 was added, and, after a
minimum of 15 min at 00C, the precipitate contain-ing DNA and KC104 was removed by centrifugation.The relative proportions of acid-insoluble and -solu-ble radioactivity were determined by measuringCerenkov radiation of the HCIO4 precipitate andsupernatant. The ClO4- ions were removed fromthe supernatant fraction by neutralizing with KOH,chilling, and centrifuging. The supernatant wasconcentrated by a stream of air and analyzed bydescending chromatography on Whatman 3MM pa-per in isobutyric acid-water-concentrated ammo-nium hydroxide (66:33:1, vol/vol/vol), together withmarkers of the 2'-, 3'-, and 5'-rNMPs and the 3'-and 5'-dNMP's. Radioactivity in the segments inand between the nucleotide spots (detected by ultra-violet light) and the oligonucleotide spot at theorigin was measured in toluene scintillation fluid.The scintillation fluid was then removed withethanol, and the labeled nucleotides were elutedwith 3 M NH4OH and reanalyzed by paper chroma-tography in isopropanol-concentrated HCl-water(65:17:18, vol/vol/vol). With the combined use ofboth chromatographic steps, the 2'(3')-rNMPs canbe separated from the 5'-rNMP's and the 3'and 5'-dNMP's. The percentage of transfer to 2'(3')-rNMPswas expressed relative to total incorporation intoDNA, determined from HCIO4 precipitate and theoligonucleotide spot on chromatography.
In experiments that included a KI density gra-dient step, pooled fractions were desalted on Sepha-dex G-100 and precipitated with ethanol (with 100,ug of yeast carrier RNA) before alkaline hydrolysis,as described above. Subsequent procedure was asdescribed except for omission of the last (isopropa-nol-water-HCl) chromatographic step.
Ultracentrifugation. Velocity sedimentation wascarried out in a Beckman SW40 rotor. Alkalinesucrose gradients were 5 to 20% (wt/vol) sucrose,0.3 M KOH, 0.7 M KCI, and 1 mM EDTA. Neutralsucrose gradients were 5 to 20% (wt/vol) sucrose in1 M KCI, 10 mM Tris-hydrochloride (pH 7.5), and 1mM EDTA. All gradients were layered over a 1-mlcushion of60% sucrose. The molecular weights wereestimated from S values by the empirical formulasof Studier (48).
Equilibrium density gradient centrifugation wascarried out in a Beckman type 65 angle rotor. Forseparation of nuclear and mitochondrial DNA, sam-ples were centrifuged to equilibrium in a mixture of10 g of CsoCl plus an 8-ml sample in 10 mM Tris-hydrochloride (pH 7.5)-l mM EDTA (p = 1.68 g/cm3); for DNA containing BrdUMP, samples werecentrifuged to equilibrium in a mixture of 11 g ofCsCl plus 8 ml of solution (p = 1.74 g/cm3). KIsolutions were prepared by dissolving 8.40 g ofpowdered 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. Unlessstated otherwise, alkali-stable, acid-insoluble radio-activity was determined ior each fraction of thegradients as described above. Of the radioactivities,64 to 78% were recovered from the CsCl gradients,and >80% was recovered from the alkaline sucrosegradients. Further details ofthe procedure are givenin the figure legends.
Incorporation of [32P]dTMP into DNA in vivo.tup po cells were grown at 23°C in 300 ml of YMMmedium (1 mM KH2PO4) to a density of about 3 x107/ml. The cells were recovered by centrifugation,suspended in 250 ml of YMM-SAT medium, andincubated with aeration for 4 h to allow adaptationto the sulfanilamide, aminopterin, and dTMP inthat medium. The cells were collected again (with-out cooling) by centrifugation (23°C) and suspendedin 3 ml of fresh YMM-SAT medium at 23°C. Incor-poration 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 of0.75 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 solutioncontaining 50 mM Tris-hydrochloride (pH 8.2), 50mM EDTA, 1 M sorbitol, and was centrifuged (6,300x g for 5 min at 0°C). The cells were washed oncewith Tris-EDTA-sorbitol, treated with A. luteusenzyme, and processed for alkali-stable, acid-insol-uble radioactivity and alkaline sucrose gradientcentrifugation, as described above.
Other procedures. Cells were counted with amicroscope with a counting chamber. Unless speci-fied, all centrifugations of whole yeast cells were at3,500 x g for 10 min at 20°C and at 1,200 x g for 5min at 00C for spheroplasts or cells treated with A.luteus enzyme.
