Vol. 135, No. 2
JOURNAL OF BACTERIOLOGY, Aug. 1978, p. 436-444 0021-9193/78/0135-0436$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Replication of Bromodeoxyuridylate-Substituted Mitochondrial DNA in Yeast JUDITH LEFFt* AND T. R. ECCLESHALL Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 Received for publication 14 March 1978
The DNA of several strains of Saccharomyces cerevisiae was labeled by growing the culture in medium supplemented with thymidylate and bromodeoxyuridylate. It was thus possible to follow the course of mitochondrial DNA replication in density shift experiments by determining the buoyant density distribution of unreplicated and replicated DNAs in analytical CsCl gradients. DNA replication was followed for three generations after transfer of cultures from light medium to heavy medium and heavy medium to light medium. Under both conditions, the density shifts observed for mitochondrial DNA were those expected for semiconservative, nondispersive replication. This was further confirmed by analysis of the buoyant density of alkali-denatured hybrid mitochondrial DNA. With this method, no significant recombination between replicated and unreplicated DNA was detected after three generations of growth. It is generally believed that all duplex DNA replication is semiconservative. However, this mode of replication can be obscured in density transfer experiments if hybrid DNA molecules arise which do not have the predicted buoyant density. When this occurs, the DNA is said to replicate dispersively. This phenomenon could be due either to the presence of large precursor nucleotide pools or to extensive recombination between replicated and unreplicated DNA. Previous workers studying the replication of mitochondrial DNA (mtDNA) have obtained buoyant density patterns for newly replicated DNA which seem dependent on the organism studied or on the method of labeling used. In mammalian cells such as rat liver (13) and HeLa cells (9), mtDNA buoyant densities were those expected for semiconservative replication of mtDNA. In Euglena, however, only dispersive patterns were observed; this was attributed to recombination between hybrid DNA strands, possibly accompanied by turnover of the mtDNA (26). The results for Neurospora crassa were interpreted to indicate a semiconservative pattern of replication of mtDNA (25), but the density labeling experiments using 15N showed a large pool of nucleotides in the cells. The presence of this pool made the interpretation of the results more difficult. In cells of Ustilago maydis made permeable to deoxyribonucleotides, no dispersive replication of mtDNA was observed (1). t Present address: 5829 Liebig Avenue, Riverdale, NY 10471.
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Williamson and Fennel (33), as well as Sena et al. (29), reported that the replication of yeast (Saccharomyces cerevisiae) mtDNA is apparently dispersive as a consequence of extensive and rapid recombination of the mtDNA molecules after replication. However, when mtDNA replication was studied in isolated S. cerevisiae
mitochondria, using bromodeoxyuridine triphosphate, no evidence for recombination was observed (20). These differences may reflect the inherent difficulties in studying the replication of yeast mtDNA. One problem is that yeast mtDNA is unusually susceptible to enzymatic degradation by specific nucleases associated with an mtDNA membrane complex (24). Another difficulty, until recently, was the inability to use radioactive thymidine or its heavy analog, 5-bromodeoxyuridine (BrdU), in studying yeast DNA replication. This is because yeast lacks the enzyme thymidine kinase to phosphorylate the nucleoside for use in DNA synthesis (12). As a result, previous studies in vivo have been carried out using 15N as a density label. The drawback of this method is that the presence of significant pools of nucleotide precursors in the cell may obscure the pattern of mtDNA replication. In recent years several workers (5, 32) have isolated yeast mutants that could incorporate thymidylate (TMP), either because they were auxotrophic for TMP or because de novo synthesis of TMP was inhibited by drugs. These mutants permit the specific labeling of yeast DNA with radioactive TMP or with its heavy analog, bromodeoxyuridylate (BrdUMP). In a previous study (17), the incorporation of BrdUMP into
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yeast nuclear DNA and mtDNA was investigated. In this report, further work is descibed using this method for the study of mtDNA rep-
