Vol. 140, No. 2
JOURNAL OF BACTERIOLOGY, Nov. 1979, p. 333-341 0021-9193/79/11-0333/09$02.00/0
Deoxyribonucleic Acid Synthesis in Saccharomyces cerevisiae Cells Permeabilized with Ether WOLFGANG OERTELt AND MEHRAN GOULIAN* Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093
Received for publication 3 August 1979
Cells of Saccharomyces cerevisiae permeabilized by treatment with ether take up and incorporate exogenous deoxynucleoside triphosphate into deoxyribonucleic acid (DNA). With p+ strains, more than 95% of the product was mitochondrial DNA (mtDNA). This report characterizes ether-permeabilized yeast cells and describes studies on the mechanism of mtDNA synthesis with this system. The initial rate of in vitro mtDNA synthesis with one strain (X2180-lBp+) was close to the rate of mtDNA replication in vivo. The extent of synthesis after 45 min was sufficient for the duplication of about 25% of the total mtDNA in the cells. The incorporated radioactivity resulting from in vitro DNA synthesis appeared in fragments that were an average of 30% mitochondrial genome size. Densitylabeling experiments showed that continuous strands of at least 7 kilobases after denaturation, and up to 25 kilobase pairs before denaturation, were synthesized by this system. Pulse-chase experiments demonstrated that a large proportion of DNA product after short labeling times appeared in 0.25-kilobase fragments (after denaturation), which served as precursors of high-molecular-weight DNA. It is not yet clear whether the short pieces participate in a mechanism of discontinuous replication similar to that of bacterial and animal cell chromosomal DNA or whether they are related to the rapidly turning over, short initiation sequence of animal cell mtDNA. In p0 strains, which lack mtDNA, the initial rate of nuclear DNA synthesis in vitro was 1 to 2% of the average in vivo rate. With temperaturesensitive DNA replication mutants (cdc8), the synthesis of nuclear DNA was temperature sensitive in vitro as well, and in vitro DNA synthesis was blocked in an initiation mutant (cdc7) that was shifted to the restrictive temperature before the ether treatment. In vivo studies of DNA replication in yeast are limited by the general unsuitability of thymidine as a precursor (15) and, in dTMP uptake or auxotrophic mutants (10, 37), by the very slow equilibration of large nucleotide pools (19, 24). This problem is avoided with in vitro systems by using deoxynucleoside triphosphate (dNTP) substrates for DNA synthesis. Systems in which only one of the three kinds of DNA in yeast cells (nuclear DNA [nDNA], mitochondrial DNA [mtDNA], and 2-pum DNA [o-DNA]) is synthesized would be most useful. Several in vitro systems from Saccharomyces cerevisiae have been described (1, 20, 25, 39, 41, 42) in which mtDNA or nDNA, or both, are synthesized. Experiments using isolated mitochondria as an in vitro system to investigate mDNA replication (23, 39, 42) have been suct Present address: Universitiit Wurzburg, Institut fur Genetik und Mikrobiologie, Lehrstuhl fiir Mikrobiologie, D-8700 Wurzburg, West Germany.
cessful only very recently (23). To study details of chromosomal DNA replication, we have recently developed a permeable cell system from yeast spheroplasts which, with some modification, may also be used to study mtDNA synthesis (24, 25). In this report we describe another perneable cell system from yeast, in which mtDNA is replicated with particularly high efficiency and which has significant advantages over the permeable spheroplast system. With suitable strains this system may also be used to study nDNA synthesis. MATERIALS AND METHODS Cells. S. cerevisiae A364p+ and its temperaturesensitive derivatives 198 and 13052 [cdc8(Ts)], 4008 [cdc7(Ts)], and 146-2-3 [cdc2l(Ts) thymidylate synthetase-] were obtained from L. Hartwell (16, 17, 18);
strain X2180-lBp+ (8) was provided by L. Hereford; JL125-1OC (a p+ ura) was from G. Michaelis; and Y379-5D (a p+ Cyhr o-DNA-) was from C. P. Hollenberg. From these p+ strains, A364Apo-3, 198p°-4,
333
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OERTEL AND GOULIAN
13052p°-14, 4008p°-9, and X2180-lBp°-2 lacking mtDNA were derived by treatment with ethidium bromide (14, 24). A364Ap--2 is a spontaneous petite mutant containing mtDNA. JL125-lOCp--4 (Eryr) was prepared from a spontaneous erythromycin-resistant derivative of the p+ strain by treatment with ethidium bromide and selection for the cytoplasmic Eryr marker
(32).
Nucleic acids. 32P-labeled nDNA and mtDNA (0.1 to 1 Ci/mmol of P) were prepared from strain A364Ap+ (24). 3H- and 3P-labeled bacteriophage fd DNA was prepared by the procedure of Schaller (31). Preparation of the 3P-labeled restriction fragments R1-HpaIIC (252 base pairs) and Rl-HpaH-D (200 base pairs), used as size markers, was as described previously (26). Preparation of ether cells. Cells were grown with moderate aeration in 500 ml of YMM medium (24) at 300C (23°C for temperature-sensitive mutants) to a density of 3 x 107 to 5 x 107/ml. If required, the DNA in the cells was prelabeled by replacement ofunlabeled uracil in the medium with [3H]uracil (0.01 mM; 0.5 Ci/ mmol) (24). The cells were collected by centrifugation, suspended in 50 ml of buffer A [50 mM Tris-hydrochloride (pH 7.5), 100 1nM KCI, 10 mM MgCl2, 1 mM dithioerythritol, 1 mM ethylene glycol-bis(46-aminoethyl ether)-N,NY-tetraacetic acid); 1 M D-(+)-sorbitol] at 00C, and shaken vigorously, in a funnel, with 50 ml of freshly prepared water-saturated diethyl ether (000) for 30 a. The phases were allowed to separate for about 30 s; the aqueous phase was then transferred to centrifuge tubes and underlayered with about 0.25 volume of fresh (ether-free and, therefore, denser) buffer A. The tubes were centrifuged immediately for 2 min at 12,000 x g. After immediate removal of the supernatant and suspension in 50 ml of fresh buffer A (00C), centrifugation was repeated in the same way and the pellet was finally suspended in 10 ml of buffer A. Portions (1 ml each) of this suspension were quickfrozen at -70°C and kept at this temperature until use. No difference was detected between ether-permeabilized cells (hereafter called "ether cells") that were used immediately after preparation and those used after 3 months of storage at -70°C. In vitro incororation of nucleotides into DNA. A freshly prepared or freshly thawed suspension of ether cells from the stock was adjusted to a density of 1 x 109 to 3 x 10" cells per ml by dilution with buffer A. A 0.9-volume of this suspension was brought to the desired incubation temperature (250C unless stated otherwise), and the reaction was started by the addition of 0.1 volume of a mixture (xlO concentrated) containing: 1 mM each dATP, dCTP, dGTP, UTP, GTP, and CTP; 20 mM ATP; 0.1 mM [3H]dTTP (4 Ci/mmol or 47 Ci/mmol); and 100 mM phosphoenolpyruvate (standard conditions). For density-labeling experiments, dTTP in the concentrated nucleotide mixture was replaced by 5-bromodeoxyuridine 5'-triphosphate at 1 mM and [3H]dCTP (0.1 mM; 16 Ci/ mmol). The reaction in the whole sample or a portion was stopped by dilution with an at least 10-fold volume of 50 mM Tris-hydrochloride (pH 8.2)-50 mM EDTA50 mM KXP207-1 M D-(+)-sorbitol and centrifuged at 3,500 x g for 5 min at 0°C. The supernatant was discarded. Sample processing. Incorporation of radioactive
J. BACTERIOL. label into DNA was assessed by determination of alkali-stable, acid-insoluble radioactivity in the cell pellets (24). When native DNA was to be analyzed by ultracentrifugation, the ether cells were suspended and briefly incubated with Arthrobacter luteus enzyme before further treatment with Sarkosyl, pronase, and phenol, as described previously (24). To analyze denatured DNA, the cell pellet after treatment with A. luteus enzyme was lysed by suspension in 0.5 ml of 0.3 M KOH and left for 15 h at 230C. Insoluble material was removed by centrifugation (15,000 x g, 00C; 15 min). The supernatant containing all acid-insoluble radioactivity either was used directly for centrifugation in alkaline sucrose gradients or was neutralized before analysis in CsCl gradients. Ultracentrifugation. Sucrose gradients, prepared as described previously (24), were centrifuged for 3.5 h at 40,000 rpm and 200C (neutral) or for 14.5 h at 32,000 rpm and 150C (alkaline), in a Beckman SW40 rotor, with appropriate internal or external size markers for evaluation of molecular weights (33). Isopycnic centrifugations were carried out in a Beckman 65 angle rotor, using CsCl solutions of density 1.68 g/ ml (32,000 rpm, 60 h, 2000) for separation of nDNA and mtDNA and 1.75 g/ml (40,000 rpm, 48 h, 2500) for bromodeoxyuridine-containing DNA (24). Other procedures. Other procedures, sources, and reagents have been described previously (24, 25).