Ethanol precipitation of nucleic acid was carriedout by making the solution 1 M in LiCl followed by2.5 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 with95% ethanol, and redissolved in 10 mM Tris-hydro-chloride (pH 7.5) and 1 mM EDTA.
For analysis of nucleotides in the incubationmixture, polyethyleneimine chromatography wascarried out on thin-layer plates of polyethyleneiminecellulose (Brinkmann Instruments Inc., Westburg,N.Y.) with 1 M LiCl as solvent.
RESULTSProperties of yeast spheroplasts. Cells of S.
cerevisiae, harvested in mid-log phase and con-verted to spheroplasts by treatment with snailgut juice (30), enter into a resting phase, prob-ably due to starvation. When spheroplasts arereturned to medium with sorbitol for osmoticsupport, the rate of DNA synthesis resumesand reaches a maximum of 50% that of un-treated cells in the same medium or about 10%that of untreated cells in the medium withoutsorbitol (30; W. Oertel, unpublished data). Thereason for diminished incorporation of [3Hluracilby intact cells in the presence of sorbitol is notknown, but it may result from interference ofuracil uptake by sorbitol. DNA synthesis inthe spheroplasts continues for at least 18 h,and under these conditions more than doublestheir DNA content (30). The DNA product inp0 spheroplasts is indistinguishable from nu-
clear DNA synthesized in intact cells, by sedi-mentation analysis and CsCl density gradientcentrifugation (data not shown).Requirements for incorporation of nucleo-
tides into permeabilized spheroplasts. The invitro system utilizes spheroplasts made perme-
able ito nucleotides by brief exposure to a hypo-tonic buffer. p0 mutants were used for examin-ing nuclear DNA synthesis without concurrentmitochondrial DNA synthesis. Results with p0
and p+ strains are compared below.Optimal DNA synthesis in permeable spher-
oplasts prepared from p0 strains of yeast re-
quired all four dNTPs, rATP, phosphoenolpyr-uvate, and Mg2+ (Table 1) and a pH between7.0 and 8.0. rATP can be replaced partially byanother rNTP, e.g., rUTP. In the absence ofphosphoenolypyruvate, rATP and the otherNTPs were degraded very rapidly, as deter-mined by chromatography (polyethyleneimine)of the incubation mixture after various incuba-tion times (data not shown). Inclusion of theother three rNTPs (rUTP, rGTP, and rCTP)did not stimulate the system to a significantextent when ATP was present at a high concen-tration (1 mM). Spermidine and CaCl2 wereincluded to stabilize the cytoplasmic mem-branes and help prevent lysis of the permeablespheroplasts, and sorbitol provided a nonfer-mentable osmotic support.In vitro DNA synthesis is inhibited by
araCTP, a klnown inhibitor of DNA replicationbut not DNA repair in E. coli and mammaliancells (9, 34). The in vitro system described hererequires relatively high ratios of araCTP/dCTPfor inhibition, resembling, in this respect, theyeast polymerase B (60). In the presence ofDNase, the product is greatly reduced; a simi-lar effect of DNase has been observed withtoluenized E. coli (36). The sensitivity to ethid-ium bromide contrasts the resistance of nuclearDNA replication to the drug in vivo, where itonly affects replication of mitochondrial DNA(19). The system is also sensitive to the sulfhy-dryl inhibitor, N-ethylmaleimide.
Kinetics of incorporation into DNA. In vitrosynthesis ofDNA in permeable spheroplasts ofthe Wt p0 strain proceeded at 23°C in approxi-mate linear fashion for the first 5 to 10 minand then at a slowly decreasing rate for another40 to 50 min (Fig. 1A). At 38°C the initial rateof DNA synthesis with the Wt p0 used in thesestudies (A364A p0-3) was 1.5 times as high asat 23°C. With some other p0 derivatives ofotherwise normal wild-type strains, the initialrate of DNA synthesis at 38°C was more thantwice the rate at 230C. With Wt p+ cells contain-ing intact mitochondria, the rate and extent of
TABLE 1. Requirements and inhibitors of the invitro systema
a The complete reaction mixtures (0.3 ml) wereprepared as described in Materials and Methods,giving a final composition of 50 mM Tris-hydrochlo-ride, (pH 7.5); 10 mM MgCl2; 1 mM CaCl2; 100 mMKCl; 1 M sorbitol; 5 mM spermidine-hydrochloride;1 mM dithioerythritol; 10 mM trisodium phos-phoenolpyruvate; 1 mM rATP; 0.1 mM each dATP,dGTP, dCTP, rGTP, rCTP, and rUTP; 0.01 mM[3H]dTTP (1.8 mCi/,umol), and 4 x 108 permeabilizedspheroplasts (Wt po). Dithioerythritol was omittedwhen N-ethylmaleimide was present. Incubationswere carried out in duplicate for 10 min at 23°C.