lication. MATERIALS AND METHODS Yeast strains. A strain auxotrophic for TMP, tnpl (tmpl-6a hisl,7 ilvl-92 lys tiyp [p+J), was obtained from J. Iittle. The wild-type strain D273-1OB was obtained from F. Sherman. Strain cdc7-2, a cell division cycle mutant of A364A (a adel ade2 ural his7 1l lys2gall), wa obtained from the Yeast Genetic Stock Center at Berkeley. Colonies that could incorporate TMP when grown in the presence of drug were selected from strain cdc7 and D273-10B as decribed by Leff and Lam (17). Mi1ds. Strain tpl was grown on complete medium containing 1% Difco yeast extract, 2% Difco peptone, 1.5 mg of KH2P04 per mL 2% ethanoL and 3% glycerol (YPPEG). The pH was adjusted to 5.7. This medium was supplemented before use with TMP (30 pg/mI) (Sigma Chemical Co.) and variable amounts of BrdUMP (7 to 20 pg/ml), synthesized by the method of Michelson et aL (22). Strains D273-10B and cdc7 were grown on a medium described by Brendel and Haynes (6) containing 0.25% Difco yeast extract, 1% Difco peptone, sulfanilamide (Sigma Chemical Co.) (6 mg/ml), opterin (K and K Laboratories) (50 pg/mI), and a carbon source which was 3% glycerol and 2% ethanol for strain D273-10B (SATEG medium) and 2% dextrose for strin cdc7 (SATD medium). The medium used to label DNA with "'N contained 0.117 g of yeast carbon base (Difco) per mL 3% glyceroL and 2% ethanol (CBGE medium), supplemented with 300 pg of ("NH)2S04 (Merck, Sharpe and Dohme, Canada; 96% isotope substitution) per ml. Denity transfer experiments. Transfer of cultures from light medium to heavy medium (upshift experiments) was carred out by growing the cultures in the presence of TMP alone until early exponential phase and then adding BrdUMP. Samples for analysis were withdrawn at various times. Transfer of the cultures from heavy medium to light medium (downshift experiments) were carried out by growing the culture in the presence of BrdUMP and TMP until either midexponentia or statioy phase, washing the ceUls twice with YPPEG medium, and resupendim contining TMP. The ing them in YPPEG labeling of strain D273-1OB with "N was performed by growng a culture to stationary phase in CBGE medium having ("NHO4SO4 as the only nitrogen source. A portion (0.5 ml) of that culture was then used to inoculate a new 10-ml culture. The procedure was repeated three times to ensure that the DNA was fully substituted with 15N. The downshift experiment consisted of tansferring the cells, after washing them with CBGE medium, to fresh medium supplemented with 0.4 mg of (NH4)2S04 per mL This concentration of (NHJ2SO permitted optimal growth of the culture with a doubling time of 90 min at 300C. All cultures were grown at 300C unless specified otherwise. Growth was monitored with a Klett-Summerson photometer equipped with a red filter. DNA isolaon and analysis. DNA was isolated
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as described by Goldthwaite et al. (10), using zymolyase (Kirin Brewery Co.) to prepare spheroplasts. Since BrdUMP-containing DNA is very susceptible to degradation by 330-nm light (16), the isolation procedure was carried out exercising great care to avoid exposure to fluorescent light. In addition, the DNA was analyzed by analytical centrifugation as soon as possible after isolation because BrdUMP-containing DNA is unstable and because yeast mtDNA degrades on storage. It was more appropriate to prepare DNA from whole cells than from isolated mitochondria because it is impossible to prepare intact yeast mitochondria and to free mitochondria of nuclear DNA without extensive DNase treatment. Preparation of bromouracil (BU)-substituted mtDNA from mitochondrial preparations yielded mtDNA of variable quality. Analytical density gradient centrifugation was carried out in a Spinco model E ultracentrifuge at 44,770 rpm for at least 16 h at 250C as described by Meselson and Stahl (21). UV photographs were scanned with a Joyce-Loebl microdensitometer. Bacillus subtilis phage SP8 DNA (p = 1.742 g/ml) and Celulomonas sp. DNA (p = 1.734 g/ml) were used as density markers. Single-standed DNA was obtained by alkali denaturation and neutralization of native DNA; 0.01 ml of 10 N NaOH was added to 0.35 ml of DNA solution and neutrlized after 2 min with 0.05 ml of 2 N HCl in 1 M tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.5). The DNA was then examined in a neutal CsCl grdient uXsin phage SP8 DNA as a density marker.