RESULTS General properties of the permeable cell system. Brief treatment with diethyl ether at low temperature, similar to the procedure of Vosberg and Hoffman-Berling (35) for Escherichia coli, rendered cells of S. cerevisiae permeable to nucleotides. Ether-treated yeast cells are nonviable (7) but retained their normal shape and incorporated externally added radioactive dNTP's into DNA. DNA synthesis in ether cells prepared from wildtype strains containing normal mtDNA (p+ strains) proceeded for at least 50 min with a slowly decreasing rate (Fig. 1). For optimal results, the system was prepared from the middle logarithmic growth phase, with glucose as the carbon source, under moderate aeration. When cells were used that had been grown under vigorous aeration with ethanol as the carbon source, conditions under which the mitochondria are optimally active metabolically, the initial fast in vitro DNA synthesis was soon followed by degradation of the products, especially when the incubation was carried out at elevated temperatures (>250C) (not illustrated). Optimal DNA synthesis in ether celLs required the presence of all four dNTP's, Mg2+, an SH reagent and, in the absence of phosphoenolpyruvate, a high concentration of ATP (Table 1). ATP could be replaced only partially by another NTP, e.g., UTP. The presence of residual nu-
VOL. 140, 1979
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DNA SYNTHESIS IN S. CEREVISIAE
30
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0
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.
10 n
20
30
~~~TlME,
40
50
MIN
FIG. 1. Kinetics of DNA synthesis in ether cells. Ether cells were prepared from strain X2180-lBp+ and incubated in a 1-ml reaction volume (10O cells per ml) (see text). After 3 min ofprewarming at 25°C, the reaction was started by the addition of a concentrated nucleotide mixture containing the 3H label in dTTP (see text). After incubation at the same temperature at the times indicated, 50-,il portions were removed and analyzed for alkali-stable, acid-insoluble radioactivity.
cleotide pools was suggested by a low level of DNA synthesis after omission of three dNTP's. Analysis of the incubation mixture after various reaction times by thin-layer chromatography revealed that in the absence, but not in the presence, of phosphoenolpyruvate all NTPs and dNTP's were dephosphorylated rapidly. UTP, GTP, and CTP were not necessary for optimal synthesis, but residual pools may have been sufficient to satisfy a possible ribonucleotide requirement. Label from radioactive ribonucleoside triphosphates was incorporated into RNA, but this has not yet been studied in detail. Sorbitol stabilized the system, perhaps by preventing inactivation of sensitive proteins or acting as osmotic support. Inhibition (70%) was observed with arabinosyl-CTP, at a concentration 7.5-fold that of dCTP during the incubation. mtDNA synthesis was highly sensitive to dideoxy-TTP (Table 1), whereas nDNA synthesis was unaffected even at much higher levels (20 ,uM) (not shown), paralleling the sensitivity of the mammalian mitochondrial (y) DNA polymerase and insensitivity of the a polymerase (9, 36). At pH 6.5 the initial rate was lower than that at pH 7.5, but the synthesis proceeded for a longer time, finally reaching the same level. At
335
pH 8.5, the initial rate was also lower than that at 7.5 but, in addition, the incorporation ceased prematurely and was followed by considerable degradation (not illustrated). A high concentration of externally added DNase did not affect DNA synthesis in the ether cells, indicating that the DNA was not accessible to externally added proteins. Properties of ether cells prepared from different yeast strains. Modifications of the ether treatment, e.g., moderate changes in the composition of the buffer or time of exposure to the solvent, had little influence on the activity. However, it was observed that the rate and extent of in vitro synthesis of nDNA as well as of mtDNA varied considerably depending on the strain used. With strains containing normal mtDNA (p+ strains) the product was mainly mtDNA. This was confirmed by density gradient centrifugation analysis of the in vitro product from p+ and p0 strains (Fig. 2A and B). In A364Ap+ ether cells (and also all other p+ strains tested), label was primarily or exclusively in material with the same density as that of native mtDNA (1.683 g/ml [18]). As expected, most of the product synthesized with a p0 strain (lacking mtDNA) had the same density as that of nDNA TABLE 1. Requirements and inhibitors of the in vitro system Condition(s)
Incorporation relative to standard conditions (%)
Complete (standard conditions) . ....... 100 -dCTP ................... 27 -dCTP, -dGTP, -dATP ........... 14 -ATP ............................ 102 -ATP, -phosphoenolpyruvate ...... 27
-Phosphoenolpyruvate ............. -Phosphoenolpyruvate, +10 mM ATP
64 94
-Phosphoenolpyruvate, -ATP, +10 mM UTP ........................ 45 +0.75 ,uM araCTP .......... ........ 30 +0.05 ,uM dideoxy-TTP ....... ...... 59 22 +0.5 ,iM dideoxy-TTP ........ ...... -Dithioerythritol, +5 mM N-ethylmal4.5 eimide ............ +500,ug of pancreatic DNase per ml 93 a The complete standard reaction mixtures (200 pi) were prepared from ether cells (strain A364Ap+) as described in the text. The final concentrations were: 50 mM Tris-hydrochloride (pH 7.5); 10 mM MgC92; 100 mM KCI; 1 M D-(+)-sorbitol; 1 mM EDTA; 1 mM dithioerythritol; 10 mM phosphoenolpyruvate; 2 mM ATP; 0.1 mM each dATP, dGTP, dCTP, GTP, CTP, UTP; 0.01 mM [3H]dTTP (4 mCi/mmol); and ether cells, 1.5 x 109/ml. Incubations were carried out in duplicate for 10 min at 25°C, followed by analysis for alkali-stable, acid-insoluble radioactivity.