DNA synthesis were generally two to threetimes as high as for p0 strains, but the patternresembles that described for Wt po, with 5 to 10min at a sustained rate followed by 40 to 50 minat a decreased rate. Ts cdc8 p0 (Ts 198 p0-4), astrain temperature sensitive in vivo in a func-tion necessary for propagation of DNA replica-tion (24), was temperature sensitive in vitro aswell (Fig. 1B). An initial burst of in vitrosynthesis occurred with the mutant at the re-strictive temperature and could not be pre-vented by preincubation (without nucleotides)for 5 to 15 min. A p0 derivative of another cdc8mutant isolated by Hartwell (25) (Ts 13052)behaves in this system in the same way as Ts198. We believe, therefore, that the type ofkinetics observed at the restrictive temperatureis related to the cdc8 lesion of Ts 198 po. Onepossible explanation is that the temperature-sensitive gene product may be protectedagainst inactivation as long as DNA synthesiscannot take place and is slowly inactivatedonce synthesis resumes.Another temperature-sensitive DNA replica-
tion mutant, Ts cdc2l pO (propagation-) (25)also showed temperature sensitivity in vitro(Fig. 1C) when compared to the non-tempera-ture-sensitive parent cells, but this is lessmarked than for Ts cdc8 po (Fig. 1A). The
TIME ,MINFIG. 1. Kinetics of deoxynucleotide incorporation
into permeable spheroplast DNA. Permeable sphero-plasts from the strains Wt po, Ts cdc8 po, and Tscdc2l po were prepared and incubated (2 ml) as
described in Materials and Methods, except that thecell suspensions (1.8 x 109 cells per ml) were prein-cubated for 5 min at the same temperature (23 or
38°C) before beginning the reaction by adding thenucleotide mixture (see Materials and Methods).After incubation at the same temperature for thetimes indicated in the figure, 200-pl samples were
removed and analyzed for alkali-stable, acid-insolu-ble radioactivity, which is expressed as pmol of totaldeoxynucleotide incorporated at 23 (0) or 38°C (a).(A) Wt po; (B) Ts cdc8 pO; (C) Ts cdc2l po.
cdc2l defect, recently shown to be in thymidyl-ate synthetase (17), should not be expressed inthis in vitro system in which dNTPs are pro-vided. However, it is possible that the slighteffect of temperature is not a direct result ofthe thermolability of the gene product, but,rather, the unusual sensitivity of the replica-tion point to degradation may be a result of
thymidylate starvation (17) during the preincu-bation.Ts cdc8 po permeable spheroplasts incorpo-
rated more than twice as much nucleotide percell at 23°C as the parent cells or the Ts cdc2lp0 mutant (compare Figs. 1A and C with B).This result may be related to the unusuallyhigh concentration of DNA polymerase in Tscdc8 p0 cells (60).On the basis of the in vitro kinetic data, it is
estimated that the initial rates of nucleotideincorporation at 23°C into the petite strains,Wt po and Ts cdc8 pO, were ca. 700 and 1,650nucleotides per cell per min, respectively. Forthe strains containing intact mitochondria, thecorresponding in vitro rates of incorporation(230C) were 1,900 (Wt p+) and 2,600 (Ts cdc8p+) nucleotides per cell per min. Based on anaverage DNA content of 3 x 107 bases in thehaploid cell and a generation time of about 2.5h (YMM medium, 230C) for p+ cells and about3.5 h for p0 cells, these in vitro rates of DNAsynthesis are equivalent to ca. 0.5% (Wt pO),1.2% (Ts cdc8 pO), 1% (Wt p+), and 2% (Ts cdc8p+) of the in vivo rates observed for the wholecells in medium without osmotic support. Themaximum in vitro incorporation per cell was6,600 nucleotides for Wt po, 15,600 for Ts cdc8p0, 18,500 for Wt p+, and 24,300 for Ts cdc8 p+.Nature of the product of DNA synthesis in
vitro. Nuclear and mitochondrial yeast DNAcan be distinguished by their different densitiesin CsCl density gradients. The products synthe-sized in the permeable spheroplasts preparedfrom p0 strains (Fig. 2A and C) banded in CsClat 1.699 g/cm3 (26), the density of double-stranded nuclear DNA. With an in vitro systemwith p+ cells containing normal mitochondrialDNA (Fig. 2B and D), 35 to 45% of the productbanded as double-stranded nuclear DNA,whereas the rest banded with the density ofdouble-stranded mitochondrial DNA (1.683 g/cm3) (26). The possibility that the peak withthe density of 1.699 g/cm3 is denatured mito-chondrial DNA is ruled out by CsCl gradientanalysis after alkali denaturation (Fig. 2E andF).