RESULTS Labeling with BrdUMP. To achieve maximum separation between replicated and unreplicated DNA in the presence of BrdUMP, complete substitution of thymine by BU would be desirable, as has been achieved with mammalian cells (3). However, strain tmpl reverted to prototrophy after several generations of growth in YPPEG medium supplemented only with BrdUMP. However, for at least two generations BrdUMP was incorporated into newly synthesized DNA as shown in Fig. 1 for an ethidium bromide-treated derivative of strain tmpl, ie., a derivative lacking mtDNA. After 4 h, a time corewponding to one generation, the majority of the nuclear DNA banded at a density of 1.755 g/ml. This value is that expected for hybrid DNA (HL) in which one strand is fully substituted with BU (19). (The substituted DNA
strand is designated H and the unsubstituted strand is designated L.) Unsubstituted nuclear DNA (LL), having a buoyant density of 1.698 g/ml, was still present at this time. Growth of cells in the presence of BrdUMP and in the absence of TMP normally leads to a lag in DNA replication. After 6 h of growth, nuclear DNA banded at densities of 1.755 and 1.812 g/ml, corresponding to hybrid (HL) and fully substi-
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ments. Strain tmpl was grown in YPPEG medium supplemented with TMP, and then BrdUMP (7 ,ug/ml) was added at early logarithmic phase. Samples were taken at 0, 1, 2, 4, 8, and 12 h following the addition of BrdUMP. Analytical density gradient centriftgation of the isolated DNA gave the CsCl density gradient profiles shown in Fig. 2 and 3. The yield of mtDNA relative to nuclear DNA is unusually high using the growth conditions and the isola4 tion method described in Materials and Methcr ~~~~~~~6ods. This is important because it permits an 0 improved resolution of the DNA species. After growth for only 1 h in the presence of BrdUMP, BrdUMP substitution in the DNA was clearly visible. Both nuclear and mtDNA showed distinct bands at the densities corresponding to hybrid DNA (HL). This rapid incorporation of the nucleotide analoges indicated an extremely small pool of TMP precursors, and it was especially evident in strain tmpl, which lacks both thymidine kinase and thymidine synthetase (4). After 2 h of growth following the addition of 1.811 734 1.755 1.698 BrdUMP, the hybrid bands were well separated from the corresponding unsubstituted DNA speDENSITY (g/cc) cies. The sharpness of the peaks and their resoFIG. 1. Buoyant density profiles of the DNA of an lution were such as to permit the conclusion that ethidium bromide derivative of strain tmpl grown in for both DNA species there was very little, if YPPD medium in the presence of 30 pg of TMP per any, DNA of intermediate buoyant density beml and transferred to a medium containing 20 jg of tween the light (LL) and hybrid (HL) species. BrdUMP per ml. Samples were taken at 4 and 6 h Figure 3 shows the incorporation of BrdUMP after the transfer. The reference is Celulomonas sp. into yeast DNA at various times up to three DNA, which has a buoyant density of 1. 734 g/ml. generations. It is interesting to note, as reported by Wells (31), that mtDNA replication was completed sooner than that of nuclear DNA. After tuted (HH) nuclear DNA, respectively. Unfortunately, in the case of mtDNA, which 8 h of growth (corresponding to two generahas long adenine-thymine-rich regions (2), ex- tions), the mtDNA peak was at the heavy dentensive BrdUMP substitution causes extensive sity (1.696 g/ml), with a slight skewing towards degradation of the genome. Moreover, since the hybrid density. Nuclear DNA was still mtDNA has a higher content of adenine-thy- mostly at hybrid buoyant density (1.705 g/ml). mine than nuclear DNA, growth of cells in in- The presence of a small peak of light unreplicreasing concentrations of BrdUMP causes the cated mtDNA after the first doubling was not buoyant-density mtDNA to increase at a faster the result of a nonreplicating fraction the rate than that of nuclear DNA (17). As a result, mtDNA molecules, as will be shown below. Unthe difference in density between the two DNA replicated DNA has been reported in other studspecies becomes smaller until it becomes diffi- ies involving incorporation of BrdUMP into cult to distnguish them in CsCl gradients. Con- DNA (7, 14). The most likely explanation is that sequently, the percentage of BrdUMP substitu- a small fraction of the mitochondria did not tion (in the range of 11 to 12%) used in the utilize BrdUMP. following e'xperiments was the most convenient mtDNA replication in downshift experifor our present studies. This degree of substitu- ments. In the downshift experiments, cells were tion was obtained by growing the celLs in YP- first grown to stationary phase in YPPEG mePEG medium in the presence of 30 ig of TMP dium containing both TMP and BrdUMP. and 7 Ag of BrdUMP per ml. This concentration These conditions were previously shown to inof BrdUMP causes very little growth inhibition, crease the proportion of mtDNA relative to nusince the culture can grow to a cell density of 2 clear DNA (8). The culture was then transferred x 108 cells per ml with a doubling time of 4 h at to YPPEG supplemented only with TMP. The 300C. tracings in Fig. 4 summarize the results of sammtDNA replication in upshift experi- ples taken up to 6 h after the transfer. Up to 2