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proportion depended on the strain used. The initial rate of mtDNA replication in X2180-lBp+ ether cells was close to the average in vivo rate calculated on the basis of a generation time of 150 min and an mtDNA content of 10% in the cell. In contrast, with A364A the rate in vitro was only about 10% of the initial 500 0 calculated average in vivo rate. With most ~~~~~~~~~~~E strains, the average initial rate of nDNA syntheoco 1000. ,, ~~~~~~~~~~~u sis was approximately 0.5 to 2% of the calculated rate of nDNA replication in vivo (based on a generation time of 210 min in the p0 strains). In IA364A (p+ and po) after permeabilization, syn> : - 2000 thesis of nDNA could barely be measured. DNA synthesis in ether cells prepared x 500 0 1500 from temperature-sensitive DNA replica500In temperature-sensitive mumutants. tion 1000 tants, defective in the cell division cycle gene 8 (cdc8) (16, 17), which participates in the propa500 gation of DNA replication, the synthesis of 100 O nDNA in vivo stopped very rapidly and almost completely after a shift to the restrictive tem30 20 10 perature. The kinetics of nucleotide incorporaFRACTION NUMBER tion into DNA of a mutant and a wild-type FIG. 2. Analysis of DNA synthesized in vitro by strain were compared in vitro at the permissive isopycnic centrifugation. Reaction mixtures (0.5 ml) and restrictive temperatures. In ether cells pre(3 x 10' cells per ml) were prepared with ether cells pared from the p0 derivative of the mutant, the as described in the text, except that the [3H]dTTP specific activity was 18 Ci/mmol. After incubation at synthesis of nDNA at the restrictive tempera25°C for 30 min, native DNA was extracted from the ture stopped completely after a short initial cells, mixed with 32P-labeled A364A nDNA and burst of synthesis (Fig. 3B), which could not be mtDNA markers, and analyzed by CsCl density gra- prevented by preincubation without NTPs for 5 dient centrifugation (see text). (The 32p marker on the to 15 min. This was found only for the two alleles right is mtDNA; that on the left is nDNA) Symbols: (198 and 13052) (Table 2) of the mutant and 32p. (A) Strain A364Ap+; (B) strain 198p°-4. confirms earlier results with another in vitro 0, 3H; system from yeast (24, 25). In contrast to this, in (1.699 g/ml [18]) (Table 2). In addition, a portion ether cells of wild-type p0 strains of a temperaof this DNA had a density higher than that of ture-sensitive mutant in the thymidylate synthenormal nDNA (Fig. 2B), which may represent tase gene (cdc2l) (13), nDNA synthesis propreferentially synthesized so-called "y" DNA, a ceeded at the high temperature at the same or nuclear satellite DNA containing the ribosomal a higher level than at the low temperature (Tagenes (6), and may be further accentuated by ble 2, Fig. 3A). association with RNA. In p+ ether cells derived from DNA replication Results with a series of strains are summarzed mutants 198p+ [cdc8(Ts)] (not illustrated) and in Table 2. Generally, the initial rate of DNA 13052p+ [cdc8(Ts)] (25), mtDNA synthesis was synthesis with a p+ strain was at least six to eight not temperature sensitive. However, the delayed times as high as with its derivatives lacking and incomplete effect of this mutation on mtDNA. A maximum of 5% of the product after mtDNA observed in vivo (3, 40) may have been a 40-min incubation had the same density as due to a more indirect effect of mutation in the that of nDNA (1.699 g/ml), and the rest had cdc8 gene on mtDNA synthesis. nDNA synthethat of mtDNA (1.683 g/ml). In vitro-labeled sis was blocked in ether cells prepared from a DNA with the density 1.699 g/ml (5% of the p0 derivative of the DNA initiation mutant cdc7 total) was also obtained with strain Y379-5Dp+ (16, 17) when the cells were shifted to the nonlacking o-DNA (C. P. Hollenberg, personal com- permissive temperature for a period equivalent munication), supporting the chromosomal origin to at least one generation time before harvest of the product with this density, at least for this (Fig. 30), indicating that in vitro DNA synthesis strain. In the in vitro system from petite mutants is closely related to the in vivo process. The lack containing an altered mtDNA (p-), both nDNA of effect of temperature in vitro on a preparation and mtDNA were made at a low rate, and their of the same mutant grown at the permissive -j
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VOL. 140, 1979
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TABLE 2. Properties of ether cells prepared from different yeast strainsa Ether cells
Incubation (0nc temp
Initial rate [(pmol of
dTMP//l0
cells)/ mi]
Etn (mo (nuo Etn of dTMP/108 cells)/40 min]
nDNA(%)
mtDNA(%)
25 0.28 3.50 97 37 0.44 4.88 25 0.75 6.25 5 95 37 1.31 5.35b 25 0.79 7.50 5 95 37 1.79 8.25b 25 0.80 5 3.80 95 37 1.80 5.38 25 2.75 25.5 -3 97 25 0.63 8.00 5 95 Y379-5Dp+ (o-DNA-) 25 0.04 0.38 70 30 A364Ap--2 ................ 25 0.04 1.25 30 70 JL125p--5 (Eryr) .......... 0.01 25 0.08 >97 A364Apo-3 ................ 25 0.13 0.94 >97 X2180-lBp0-2 ............. 37 0.13 1.00 X2180-lBpo-2 ............. 25 0.06 0.83 >97 198p°-4 (cdc8) ............. 37 0.05 0.44 198p°-4 (cdc8) ............. 25 0.14 >97 13052p0-14 (cdc8) .......... 1.09 37 0.10 0.68 13052p°-14 (cdc8) ........... aThe initial rate and the extent of the DNA synthesis in vitro after 40 min were derived from kinetic data as described in the legend to Fig. 3. The proportions of nDNA and mtDNA were determined by isopycnic in CsCl gradients (see text) (cf. Fig. 2). centrifugation b With these strains, at 370C by 40 min some of the initially synthesized DNA was already degraded.
A364Ap ................. A364Ap+ .................. 198p+ (cdc8) .............. 198p+ (cdc8) .............. 13052p+ (cdc8) ............ 13052p+ (cdc8) ............ 146-2-3p+ (cdc2l) .......... 146-2-3p+ (cdc2l) .......... X2180-lBp ..............
temperature (Fig. 3C) is consistent with there being few or no new initiations in nDNA synthesis in the ether-treated cells. In vitro synthesis of mtDNA in the corresponding cdc7 (4008) p+ strain was not influenced by in vivo exposure to restrictive temperature, and control experiments with p0 and p+ nonmutant strains (X2180-1B) showed that in vitro synthesis of nDNA was not affected by the shift to higher temperature during growth of the cells. Analysis of mtDNA pulse-labeled in vitro. mtDNA, pulse-labeled in vitro for 10 min with [3H]dTTP in X2180-lBp+ ether cells, sedimented in neutral sucrose gradients in a broad peak with the maximum at about 25S (30% mitochondrial genome size), with a significant portion up to 39S (full genome size) (Fig. 4A). With longer incubation times (up to 45 min), no significant change in this size distribution was noticed. In vivo prelabeled DNA sedimented faster than the in vitro products and consisted mainly of sheared chromosomal DNA (458) (data not shown; 25). After X2180-lBp+ ether cells were pulse-labeled with [3H]dTTP for 15 s at 180C followed by a chase with unlabeled dTTP of up to 5 min, the main product observed in alkaline sucrose gradients consisted of short pieces, initially 0.1 to 0.15 kilobase (kb) and growing to about 0.25 kb (Fig. 4B). When this experiment was per-
formed at 25°C (with 5-bromodeoxyuridirie 5'triphosphate in place of dTTP to allow subsequent density analysis), a portion of the newly synthesized DNA achieved the size of 0.25 kb within 20 s while, in addition, a second broad peak of label sediimented principally at 13S (Fig. 40). After a chase with unlabeled dNTP for 15 min, about 40% of the short pieces had joined to the large material, which had grown to about 14S. With labeling times longer than 20 s (without chase), a fraction of the 0.25-kb pieces accumulated (Fig. 4D). Analysis of density-labeled mtDNA. Density-labeled 0.25-kb pieces recovered from the alkaline sucrose gradients (Fig. 4C and D, pool I) consisted primarily or completely of bromodeoxyuridine-labeled, and thus completely in vitro synthesized, mtDNA, independent of the pulse length (Fig. 5A) (the density distribution was broad because of the small size of the material). In contrast, approximately 50% of the DNA pieces of the 12 to 18S size class (3 to 6 kb) (Fig. 4C and D, pool II) grew to the whole fragment length within 5 min (Fig. 5B). After 15 min, more than 75% of the 12 to 18S product and 50% of the longest fragments (Fig. 4D, fractions 9 through 12) (ca. 7 kb) were fully substituted with bromodeoxyuridine (not illustrated). These experiments show that continuous stretches of greater than 10% mitochondrial genome length
338 Xn -i tLJ _ O
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OERTEL AND GOULIAN
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(72 kb) can be synthesized in ether cells of yeast. To demonstrate that the synthesis of mtDNA in vitro proceeded by a semiconservative mechanism, native X2180-lBp+ DNA, density-labeled with 5-bromodeoxyuridine 5'-triphosphate for 45 nmi, was analyzed directly by isopycnic centrifugation in CsCl gradients. About 25% of this material banded with "hybrid" density,
A
most of
sthe remainder containing additional proportions of preexisting (unsubstituted) mtDNA and a ci r W small fraction with a density higher than that of hybrid mtDNA (Fig. 50). The hybrid-density B 4 reisolated from CsCl gradients had a broad DNA .0 O size distribution as measured by velocity sedimentation in neutral sucrose gradients with the O majority between I and 6 kilobase pairs, and a 0.5 z _ significant portion up to 30% genome size (27S, 25 kilobase pairs). After sonication (average chain H , . ...length, 0.9 kb), at least 75% of the product 40 10 20 30 banded in CsCl gradients at hybrid density and, in addition, approximately 15% banded as fully bromodeoxyuridine-substituted mtDNA (Fig. have arisen dense material may The fully before 5D). id completion of a full round by reinitiation o of replication, but other possibilities have not 0 0.8 c been excluded, e.g., recombination (38) or deo tachment of short, newly synthesized DNA by branch migration (27) and renaturation, both / 0 perhapsfavored by the highpolydeoxyadenylate a o polydeoxythymidylate content (2). Further E studies will be required to determine its origin. ,X,-' cr uJ 0.4 / DISCUSSION ~ 4 / ,,0 observed in vitro mtDNA the -, In favor of DNA synthesis being replicative synthesis ir 01/ O rather than some form of repair are several ,/, observations that indicate important similarities z t.' to the in vivo process. The continuous length of A ICA product strands, 7 kb after denaturation and up 40 to 25 kilobase pairs before denaturation, is 30 20 X0 10 strongly against a repair mechanism. The initial TIME, MIN rate of synthesis was on the order of the in vivo rate of mtDNA synthesis, and the extent of FIG. 3. KneticsofchromosomalpandmstDNA s ynA thesis in etheraces from temperature-sensite DNA synthesis after 45 min was sufficient for the replication mutants. Thereactnmixtures(0.5ml) duplication of 25% of the mtDNA in the cell. were prepared with ether cells (2 x 109/ml) as demtD by the syn scribed in the text, except that the samples were The synthesis could be inhibited by araCTP, an not repair, DNA but replicative, of inhibitor temperature preincubated for 5min at the incubation (230C in vitro or 37°C in vitro) before the reaction synthesis in other eucaryotic and procaryotic was started by addition of the concentrated nucleo- systems (5, 22). By density-labeling experiments it was shown tide mixture with [3HJdTTP. The cells were grown at 23°C (230C in vivo), but for (C) a separate portion that the native DNA product with "hybrid" of cells was shifted to 37°C for the 3 h just before density had a size between 5 and 30% that of the a.