In S. cerevisiae there is a species of non-nu-clear DNA that bands in CsCl gradients withthe same density as nuclear DNA. This non-nu-clear DNA consists of small, superhelical cir-cles with a contour length of ca. 2 ,um (7, 8, 21,22). Recent results of Clark-Walker stronglysuggest that this DNA is associated with acharacteristic cytoplasmic membrane fraction(7, 8). To test the possibility that the in vitrosynthesized DNA with a density of 1.699 g/cm3is the 2-,gm circular DNA rather than nuclear
FIG. 2. Equilibrium centrifugation analysis ofDNA synthesized in vitro. Reaction mixtures (2 ml) were
prepared with permeable spheroplasts (1.8 x 101/ml) of strains Wt po, Wt p+, Ts cdc8 po, and Ts cdc8 p+ as
described in Materials and Methods, except that the specific activity of [3H]dTTP was 18 mCi/Imol.Incubations were kfor 30 min at 23°C. After density markers of 32P-labeled yeast DNA were added, half ofeach mixture was treated with Sarkosyl, Pronase, and phenol (nondenatured) and the other half was dena-tured with alkali (see Materials and Methods). The denatured and nondenatured samples were analyzed byCsCl equilibrium density gradient centrifugation (35,000 rpm for 60 h at 20°C) (see Materials andMethods). The larger of the two 32P-marker peaks (left-hand side) is nuclear DNA (1.699 glcm), whereas thesmaller peak (right-hand side) is mitochondrial DNA (1.683 g/cm3). For technical reasons, the amountanalyzed for the nondenatured sample of the Wt p0 production was less than for the other samples. (A) Wt p0
DNA, po-spheroplasts, prelabeled in vivo with[3H]uracil and pulse-labeled in vitro with[a-32P]dTTP, were lysed with saponin by themethod of Udem and Warner (53) and frac-tionated by differential centrifugation into a
nuclear and a cytoplasmic fraction. Most of the32P and alkali-stable, acid-insoluble 3H countswere detected in the nuclear fraction. Theproportion of 3H/32P in the nuclear fraction wasthe same as in the whole cells. When the cyto-plasmic fraction was further fractionated bydensity in a sucrose gradient as described byClark-Walker (7) to obtain the circular DNA-containing membrane fraction, 3H-prelabeledDNA and a small amount of 3P-labeled DNA(<1% of the total incorporated "2P) was detectedat the expected density. The 3H/32P ratio wassimilar to that of whole cell DNA and indicated
no preferential synthesis of the DNA in thisfraction in vitro. In contrast to this result, a
control experiment with cells containing mito-chondria resulted in preferential incorporationof 32P label into a cytoplasmic membrane frac-tion, presumably the mitochondria. This proce-dure does not firmly exclude the possibility thata part, or even all, of the in vitro nuclear prod-uct consists of the 2-,um DNA, if we assumethat the 2-,um DNA is replicated in the nucleusand subsequently transported to the cytoplasm.Additional evidence supporting the chromo-somal nature ofthe in vitro product comes fromneutral sucrose gradient velocity centrifuga-tion. The bulk of the undenatured in vitro-labeled DNA sedimented more rapidly than the21 to 23S expected for 2-I,m DNA (data notshown).
We conclude from these experiments thatthe permeable spheroplast system preparedfrom po cells synthesizes only nuclear DNA;whereas, the system prepared from p+ cellssynthesizes both nuclear and mitochondrialDNA, although the proportions of the twoforms of DNA are greatly altered comparedwith intact cells. It is also evident that in thein vitro system from the temperature-sensitivemutant Ts cdc8 p+, relatively less mitochon-drial DNA is synthesized than in the parentstrain Wt p+ (compare Fig. 2B and D and F).Discontinuous synthesis. Spheroplasts were
prelabeled in vivo with [3H]uracil and, afterpermeabilization, pulse-labeled for variouslengths of time with [a-32P]dTTP; after this,the DNA was analyzed in alkaline sucrosegradients (Fig. 3). After very short in vitropulses, the product consisted of small frag-ments sedimenting at about 4S (Fig. 3A). Withlonger pulse times, these pieces accumulatedand grew to a larger and more heterogeneoussize class, most of which sedimented at ca. 6 to8S, but with a significant portion up to 15S(Fig. 3B and C). A small fraction of the pulse-labeled material sedimented with the bulkDNA to the cushion at the bottom of the gra-dient (>30S), but the amount of in vitro-syn-thesized DNA in the rapidly sedimenting frac-tion increased at a rate slower than that of thesmall pieces. Pulse-chase experiments con-firmed the growth of the short pieces and theirlimited integration into high-molecular-weightDNA (Fig. 3D); this result was not altered byinclusion of NAD+ (1 mM). In the experimentsdescribed above (Fig. 2), most of the materialwas double stranded after longer incubationtimes (15 to 30 min); thus it can be assumedthat at least the longer fragments must havebeen hydrogen bonded to preexisting DNA be-fore treatment with alkali.