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cept that some unreplicated mtDNA was still present. After an additional 2 h, most of the mtDNA banded at its hybrid density. There was no evidence of any remaining heavy mtDNA. This lends support to the conclusion that all the mtDNA molecules replicate (33), so that the persistence of the light peak in the upshift experiments must be because some mitochondria cannot utilize BrdUMP. This set of experiments
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DENSITY (g/cc) FIG. 2. Buoyant density profiles of the DNA of a culure ofbnpl grown in the presence of 3Opg of TMP and 7 pg of Brd UMP per ml. Samples were taken at 0,1, and 2 h after the addition of Brd UMP. The shoulder on the hcavy side of the nuckar DNA at zero time represents the y sateUite band. Nuclear DNA bands are at 1.698 glml (LL) and 1.705 g/ml (HL). mtDNA bands are at 1.682 g/ml (LL) and 1.689
g/ml (HL). The reference is B. subtiis phage SP8 DNA, which has a buoyant density of 1.742 g/ml
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h after the transfer to light medium, there was FIG. 3. Buoyant density tracings of DNA isolated no change in the DNA buoyant densities befrom a culture of tnpl grown for 4, 8, and 12 h in cause cells emerging from, the stationary phase medium containing TMP and BrdUMP. The referexperience a time lag in their growth. After 4 h, ence DNA was from phage SP8. Nuclear DNA bands most of the nuclear DNA banded at a hybrid are at 1.698 glml (LL), 1.705 g/ml (HL), and 1.711 density but having a shoulder at heavier buoyant g/ml (HI). mtDNA bands are at 1.683 glml (LL), density. mtDNA showed a similar pattern, ex- 1.689 g/li (HL), and 1.696 glml (H11.
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LEFF AND ECCLESHALL
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DENSITY ( g/cc) FIG. 4. Buoyant density profiles of DNA from a culture of tmpl grown in the presence of TMP and BrdUMP for six generations and transferred to light medium. Samples were taken at 2,4, and 6 h after the transfer. The reference was phage SP8 DNA. Nuclear DNA bands are at 1.698g/ml (LL), 1.705g/ml (HL), and 1.711 g/ml (HH). mtDNA bands are at 1.683g/ml (LL), 1.689 g/ml (HL), and 1.696 g/ml (HI).
again points out the parallels between nuclear and mtDNA replication. In both cases, whether in upshift or in downshift experiments, the buoyant densities of the DNA are distributed among bands corresponding in density to light hybrid and heavy DNA, in accordance with a mode of replication that is semiconservative. The lack of dispersion indicates that the use of BrdUMP
overcomes any posiible pool problem and that little detectable recombination occurs between replicated and unreplicated mtDNA genomes. Density of denatured replicated DNA. Separation of the strands of DNA of hybrid density (HL) provided another means to detect the extent of recombination after semiconservative replication. DNA samples from the upshift experiments described in Fig. 2 and 3 (0 and 2 h and 8 h) were denatured as described in Materials and Methods. Figure 5 shows the DNA profiles after analytical centrifugation in neutral CsCl gradients. Two hours after the addition of BrdUMP to the culture, mtDNA gave rise to two peaks, the light peak banding at 1.696 g/ml, corresponding to the buoyant density expected for denatured light DNA, and the heavier peak banding at 1.703 g/ml. After 3 h of growth in the presence of BrdUMP, the resolution of the heavy mtDNA strand from the light mtDNA strand was very clear. The buoyant density of the heavy strand of mtDNA (1.703 g/ml) was significantly lower than that expected for mtDNA with 11% BrdUMP substitution (1.709 g/ml). However, in some experiments in which cultures were grown for five generations or more in the presence of BrdUMP, the buoyant density of single-stranded mtDNA was 1.709 g/ml. The lower buoyant density of the substituted mtDNA strands encountered in most experiments might be due to the presence of a region of neighboring BU bases which were preferentially degraded during alkali