------ 0
tPe
harvest for ether treatment (37°C in vivo). Samples (50 ul each) were removed after incubation for the times indicated and analyzed for incorporation of radioactivity into DNA. (A) Strain X2180-lBpo-2; (B) strain 198p°-4; (C) strain 4008p°-9. Symbols: 0, m v 23°C; in vitro, 23°C; 0, in vivo, 23°C; in vitro, 37°C; A, in vivo, 37°C; in vitro, 23°C; A, in vivo, 37°C; in vitro, 37°C.
an
mitochondrial genome. The majority of the ma-
terial synthesized in vitro (15-min incubation)
consisted of DNA chains between
3 and 7 kb to 5 to 10% of mtDNA size. The chain elongation rate corresponded to at least 0.4 kb/min, a rate sufficient for the replication of the mitochondrial genome
b eequivalent 3a7k etw aftedeNAturain, after denaturation, en
DNA SYNTHESIS IN S. CEREVISIAE
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within one generation time (150 min, 25°C). Label appeared early in 0.25-kb pieces which consisted of DNA completely synthesized in vitro and was observed growing to this size. Pulsechase experiments showed that the 0.25-kb fragments served as precursors of high-molecularweight DNA, but, in addition, with labeling for longer periods a portion of the short pieces accumulated. The significance of the 0.25-kb precursor is not yet clear. One possibility is that it reflects a mechanism of discontinuous synthesis, with multiple tandem initiations on one or both 400 arms of the replication fork, as in bacterial and animal cell chromosomal replication (11, 21, 29). 300 Incomplete maturation of short nascent pieces of DNA has been commonly observed in eucar200 yotic in vitro systems for DNA synthesis (12, 28, E 34), and a similar presumed defect in joining 100 f could account for accumulation of the fragments in the yeast in vitro system. A second possibility w is that the 0.25-kb pieces observed here are X 600 . related to the 7S initiation sequence of animal 500 xa. cell mtDNA, which is made in excess over the rest of the mtDNA (4). The accumulation could 1400 ^ reflect this turnover process, possibly enhanced 300 by nicks in the mtDNA molecule, which have been shown to result in separation of the 7S 200 segment from the rest of the molecule by branch migration (30). 100 The rate and extent of in vitro nDNA synthe° sis in ether cells were low compared with those of mitochondrial (p+) DNA, but with most strains it is still in the same order of magnitude as with other permeable systems from yeast (20, 24, 41). The in vivo temperature sensitivity of nDNA synthesis in cdc8 mutants was expressed in the ether cell system, similar to results with the permeable spheroplast system (24). Further support for the replicative nature of nDNA syn-
E
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3H, 15-s pulse, 5-min chase; 32p size marker. (C, D) The incubation was performed at 25°C in a 2.0-ml volume with the 3H label in dCTP (16 Ci/mmol) and with 5-bromodeoxyuridine 5'-triphosphate instead of dTTP. After 20 s, two 400-tl portions were removed: in one the reaction was terminated immediately; to the other a 200-fold excess of unlabeled dCTP (2 mM) was added, and incubation continued for an additional 15 min. In additional portions (400 sl), the reactions were terminated after 60 s, 5 min, and 15 min. Further sample processing and centrifugation were as for part (B), except that the 32P-labeled size marker (R1-HpaII-C) was 252 base pairs long. Radioactivity in DNA was determined in 50-il portions of the fractions, and the remainder was pooled as indicated by "I" and "II." (C) 0, 3H, 20-s pulse; +, 3H, 20-s pulse, 15-min chase; 32p size marker. (D) x, 3H, 1-min pulse; *, 3H, 5-min pulse; A, 3H, 15-min pulse. (M, 32p 252-base pair size marker). 32P-labeled fd DNA was centrifuged in a duplicate tube as an external 30S (neutral) or 20S (alkaline) size marker. ,
30 20 10 FRACTION NUMBER
FIG. 4. Size determination of in vitro pulse-labeled native and denatured mtDNA by sucrose gradient centrifugation. The reaction mixtures with ether cells (X2180-lBp+, 3 x 109/ml) (see text) were incubated as follows. (A) Reaction mixture of 400 ,d; 3H label in dTTP (46 Ci/mmol); 25°C. After 10 min, DNA was isolated and analyzed in neutral sucrose gradients (see text). (B) Reaction mixture of 1.5 ml; 3H label in dTTP (46 Ci/mmol); 18°C. After 15 s, the reaction in a 400-,ul portion was terminated. Incubation of the remainder was continued after the addition of a 200fold excess of unlabeled dTTP (2 mM), with removal of additional portions (400 IlI) at 60 s (not illustrated) and 5 min (pulse-chase). DNA was analyzed in alkaline sucrose gradients (see text) after addition of 32P-labeled R1-HpaII-D DNA (200 base pairs) as the internal size marker. Symbols: 0, 3H, 15-s pulse; +,
,
340
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OERTEL AND GOULIAN
thesis in ether cells was provided by the elimination of nDNA synthesis (in vitro) in an initiation mutant (cdc7) by exposure to the restrictive temperature in vivo. An advantage of the ether cell system in comparison to the permeable spheroplast system developed recently in our laboratory for investigation of nDNA synthesis (24) is that it can be prepared from cells in their normal physiological state and any growth cycle phase. In addition, the joining of replication intermediates seems to be less impaired in ether cells than in permeable spheroplasts (25). By selection of the proper yeast strain for preparation of ether cells, it is possible to study the replication in vitro of normal mitochondrial, as well as chromosomal, DNA separately from the synthesis of other species of DNA normally present. Particularly for study of early nascent intermediates in DNA replication, the system allows experiments difficult or impossible with intact cells because of their precursor pools (19, 24). The possibility that mitochondrial reinitiation may be occurring in ether cells is particularly provocative, since this has been an almost uniform deficiency of in vitro systems for DNA synthesis, both from procaryote and eucaryote sources. Resolution of this, as well as of other questions on the mechanism of DNA synthesis, awaits results of studies now in progress.