Density-labeling experiments. To determinehow much of a particular DNA fragment isactually synthesized in vitro, incorporation wascarried out with BrdUTP in place of dTTP todensity-label the DNA. The undenatured DNAwas sonified, and the average. chain lengthwas determined to be ca. 1,000 nucleotides byvelocity sedimentation in a neutral sucrosegradient with phage fd DNA as an internalmarker. Equilibrium density gradient centrifu-gation of the BrdUMP-containing DNA gave adistribution of densities between "hybrid" and"light" DNA (Fig. 4A). For the Wt po, the peakmaterial corresponded to about 30% substitu-tion in one of the chains in the double strand.Approximately 20 to 25% of the native producthad fully hybrid density (i.e., one strand fullysubstituted with BrdUMP). Analysis of a sam-
of DNA pulse-labeled in vitro. Reaction mixtures(1.65 ml) were prepared with permeable spheroplastsof Wt po (2 x 109/ml) prelabeled in vivo with[3H]uracil (see Materials and Methods) and incu-bated at 23°C with [a_32P]dTTP (100 mCiI,Amol)instead of[3H]dTTP. Samples (400 j) were removedat 30 s, 3 and 15 min. An additional sample takenat 30 s was made 2.5 mM in unlabeled dTTP (250-fold excess over [a32P]dTTP) followed by an addi-tional 15-min incubation (pulse-chase). Cell pelletswere lysed and centrifuged in alkaline sucrose gra-dients (see Materials and Methods) at 32,000 rpmfor 12.5 h at 20'C. The position of20S (arrow) wasfrom marker 14C phage fd DNA included in sampleA. Direction of sedimentation is from right to left.(A) 30-s pulse; (B) 3-min pulse; (C) 15-min pulse;(D) 30-s pulse followed by a 15-min chase. Symbols:0, 32p; 0, 3Hj
FIG. 4. Equilibrium centrifugation ancDNA density-labeled (BrdUTP) in vitro. Ino(2 ml) ofpermeable spheroplasts of Wt p0 (2cells per ml) was carried out at 23°C with .(0.1 mM) instead ofdTTP and with the ralabel in [3H]dCTP (0.01 mM; 1.62 mCilp.nincubation was stopped after 20 min, and, c
labeled yeast nuclear DNA had been addednal density marker, half of the material uwithout denaturation, briefly sonicated, aprocessed with Sarkosyl, Pronase, and phwhile the other halfwas treated with alkali Ineutralized (B) (see Materials and Methodstion ofeach sample was used to determine tthe DNA fragments by neutral sucrosecentrifugation (see Materials and Methodadditional incubations (each 1 ml) were caiwith permeable spheroplasts of Ts cdc8 po (2cells per ml) at 23C (C) and 38°C (D), resjThe incubations for samples C and D weimin, after a 5-min preincubation of the 8spsuspensions, and both were treated with althen neutralized. All four sampks were cer
ple of the BrdUMP-labeled DNA that had been200 alkali denatured but not sheared showed that
most of the label was added to preexistingchains, but ca. 20% banded with the densityof DNA fully substituted with BrdUMP (Fig.
100 4B). Only the latter fraction represents strandsfully synthesized in vitro.Combined with the information from alka-
line sucrose gradients (Fig. 3), these resultsindicate that, after the initial appearance of
iooo the product in very short pieces (ca. 4S), furthergrowth probably occurs primarily by condensa-tion ofthe initial pieces with preexisting chainsof as yet undefined length. The alternative
500 possibility for the apparent growth, elongationE by nucleotide addition, cannot be firmly ex-Q cluded at present, but seems less likely for the0 large proportion of the product that consists2 predominantly of preexisting material (Fig. 4).