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JOURNAL OF BACTERIOLOGY, Nov. 1979, p. 333-341 0021-9193/79/11-0333/09$02.00/0
Deoxyribonucleic Acid Synthesis in Saccharomyces cerevisiae Cells Permeabilized with Ether WOLFGANG OERTELt AND MEHRAN GOULIAN* Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093
Received for publication 3 August 1979
Cells of Saccharomyces cerevisiae permeabilized by treatment with ether take up and incorporate exogenous deoxynucleoside triphosphate into deoxyribonucleic acid (DNA). With p+ strains, more than 95% of the product was mitochondrial DNA (mtDNA). This report characterizes ether-permeabilized yeast cells and describes studies on the mechanism of mtDNA synthesis with this system. The initial rate of in vitro mtDNA synthesis with one strain (X2180-lBp+) was close to the rate of mtDNA replication in vivo. The extent of synthesis after 45 min was sufficient for the duplication of about 25% of the total mtDNA in the cells. The incorporated radioactivity resulting from in vitro DNA synthesis appeared in fragments that were an average of 30% mitochondrial genome size. Densitylabeling experiments showed that continuous strands of at least 7 kilobases after denaturation, and up to 25 kilobase pairs before denaturation, were synthesized by this system. Pulse-chase experiments demonstrated that a large proportion of DNA product after short labeling times appeared in 0.25-kilobase fragments (after denaturation), which served as precursors of high-molecular-weight DNA. It is not yet clear whether the short pieces participate in a mechanism of discontinuous replication similar to that of bacterial and animal cell chromosomal DNA or whether they are related to the rapidly turning over, short initiation sequence of animal cell mtDNA. In p0 strains, which lack mtDNA, the initial rate of nuclear DNA synthesis in vitro was 1 to 2% of the average in vivo rate. With temperaturesensitive DNA replication mutants (cdc8), the synthesis of nuclear DNA was temperature sensitive in vitro as well, and in vitro DNA synthesis was blocked in an initiation mutant (cdc7) that was shifted to the restrictive temperature before the ether treatment. In vivo studies of DNA replication in yeast are limited by the general unsuitability of thymidine as a precursor (15) and, in dTMP uptake or auxotrophic mutants (10, 37), by the very slow equilibration of large nucleotide pools (19, 24). This problem is avoided with in vitro systems by using deoxynucleoside triphosphate (dNTP) substrates for DNA synthesis. Systems in which only one of the three kinds of DNA in yeast cells (nuclear DNA [nDNA], mitochondrial DNA [mtDNA], and 2-pum DNA [o-DNA]) is synthesized would be most useful. Several in vitro systems from Saccharomyces cerevisiae have been described (1, 20, 25, 39, 41, 42) in which mtDNA or nDNA, or both, are synthesized. Experiments using isolated mitochondria as an in vitro system to investigate mDNA replication (23, 39, 42) have been suct Present address: Universitiit Wurzburg, Institut fur Genetik und Mikrobiologie, Lehrstuhl fiir Mikrobiologie, D-8700 Wurzburg, West Germany.
cessful only very recently (23). To study details of chromosomal DNA replication, we have recently developed a permeable cell system from yeast spheroplasts which, with some modification, may also be used to study mtDNA synthesis (24, 25). In this report we describe another perneable cell system from yeast, in which mtDNA is replicated with particularly high efficiency and which has significant advantages over the permeable spheroplast system. With suitable strains this system may also be used to study nDNA synthesis. MATERIALS AND METHODS Cells. S. cerevisiae A364p+ and its temperaturesensitive derivatives 198 and 13052 [cdc8(Ts)], 4008 [cdc7(Ts)], and 146-2-3 [cdc2l(Ts) thymidylate synthetase-] were obtained from L. Hartwell (16, 17, 18);
strain X2180-lBp+ (8) was provided by L. Hereford; JL125-1OC (a p+ ura) was from G. Michaelis; and Y379-5D (a p+ Cyhr o-DNA-) was from C. P. Hollenberg. From these p+ strains, A364Apo-3, 198p°-4,
333
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OERTEL AND GOULIAN
13052p°-14, 4008p°-9, and X2180-lBp°-2 lacking mtDNA were derived by treatment with ethidium bromide (14, 24). A364Ap--2 is a spontaneous petite mutant containing mtDNA. JL125-lOCp--4 (Eryr) was prepared from a spontaneous erythromycin-resistant derivative of the p+ strain by treatment with ethidium bromide and selection for the cytoplasmic Eryr marker
(32).
Nucleic acids. 32P-labeled nDNA and mtDNA (0.1 to 1 Ci/mmol of P) were prepared from strain A364Ap+ (24). 3H- and 3P-labeled bacteriophage fd DNA was prepared by the procedure of Schaller (31). Preparation of the 3P-labeled restriction fragments R1-HpaIIC (252 base pairs) and Rl-HpaH-D (200 base pairs), used as size markers, was as described previously (26). Preparation of ether cells. Cells were grown with moderate aeration in 500 ml of YMM medium (24) at 300C (23°C for temperature-sensitive mutants) to a density of 3 x 107 to 5 x 107/ml. If required, the DNA in the cells was prelabeled by replacement ofunlabeled uracil in the medium with [3H]uracil (0.01 mM; 0.5 Ci/ mmol) (24). The cells were collected by centrifugation, suspended in 50 ml of buffer A [50 mM Tris-hydrochloride (pH 7.5), 100 1nM KCI, 10 mM MgCl2, 1 mM dithioerythritol, 1 mM ethylene glycol-bis(46-aminoethyl ether)-N,NY-tetraacetic acid); 1 M D-(+)-sorbitol] at 00C, and shaken vigorously, in a funnel, with 50 ml of freshly prepared water-saturated diethyl ether (000) for 30 a. The phases were allowed to separate for about 30 s; the aqueous phase was then transferred to centrifuge tubes and underlayered with about 0.25 volume of fresh (ether-free and, therefore, denser) buffer A. The tubes were centrifuged immediately for 2 min at 12,000 x g. After immediate removal of the supernatant and suspension in 50 ml of fresh buffer A (00C), centrifugation was repeated in the same way and the pellet was finally suspended in 10 ml of buffer A. Portions (1 ml each) of this suspension were quickfrozen at -70°C and kept at this temperature until use. No difference was detected between ether-permeabilized cells (hereafter called "ether cells") that were used immediately after preparation and those used after 3 months of storage at -70°C. In vitro incororation of nucleotides into DNA. A freshly prepared or freshly thawed suspension of ether cells from the stock was adjusted to a density of 1 x 109 to 3 x 10" cells per ml by dilution with buffer A. A 0.9-volume of this suspension was brought to the desired incubation temperature (250C unless stated otherwise), and the reaction was started by the addition of 0.1 volume of a mixture (xlO concentrated) containing: 1 mM each dATP, dCTP, dGTP, UTP, GTP, and CTP; 20 mM ATP; 0.1 mM [3H]dTTP (4 Ci/mmol or 47 Ci/mmol); and 100 mM phosphoenolpyruvate (standard conditions). For density-labeling experiments, dTTP in the concentrated nucleotide mixture was replaced by 5-bromodeoxyuridine 5'-triphosphate at 1 mM and [3H]dCTP (0.1 mM; 16 Ci/ mmol). The reaction in the whole sample or a portion was stopped by dilution with an at least 10-fold volume of 50 mM Tris-hydrochloride (pH 8.2)-50 mM EDTA50 mM KXP207-1 M D-(+)-sorbitol and centrifuged at 3,500 x g for 5 min at 0°C. The supernatant was discarded. Sample processing. Incorporation of radioactive
J. BACTERIOL. label into DNA was assessed by determination of alkali-stable, acid-insoluble radioactivity in the cell pellets (24). When native DNA was to be analyzed by ultracentrifugation, the ether cells were suspended and briefly incubated with Arthrobacter luteus enzyme before further treatment with Sarkosyl, pronase, and phenol, as described previously (24). To analyze denatured DNA, the cell pellet after treatment with A. luteus enzyme was lysed by suspension in 0.5 ml of 0.3 M KOH and left for 15 h at 230C. Insoluble material was removed by centrifugation (15,000 x g, 00C; 15 min). The supernatant containing all acid-insoluble radioactivity either was used directly for centrifugation in alkaline sucrose gradients or was neutralized before analysis in CsCl gradients. Ultracentrifugation. Sucrose gradients, prepared as described previously (24), were centrifuged for 3.5 h at 40,000 rpm and 200C (neutral) or for 14.5 h at 32,000 rpm and 150C (alkaline), in a Beckman SW40 rotor, with appropriate internal or external size markers for evaluation of molecular weights (33). Isopycnic centrifugations were carried out in a Beckman 65 angle rotor, using CsCl solutions of density 1.68 g/ ml (32,000 rpm, 60 h, 2000) for separation of nDNA and mtDNA and 1.75 g/ml (40,000 rpm, 48 h, 2500) for bromodeoxyuridine-containing DNA (24). Other procedures. Other procedures, sources, and reagents have been described previously (24, 25).
RESULTS General properties of the permeable cell system. Brief treatment with diethyl ether at low temperature, similar to the procedure of Vosberg and Hoffman-Berling (35) for Escherichia coli, rendered cells of S. cerevisiae permeable to nucleotides. Ether-treated yeast cells are nonviable (7) but retained their normal shape and incorporated externally added radioactive dNTP's into DNA. DNA synthesis in ether cells prepared from wildtype strains containing normal mtDNA (p+ strains) proceeded for at least 50 min with a slowly decreasing rate (Fig. 1). For optimal results, the system was prepared from the middle logarithmic growth phase, with glucose as the carbon source, under moderate aeration. When cells were used that had been grown under vigorous aeration with ethanol as the carbon source, conditions under which the mitochondria are optimally active metabolically, the initial fast in vitro DNA synthesis was soon followed by degradation of the products, especially when the incubation was carried out at elevated temperatures (>250C) (not illustrated). Optimal DNA synthesis in ether celLs required the presence of all four dNTP's, Mg2+, an SH reagent and, in the absence of phosphoenolpyruvate, a high concentration of ATP (Table 1). ATP could be replaced only partially by another NTP, e.g., UTP. The presence of residual nu-
VOL. 140, 1979
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DNA SYNTHESIS IN S. CEREVISIAE
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20
30
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FIG. 1. Kinetics of DNA synthesis in ether cells. Ether cells were prepared from strain X2180-lBp+ and incubated in a 1-ml reaction volume (10O cells per ml) (see text). After 3 min ofprewarming at 25°C, the reaction was started by the addition of a concentrated nucleotide mixture containing the 3H label in dTTP (see text). After incubation at the same temperature at the times indicated, 50-,il portions were removed and analyzed for alkali-stable, acid-insoluble radioactivity.
cleotide pools was suggested by a low level of DNA synthesis after omission of three dNTP's. Analysis of the incubation mixture after various reaction times by thin-layer chromatography revealed that in the absence, but not in the presence, of phosphoenolpyruvate all NTPs and dNTP's were dephosphorylated rapidly. UTP, GTP, and CTP were not necessary for optimal synthesis, but residual pools may have been sufficient to satisfy a possible ribonucleotide requirement. Label from radioactive ribonucleoside triphosphates was incorporated into RNA, but this has not yet been studied in detail. Sorbitol stabilized the system, perhaps by preventing inactivation of sensitive proteins or acting as osmotic support. Inhibition (70%) was observed with arabinosyl-CTP, at a concentration 7.5-fold that of dCTP during the incubation. mtDNA synthesis was highly sensitive to dideoxy-TTP (Table 1), whereas nDNA synthesis was unaffected even at much higher levels (20 ,uM) (not shown), paralleling the sensitivity of the mammalian mitochondrial (y) DNA polymerase and insensitivity of the a polymerase (9, 36). At pH 6.5 the initial rate was lower than that at pH 7.5, but the synthesis proceeded for a longer time, finally reaching the same level. At
335
pH 8.5, the initial rate was also lower than that at 7.5 but, in addition, the incorporation ceased prematurely and was followed by considerable degradation (not illustrated). A high concentration of externally added DNase did not affect DNA synthesis in the ether cells, indicating that the DNA was not accessible to externally added proteins. Properties of ether cells prepared from different yeast strains. Modifications of the ether treatment, e.g., moderate changes in the composition of the buffer or time of exposure to the solvent, had little influence on the activity. However, it was observed that the rate and extent of in vitro synthesis of nDNA as well as of mtDNA varied considerably depending on the strain used. With strains containing normal mtDNA (p+ strains) the product was mainly mtDNA. This was confirmed by density gradient centrifugation analysis of the in vitro product from p+ and p0 strains (Fig. 2A and B). In A364Ap+ ether cells (and also all other p+ strains tested), label was primarily or exclusively in material with the same density as that of native mtDNA (1.683 g/ml [18]). As expected, most of the product synthesized with a p0 strain (lacking mtDNA) had the same density as that of nDNA TABLE 1. Requirements and inhibitors of the in vitro system Condition(s)
Incorporation relative to standard conditions (%)
Complete (standard conditions) . ....... 100 -dCTP ................... 27 -dCTP, -dGTP, -dATP ........... 14 -ATP ............................ 102 -ATP, -phosphoenolpyruvate ...... 27
-Phosphoenolpyruvate ............. -Phosphoenolpyruvate, +10 mM ATP
64 94
-Phosphoenolpyruvate, -ATP, +10 mM UTP ........................ 45 +0.75 ,uM araCTP .......... ........ 30 +0.05 ,uM dideoxy-TTP ....... ...... 59 22 +0.5 ,iM dideoxy-TTP ........ ...... -Dithioerythritol, +5 mM N-ethylmal4.5 eimide ............ +500,ug of pancreatic DNase per ml 93 a The complete standard reaction mixtures (200 pi) were prepared from ether cells (strain A364Ap+) as described in the text. The final concentrations were: 50 mM Tris-hydrochloride (pH 7.5); 10 mM MgC92; 100 mM KCI; 1 M D-(+)-sorbitol; 1 mM EDTA; 1 mM dithioerythritol; 10 mM phosphoenolpyruvate; 2 mM ATP; 0.1 mM each dATP, dGTP, dCTP, GTP, CTP, UTP; 0.01 mM [3H]dTTP (4 mCi/mmol); and ether cells, 1.5 x 109/ml. Incubations were carried out in duplicate for 10 min at 25°C, followed by analysis for alkali-stable, acid-insoluble radioactivity.