2 That there is chain growth and no evidence ofX. breakdown in prelabeled DNA (Fig. 3) makes
i it unlikely that repair synthesis could account1000 for the association of product with preexisting
DNA.A similar analysis of the denatured product
of in vitro synthesis with the temperature-sensitive DNA replication mutants Ts cdc8 p0
2000 at the permissive temperature gave, princi-pally, results similar to Wt p°, with most of thelabel shifted only partially from the positionof normal density DNA and, at most, one-third
1000 completely (or almost) free from preexistingDNA (Fig. 4C and D).Comparison of the density distribution of
DNA from the mutant density-labeled at thepermissive and restrictive temperatures showsa narrower density distribution and less den-sity shift for the restrictive than for the permis-
aiysis of sive conditions. A peak of fully dense DNA iscubation still observed, and the proportion of material¢.7 x 109 of intermediate density to fully dense materialBrdUTP is not altered significantly. The most likelydoal)cThe explanation for the smaller density shift is thatafter 32p_ it is simply a consequence of reduced in vitroas inter- synthesis of density-labeled DNA, most ofvas lysed which is attached to preexisting DNA ofnormalind then density. Some other explanations seem lessenol (A)and thenP). A por-he size ofgradientis). Tworried out?.3 x109pectively.re for 20keroplastfkali andItrifuged
for 48 h at 20°C (see Materials and Methods), A andB at 40,000 rpm, and C and D at 45,000 rpm. Thepositions in the gradients for fully substituted(BrdUMP), native (HH), and denatured (H) DNA,hybrid (HL), and nonsubstituted native (LL) anddenatured (L) DNA were determined from the pyc-nometrically measured densities ofgradient fractionsand the data for hybrid and fully substituted dena-tured lymphocyte DNA (human) (51), which has thesame guanine plus cytosine content as S. cerevisiae(1). Symbols: 0, 3H; , 32p
likely; e.g., inhibition of initiation would haveresulted in a relative decrease or absence of afully dense product (unattached to preexistingDNA), whereas a defect in joining 4S pieces topreexisting DNA of normal density would re-sult in a larger proportion of fully dense DNA.RNA associated with newly synthesized
DNA. To test whether DNA chains are initi-ated at the 3' end of RNA primers, synthesiswas carried out in vitro with different [a-3P]-dNTP's and analyzed for transfer of 32p to2'(3')-rNMP's after treatment with alkali (13,28, 50, 58). Since the in vitro system is defectivein joining long replication intermediates (6 to8S) to high-molecular-weight DNA, a relativelylong labeling period (15 min) was chosen toaccumulate these fragments because of thepossibility that they may retain RNA primersor fragments thereof. The results indicate thatincorporated 32p can be transferred from eachof the [a-32P]dNTP's to each of the 2'(3')-rNMP's (Table 2). There is a general preferencefor transfer to adenosine 5'-monophosphate(rAMP) and cytidine 5'-monophosphate (rOMP);however, the particularly high rate from[a-32P]dATP to rAMP may be partly the resultof other mechanisms not necessarily reflectingreplication (50). One would expect 32p to betransferred to only one 2'(3')-rNMP per DNAchain only if the latter were initiated in vitro.Considering that a maximum of about 20% ofthe DNA chains could have retained RNA-DNA primer junctions (Fig. 4B and C) andthe average chain length is several hundrednucleotides, the proportion of label transferredto ribonucleotides from dGTP and dCTP ismuch too high to be explained solely by primingnascent DNA.
TABLE 2. Transfer of incorporated label from[a-32PJdNTPs to 2'(3),_rNMP'sa
a Incubations were carried out separately for eachof the [a-32P]dNTPs with permeable spheroplasts ofWt po (see Materials and Methods), after whichnucleic acids were isolated, hydrolyzed in alkali,and analyzed for 2'(3')-rNMPs as described in Ma-terials and Methods. The data with dATP and dCTPwere derived from one experiment each, whereas,for dGTP and dTTP, they were averaged from twoindependent experiments-one with [a--"P]dNTPprepared in this laboratory and the other from acommercial source.
The product labeled with [a-32P]dTTP wasanalyzed for 32p transfer after fractionation byequilibrium centrifugation in density gradientsof KI (Fig. 5 and Table 3). Except for a some-what higher rate of transfer in the materialfrom the RNA region of the KI gradient, therate of transfer was in the expected range forchains of the size found (Fig. 3) and similar topreviously reported results in animal cells andviruses (16, 33, 50, 56).