336
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proportion depended on the strain used. The initial rate of mtDNA replication in X2180-lBp+ ether cells was close to the average in vivo rate calculated on the basis of a generation time of 150 min and an mtDNA content of 10% in the cell. In contrast, with A364A the rate in vitro was only about 10% of the initial 500 0 calculated average in vivo rate. With most ~~~~~~~~~~~E strains, the average initial rate of nDNA syntheoco 1000. ,, ~~~~~~~~~~~u sis was approximately 0.5 to 2% of the calculated rate of nDNA replication in vivo (based on a generation time of 210 min in the p0 strains). In IA364A (p+ and po) after permeabilization, syn> : - 2000 thesis of nDNA could barely be measured. DNA synthesis in ether cells prepared x 500 0 1500 from temperature-sensitive DNA replica500In temperature-sensitive mumutants. tion 1000 tants, defective in the cell division cycle gene 8 (cdc8) (16, 17), which participates in the propa500 gation of DNA replication, the synthesis of 100 O nDNA in vivo stopped very rapidly and almost completely after a shift to the restrictive tem30 20 10 perature. The kinetics of nucleotide incorporaFRACTION NUMBER tion into DNA of a mutant and a wild-type FIG. 2. Analysis of DNA synthesized in vitro by strain were compared in vitro at the permissive isopycnic centrifugation. Reaction mixtures (0.5 ml) and restrictive temperatures. In ether cells pre(3 x 10' cells per ml) were prepared with ether cells pared from the p0 derivative of the mutant, the as described in the text, except that the [3H]dTTP specific activity was 18 Ci/mmol. After incubation at synthesis of nDNA at the restrictive tempera25°C for 30 min, native DNA was extracted from the ture stopped completely after a short initial cells, mixed with 32P-labeled A364A nDNA and burst of synthesis (Fig. 3B), which could not be mtDNA markers, and analyzed by CsCl density gra- prevented by preincubation without NTPs for 5 dient centrifugation (see text). (The 32p marker on the to 15 min. This was found only for the two alleles right is mtDNA; that on the left is nDNA) Symbols: (198 and 13052) (Table 2) of the mutant and 32p. (A) Strain A364Ap+; (B) strain 198p°-4. confirms earlier results with another in vitro 0, 3H; system from yeast (24, 25). In contrast to this, in (1.699 g/ml [18]) (Table 2). In addition, a portion ether cells of wild-type p0 strains of a temperaof this DNA had a density higher than that of ture-sensitive mutant in the thymidylate synthenormal nDNA (Fig. 2B), which may represent tase gene (cdc2l) (13), nDNA synthesis propreferentially synthesized so-called "y" DNA, a ceeded at the high temperature at the same or nuclear satellite DNA containing the ribosomal a higher level than at the low temperature (Tagenes (6), and may be further accentuated by ble 2, Fig. 3A). association with RNA. In p+ ether cells derived from DNA replication Results with a series of strains are summarzed mutants 198p+ [cdc8(Ts)] (not illustrated) and in Table 2. Generally, the initial rate of DNA 13052p+ [cdc8(Ts)] (25), mtDNA synthesis was synthesis with a p+ strain was at least six to eight not temperature sensitive. However, the delayed times as high as with its derivatives lacking and incomplete effect of this mutation on mtDNA. A maximum of 5% of the product after mtDNA observed in vivo (3, 40) may have been a 40-min incubation had the same density as due to a more indirect effect of mutation in the that of nDNA (1.699 g/ml), and the rest had cdc8 gene on mtDNA synthesis. nDNA synthethat of mtDNA (1.683 g/ml). In vitro-labeled sis was blocked in ether cells prepared from a DNA with the density 1.699 g/ml (5% of the p0 derivative of the DNA initiation mutant cdc7 total) was also obtained with strain Y379-5Dp+ (16, 17) when the cells were shifted to the nonlacking o-DNA (C. P. Hollenberg, personal com- permissive temperature for a period equivalent munication), supporting the chromosomal origin to at least one generation time before harvest of the product with this density, at least for this (Fig. 30), indicating that in vitro DNA synthesis strain. In the in vitro system from petite mutants is closely related to the in vivo process. The lack containing an altered mtDNA (p-), both nDNA of effect of temperature in vitro on a preparation and mtDNA were made at a low rate, and their of the same mutant grown at the permissive -j
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VOL. 140, 1979
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TABLE 2. Properties of ether cells prepared from different yeast strainsa Ether cells
Incubation (0nc temp
Initial rate [(pmol of
dTMP//l0
cells)/ mi]
Etn (mo (nuo Etn of dTMP/108 cells)/40 min]
nDNA(%)
mtDNA(%)
25 0.28 3.50 97 37 0.44 4.88 25 0.75 6.25 5 95 37 1.31 5.35b 25 0.79 7.50 5 95 37 1.79 8.25b 25 0.80 5 3.80 95 37 1.80 5.38 25 2.75 25.5 -3 97 25 0.63 8.00 5 95 Y379-5Dp+ (o-DNA-) 25 0.04 0.38 70 30 A364Ap--2 ................ 25 0.04 1.25 30 70 JL125p--5 (Eryr) .......... 0.01 25 0.08 >97 A364Apo-3 ................ 25 0.13 0.94 >97 X2180-lBp0-2 ............. 37 0.13 1.00 X2180-lBpo-2 ............. 25 0.06 0.83 >97 198p°-4 (cdc8) ............. 37 0.05 0.44 198p°-4 (cdc8) ............. 25 0.14 >97 13052p0-14 (cdc8) .......... 1.09 37 0.10 0.68 13052p°-14 (cdc8) ........... aThe initial rate and the extent of the DNA synthesis in vitro after 40 min were derived from kinetic data as described in the legend to Fig. 3. The proportions of nDNA and mtDNA were determined by isopycnic in CsCl gradients (see text) (cf. Fig. 2). centrifugation b With these strains, at 370C by 40 min some of the initially synthesized DNA was already degraded.
A364Ap ................. A364Ap+ .................. 198p+ (cdc8) .............. 198p+ (cdc8) .............. 13052p+ (cdc8) ............ 13052p+ (cdc8) ............ 146-2-3p+ (cdc2l) .......... 146-2-3p+ (cdc2l) .......... X2180-lBp ..............
temperature (Fig. 3C) is consistent with there being few or no new initiations in nDNA synthesis in the ether-treated cells. In vitro synthesis of mtDNA in the corresponding cdc7 (4008) p+ strain was not influenced by in vivo exposure to restrictive temperature, and control experiments with p0 and p+ nonmutant strains (X2180-1B) showed that in vitro synthesis of nDNA was not affected by the shift to higher temperature during growth of the cells. Analysis of mtDNA pulse-labeled in vitro. mtDNA, pulse-labeled in vitro for 10 min with [3H]dTTP in X2180-lBp+ ether cells, sedimented in neutral sucrose gradients in a broad peak with the maximum at about 25S (30% mitochondrial genome size), with a significant portion up to 39S (full genome size) (Fig. 4A). With longer incubation times (up to 45 min), no significant change in this size distribution was noticed. In vivo prelabeled DNA sedimented faster than the in vitro products and consisted mainly of sheared chromosomal DNA (458) (data not shown; 25). After X2180-lBp+ ether cells were pulse-labeled with [3H]dTTP for 15 s at 180C followed by a chase with unlabeled dTTP of up to 5 min, the main product observed in alkaline sucrose gradients consisted of short pieces, initially 0.1 to 0.15 kilobase (kb) and growing to about 0.25 kb (Fig. 4B). When this experiment was per-
formed at 25°C (with 5-bromodeoxyuridirie 5'triphosphate in place of dTTP to allow subsequent density analysis), a portion of the newly synthesized DNA achieved the size of 0.25 kb within 20 s while, in addition, a second broad peak of label sediimented principally at 13S (Fig. 40). After a chase with unlabeled dNTP for 15 min, about 40% of the short pieces had joined to the large material, which had grown to about 14S. With labeling times longer than 20 s (without chase), a fraction of the 0.25-kb pieces accumulated (Fig. 4D). Analysis of density-labeled mtDNA. Density-labeled 0.25-kb pieces recovered from the alkaline sucrose gradients (Fig. 4C and D, pool I) consisted primarily or completely of bromodeoxyuridine-labeled, and thus completely in vitro synthesized, mtDNA, independent of the pulse length (Fig. 5A) (the density distribution was broad because of the small size of the material). In contrast, approximately 50% of the DNA pieces of the 12 to 18S size class (3 to 6 kb) (Fig. 4C and D, pool II) grew to the whole fragment length within 5 min (Fig. 5B). After 15 min, more than 75% of the 12 to 18S product and 50% of the longest fragments (Fig. 4D, fractions 9 through 12) (ca. 7 kb) were fully substituted with bromodeoxyuridine (not illustrated). These experiments show that continuous stretches of greater than 10% mitochondrial genome length
338 Xn -i tLJ _ O
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(72 kb) can be synthesized in ether cells of yeast. To demonstrate that the synthesis of mtDNA in vitro proceeded by a semiconservative mechanism, native X2180-lBp+ DNA, density-labeled with 5-bromodeoxyuridine 5'-triphosphate for 45 nmi, was analyzed directly by isopycnic centrifugation in CsCl gradients. About 25% of this material banded with "hybrid" density,
A
most of
sthe remainder containing additional proportions of preexisting (unsubstituted) mtDNA and a ci r W small fraction with a density higher than that of hybrid mtDNA (Fig. 50). The hybrid-density B 4 reisolated from CsCl gradients had a broad DNA .0 O size distribution as measured by velocity sedimentation in neutral sucrose gradients with the O majority between I and 6 kilobase pairs, and a 0.5 z _ significant portion up to 30% genome size (27S, 25 kilobase pairs). After sonication (average chain H , . ...length, 0.9 kb), at least 75% of the product 40 10 20 30 banded in CsCl gradients at hybrid density and, in addition, approximately 15% banded as fully bromodeoxyuridine-substituted mtDNA (Fig. have arisen dense material may The fully before 5D). id completion of a full round by reinitiation o of replication, but other possibilities have not 0 0.8 c been excluded, e.g., recombination (38) or deo tachment of short, newly synthesized DNA by branch migration (27) and renaturation, both / 0 perhapsfavored by the highpolydeoxyadenylate a o polydeoxythymidylate content (2). Further E studies will be required to determine its origin. ,X,-' cr uJ 0.4 / DISCUSSION ~ 4 / ,,0 observed in vitro mtDNA the -, In favor of DNA synthesis being replicative synthesis ir 01/ O rather than some form of repair are several ,/, observations that indicate important similarities z t.' to the in vivo process. The continuous length of A ICA product strands, 7 kb after denaturation and up 40 to 25 kilobase pairs before denaturation, is 30 20 X0 10 strongly against a repair mechanism. The initial TIME, MIN rate of synthesis was on the order of the in vivo rate of mtDNA synthesis, and the extent of FIG. 3. KneticsofchromosomalpandmstDNA s ynA thesis in etheraces from temperature-sensite DNA synthesis after 45 min was sufficient for the replication mutants. Thereactnmixtures(0.5ml) duplication of 25% of the mtDNA in the cell. were prepared with ether cells (2 x 109/ml) as demtD by the syn scribed in the text, except that the samples were The synthesis could be inhibited by araCTP, an not repair, DNA but replicative, of inhibitor temperature preincubated for 5min at the incubation (230C in vitro or 37°C in vitro) before the reaction synthesis in other eucaryotic and procaryotic was started by addition of the concentrated nucleo- systems (5, 22). By density-labeling experiments it was shown tide mixture with [3HJdTTP. The cells were grown at 23°C (230C in vivo), but for (C) a separate portion that the native DNA product with "hybrid" of cells was shifted to 37°C for the 3 h just before density had a size between 5 and 30% that of the a.
------ 0
tPe
harvest for ether treatment (37°C in vivo). Samples (50 ul each) were removed after incubation for the times indicated and analyzed for incorporation of radioactivity into DNA. (A) Strain X2180-lBpo-2; (B) strain 198p°-4; (C) strain 4008p°-9. Symbols: 0, m v 23°C; in vitro, 23°C; 0, in vivo, 23°C; in vitro, 37°C; A, in vivo, 37°C; in vitro, 23°C; A, in vivo, 37°C; in vitro, 37°C.
an
mitochondrial genome. The majority of the ma-
terial synthesized in vitro (15-min incubation)
consisted of DNA chains between
3 and 7 kb to 5 to 10% of mtDNA size. The chain elongation rate corresponded to at least 0.4 kb/min, a rate sufficient for the replication of the mitochondrial genome
b eequivalent 3a7k etw aftedeNAturain, after denaturation, en
DNA SYNTHESIS IN S. CEREVISIAE
VOL. 140, 1979
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within one generation time (150 min, 25°C). Label appeared early in 0.25-kb pieces which consisted of DNA completely synthesized in vitro and was observed growing to this size. Pulsechase experiments showed that the 0.25-kb fragments served as precursors of high-molecularweight DNA, but, in addition, with labeling for longer periods a portion of the short pieces accumulated. The significance of the 0.25-kb precursor is not yet clear. One possibility is that it reflects a mechanism of discontinuous synthesis, with multiple tandem initiations on one or both 400 arms of the replication fork, as in bacterial and animal cell chromosomal replication (11, 21, 29). 300 Incomplete maturation of short nascent pieces of DNA has been commonly observed in eucar200 yotic in vitro systems for DNA synthesis (12, 28, E 34), and a similar presumed defect in joining 100 f could account for accumulation of the fragments in the yeast in vitro system. A second possibility w is that the 0.25-kb pieces observed here are X 600 . related to the 7S initiation sequence of animal 500 xa. cell mtDNA, which is made in excess over the rest of the mtDNA (4). The accumulation could 1400 ^ reflect this turnover process, possibly enhanced 300 by nicks in the mtDNA molecule, which have been shown to result in separation of the 7S 200 segment from the rest of the molecule by branch migration (30). 100 The rate and extent of in vitro nDNA synthe° sis in ether cells were low compared with those of mitochondrial (p+) DNA, but with most strains it is still in the same order of magnitude as with other permeable systems from yeast (20, 24, 41). The in vivo temperature sensitivity of nDNA synthesis in cdc8 mutants was expressed in the ether cell system, similar to results with the permeable spheroplast system (24). Further support for the replicative nature of nDNA syn-
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3H, 15-s pulse, 5-min chase; 32p size marker. (C, D) The incubation was performed at 25°C in a 2.0-ml volume with the 3H label in dCTP (16 Ci/mmol) and with 5-bromodeoxyuridine 5'-triphosphate instead of dTTP. After 20 s, two 400-tl portions were removed: in one the reaction was terminated immediately; to the other a 200-fold excess of unlabeled dCTP (2 mM) was added, and incubation continued for an additional 15 min. In additional portions (400 sl), the reactions were terminated after 60 s, 5 min, and 15 min. Further sample processing and centrifugation were as for part (B), except that the 32P-labeled size marker (R1-HpaII-C) was 252 base pairs long. Radioactivity in DNA was determined in 50-il portions of the fractions, and the remainder was pooled as indicated by "I" and "II." (C) 0, 3H, 20-s pulse; +, 3H, 20-s pulse, 15-min chase; 32p size marker. (D) x, 3H, 1-min pulse; *, 3H, 5-min pulse; A, 3H, 15-min pulse. (M, 32p 252-base pair size marker). 32P-labeled fd DNA was centrifuged in a duplicate tube as an external 30S (neutral) or 20S (alkaline) size marker. ,
30 20 10 FRACTION NUMBER
FIG. 4. Size determination of in vitro pulse-labeled native and denatured mtDNA by sucrose gradient centrifugation. The reaction mixtures with ether cells (X2180-lBp+, 3 x 109/ml) (see text) were incubated as follows. (A) Reaction mixture of 400 ,d; 3H label in dTTP (46 Ci/mmol); 25°C. After 10 min, DNA was isolated and analyzed in neutral sucrose gradients (see text). (B) Reaction mixture of 1.5 ml; 3H label in dTTP (46 Ci/mmol); 18°C. After 15 s, the reaction in a 400-,ul portion was terminated. Incubation of the remainder was continued after the addition of a 200fold excess of unlabeled dTTP (2 mM), with removal of additional portions (400 IlI) at 60 s (not illustrated) and 5 min (pulse-chase). DNA was analyzed in alkaline sucrose gradients (see text) after addition of 32P-labeled R1-HpaII-D DNA (200 base pairs) as the internal size marker. Symbols: 0, 3H, 15-s pulse; +,
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J. BACTrERIOL.
OERTEL AND GOULIAN
thesis in ether cells was provided by the elimination of nDNA synthesis (in vitro) in an initiation mutant (cdc7) by exposure to the restrictive temperature in vivo. An advantage of the ether cell system in comparison to the permeable spheroplast system developed recently in our laboratory for investigation of nDNA synthesis (24) is that it can be prepared from cells in their normal physiological state and any growth cycle phase. In addition, the joining of replication intermediates seems to be less impaired in ether cells than in permeable spheroplasts (25). By selection of the proper yeast strain for preparation of ether cells, it is possible to study the replication in vitro of normal mitochondrial, as well as chromosomal, DNA separately from the synthesis of other species of DNA normally present. Particularly for study of early nascent intermediates in DNA replication, the system allows experiments difficult or impossible with intact cells because of their precursor pools (19, 24). The possibility that mitochondrial reinitiation may be occurring in ether cells is particularly provocative, since this has been an almost uniform deficiency of in vitro systems for DNA synthesis, both from procaryote and eucaryote sources. Resolution of this, as well as of other questions on the mechanism of DNA synthesis, awaits results of studies now in progress.
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