In contrast, most of the material that trans-fers radioactivity from [a-32P]dGTP to 2'(3')-rNMP banded in the position of RNA ratherthan DNA (Fig. 5, Table 3). Analysis of thematerial from the RNA region of the KI gra-dient by velocity sedimentation in dimethylsulfoxide-sucrose gradients (53) yielded S val-ues in the range of ribosomal RNA (no figureshown). The small peak of material with den-sity greater than RNA was present to variabledegrees in different experiments, and labeltransfer occurred exclusively to 2'(3')-rGMPand/or 2'(3')-rUMP (Table 3); the material hasnot yet been further characterized. As in thecase of the [a-32P]dTTP experiment, the rate oftransfer in the DNA fraction from the KI gra-dient resulting from [a-32P]dGTP incorporationwas in the expected range (ca. 0.5%). In addi-tion, the proportions of the ribonucleotides inthe RNA-DNA junctions labeled with [a-32P]dGTP showed a great disparity among thefractions of different density in contrast to thesimilar proportions after labeling with [a-32P]dTTP (Fig. 5, Table 3).Comparison with results in vivo by using a
mutant capable of taking up NMPs. Themutant tup p0 incorporates labeled dTMP spe-cifically and effectively into nuclear DNA whenthe nucleotide is present in a sufficiently highconcentration (0.5 mM) and when the metabolicpathway from rUMP to dTMP is blocked byaminopterin and sulfanilamide. Cells pulse-la-beled with [a-32P]dTMP showed slow initialkinetics of incorporation, probably due to adelay in equilibration of the nucleotide poolwith [a-32P]dTMP (Fig. 6). With a 30-s pulse, alarge part of the radioactivity was in fragmentsthat sedimented at about 2 to 7S, with a maxi-mum at 4S, and additional material up to 15Sand on the cushion of concentrated sucrose atthe bottom of the gradient (Fig. 7A). With in-creasing pulse times, the amounts of bothspecies of DNA increased, but label in high-molecular-weight DNA increased faster than inthe small pieces (Fig. 7A and C). This is con-sistent with the role of the fragments as pre-cursors of the high-molecular-weight DNA.Thus far it has not been possible to carry out asatisfactory pulse-chase experiment to confirm
FIG. 5. Transfer of incorporated label from [cva2P]dNTP`s to 2'(3')-rNMPs: fractionation of the nucleicacids in KI density gradients. The procedure (see Materials and Methods) employed spheroplasts preparedfrom Ts cdc8-p°-cells and in vitro incorporation of [C-32P]dTTP (A) and [a_32P]dGTP (B). The permeablespheroplasts used for experiment B were prelabeled in vivo with [3H]uracil to provide markers for RNA andDNA in the gradient. The nucleic acids were heat denatured (100°C, 3 min) and centrifuged to equilibriumin KI density gradients (see Materials and Methods) (48,000 rpm for 30 h at 200C). Portions from thefractions were assayed for acid-insoluble 32P (0) (A and B), and, in addition, in B, total acid-insoluble 3H(+) (prelabeled DNA and RNA) as well as alkali-stable and acid-insoluble 3H (-) (prelabeled DNA).Further processing of fractions from this experiment is given in Table 3.
TABLE 3. Transfer of incorporated label from [a-32P]dNTP to 2'(3')-rNMPs in fractions of differentdensity'32p in fraction Distribution of 2P in ribonucleotides
a-"P-labeled Pooled frac- 32P incorpo- pool trans-substrate tions rated (%) ferred to ribo- rAMP rCMP rUMP +
a Pooled fractions from the KI gradients (Fig. 5) were hydrolyzed with alkali and analyzed for transferto 2'(3')-rNMP`s as described in Materials and Methods. The third column refers to the percentage of totalincorporated 32P represented by the counts in that fraction pool, whereas the fourth column givespercentage of label in that fraction pool that was transferred to rNMPs by alkali. The sum of radioactivityin the fractions for each experiment is less than 100% because some fractions were omitted. Repeats ofboth experiments with the in vitro system from Wt po cells gave similar results (not illustrated).
this, because of the lag in equilibration of plasts from petite strains of yeast lacking mi-nucleotide pools. tochondrial DNA has characteristics associated
DISCUSSION with vivo replication.DISCUSSION Ts mutants in DNA synthesis displayed a sim-
The DNA synthesis in permeable sphero- ilar defect in vitro; most of the product con-
FIG. 6. Kinetics of [32P]dTMP incorporation intoDNA in vivo. A suspension of tup po cells in YMM-SAT medium (3 x 109 cells per ml) at 25°C wasprepared as described in Materials and Methods.After addition of[32P]dTMP, 0.75-ml portions wereremoved at the times indicated, and radioactivity(alkali-stable, acid-insoluble) was measured on 75-.d portions of each.
sisted of strands at least several hundred nu-cleotides in length; and the product first ap-peared in short chains (ca. 4S) similar in sizeto the replication intermediates in vivo inyeast, as reported here, and in other eucaryotes(29, 37, 46, 51, 56), animal viruses (11, 14, 58),and some procaryotes (32, 49). With longerincubation times, the 4S pieces grew to a longerand more heterogeneous size class (6 to 8S),resembling in this respect the products of lim-ited elongation or joining observed in some invitro systems for DNA synthesis utilizingmammalian cell nuclei (51). Although spermi-dine, which was used to help stabilize thesystem, is an inhibitor of yeast DNA ligase(W. Oertel, unpublished data), preliminary re-sults indicate that the joining step is not re-stored by simply replacing spermidine withother membrane-stabilizing agents.Other features of the in vitro product suggest
repair and/or defects in the in vitro process ofreplication. Only a small portion of the product,if any, was integrated into high-molecular-weight DNA; this has also been observed incertain other in vitro systems derived fromboth eucaryotes and procaryotes (15, 40, 43, 51).A significant portion of the labeled product
with [a-32P]dGTP as precursor was found at-
J. BACTZRIOL.
tached to high-molecular-weight RNA and wasassociated with high rates of label transfer to2'(3')-rNMP's. This has not been observedheretofore in similar experiments on mamma-lian in vitro systems (50, 56), although highlabel transfer from [a-32P]rGTP has also beenobserved wlth Physarum polycephalum (56).The significance of this result is not known. Itdoes not seem likely to result from incorpora-tion of [a-32P]rGTP contaminating the dGTP,since the results were similar with severalsources of labeled nucleotide, including our
100
50
EC. 1000
LiiI-
2 5000
z-
2000
1000
10 20
FRACTION NUMBER30
FIG. 7. Alkaline velocity sedimentation analysisofDNA pulse-labeled in vivo. The remainder ofeachsample from the experiment described in Fig. 6 waslysed as described in Materials and Methods andanalyzed by centrifugation in an alkaline sucrosegradient (40,000 rpm for 12.5 h at 200C) with 3Hphage fd DNA as an internal marker. Direction ofsedimentation is from right to left. (A) 30-s pulse;(B) 3-min pulse; (C) 5-min pulse.
own purified preparation, and the incubationmixture contained high concentrations of unla-beled rGTP. In contrast, the results with [a-32P]dITP were consistent with a primer RNAcovalently attached to the nascent DNA chainsand suggest that at least some, and possiblyall, of the 4S pieces are initiated in vitro.However, supporting evidence is needed forthis interpretation, especially considering theresults with [a-32P]dGTP suggesting anotherorigin for RNA-DNA copolymers.Compared with the two in vitro DNA repli-
cation systems from yeast reported previ-ously (2, 27), the system described here hasthe advantage of rapid and gentle lysis ofthe cells after incorporation. The permeablespheroplast was able to synthesize both nuclearand mitochondrial DNA, in contrast to thesystem described by Banks (2) that synthesizesonly mitochondrial DNA. The system of Here-ford (personal communication) was not suffi-ciently characterized to identify the product.The in vitro rate of DNA synthesis in p+ cellswas comparable to, or higher than, that of thesystem from Hereford and Hartwell (27). Arecent report by Yee et al (62) describes thepermeabilization of yeast cells by treatmentwith Glusulase and a sulfhydryl reducingagent. In that system, rate and extent of syn-thesis are similar to the results reported here,and both nuclear and mitochondrial DNA aresynthesized, but the reported characterizationof that system does not go further.
If the sensitivity of the permeable sphero-plast system to DNase results from some degreeof permeability to proteins, it may be possibleto supplement the system with fractions fromcell extracts or purified enzymes (e.g., ligase)to restore the defective joining step or to com-plement preparations from the temperature-sensitive cells with wild-type proteins at therestrictive temperature. However, permeabil-ized E. coli, which has a similar DNase sensi-tivity (36), has not proved useful for experi-ments of that type.
ACKNOWLEDGMENTSWe gratefully acknowledge the kind assistance of Leland
Hartwell in providing the mutant strains and the helpfuldiscussions with Linne Hereford.
This work was supported by grant 430/402/802/3 fromthe North Atlantic Treaty Organization, training fellow-ship (W. O.) Oe66/1/2 from the Deutsche Forschungsgemein-schaft, Public Health Service research grant CA 11705 fromthe National Cancer Institute, and grant NP-102 from theAmerican Cancer Society.
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