denaturation as the result of BU-induced single-strand breaks. Moreover, these observations, as well as those of others (15, 23), suggest that BrdUMP substitution in DNA increases its lability to alkaline treatment. It could also be argued that the density of 1.703 g/ml represents an intermediate density between the light and heavy strands due to recombinational events. This is not likely, however, for the following reasons. First, native mtDNA had buoyant densities expected for DNA replicating semiconservatively. If some recombination had occurred, the amount of the recombined DNA was too small to be detected in the buoyant density tracings of undernatured DNA. Consequently, the large peaks observed after denaturation of the hybrid mtDNA (HL) were unlikely to represent recombined strands of DNA. Moreover, extensive recombination between stands of hybrid DNA (HL) would result in DNA molecules in which both strands would be BU substituted. Denaturation of this DNA would yield two strands with very similar buoyant densities which would probably yield a single band in CsCl density gradients. The buoyant density of this band would have a value intermediate between those expected for unsubsti-
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BrdUMP, with derivatives of the strains D273lOB and cdc7-2 selected for TMP uptake (17). The strains were grown as described in Materials and Methods. The results with both strains were similar to those observed with strain tmpl. Figure 6 shows the density profile of D273-1OB 3 and 6 h after the addition of BrdUMP. The generation time for strain D273-1OB under these conditions was 8 h. mtDNA banded at discrete values corresponding to unsubstituted DNA and to hybrid DNA. Denaturation of the DNA yielded one light and one heavy band as described for strain tmpl. Density labeling with 1N. It is usually assumed that incorporation of reasonable amounts of BrdUMP into DNA does not affect replication
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DENSITY (g/cc) FIG. 5. Buoyant density profies of analytical centrifiugation in neutral CsCI of alkali-denatured DNA. DNA was isolated from samples taken from tmpl cultures grown for 0, 2, and 8 h in the presence of BrdUMP and TMP. The reference was phage SP8 DNA. Nuclear DNA bands are at 1.711 g/ml (L) and 1.727 g/mi (H). mtDNA bands are at 1.696 g/ml (L) and 1.703 g/ml (H).
tuted (L) and BU-substituted (H) singlestranded DNA molecules. In our experiments, however, the light and heavy mtDNA strands were well separated, and their buoyant density remained essentially invariant (± 0.002 g/ml) throughout the course of replication of mtDNA. mtDNA replication in other strains. We have also studied DNA replication for one generation in upshift experiments in the presence of
DENSITY (g/cc) FIG. 6. Buoyant density profile of DNA from a culture of strain D273-IOB grown in SATEG medium and containing 25 pg of TMP and 7 pg of BrdUMP per ml. Samples were taken at 3 and 6 h after the addition of BrdUMP. The reference was phage SP8 DNA. Nuclear DNA bands are at 1.698 g/ml (LL) and 1.704g/ml (HL). mtDNA bands are at 1.683g/ml (LL) and 1.689g/ml (HL).
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(11). Nevertheless, we compared the results of BrdUMP and '5N labeling by growing strain D273-1OB in the presence of ("5NH4)2SO4. This experiment could not be performed with tmpl because it would have required '5N-substituted TMP, which was not available. The results of the downshift experiment with 16N-labeled DNA are shown in Fig. 7. After 1.5 h (equivalent to one generation) in light medium, half of the nuclear and most of the mtDNA still banded at the heavy density. After 4.5 h in the light medium, a significant proportion of the nuclear DNA was still at the heavy density, whereas most of the mtDNA was unsubstituted. Only a small fraction of mtDNA remained at hybrid density (1.689 g/ml). Nevertheless, it is apparent that mtDNA banded at buoyant density values corresponding successively to heavy, hybrid, and light DNA. The separation of the peaks was not as distinct as with BrdUMP-labeled DNA, but there was clearly no single band with progressively decreasing buoyant density, as would be expected if extensive recombination had occurred. Denaturation of 15N-labeled mtDNA yielded two bands with the expected buoyant densities for light (1.696 g/ml) and heavy (1.710 g/ml) strands. No detectable DNA of intermediate buoyant density was observed. In addition, this experiment also confirmed that all the mtDNA molecules replicate. DISCUSSION In previous experiments with 15N-labeled DNA, replication mtDNA appeared as a single unimodal peak whose buoyant density decreased continuously during growth of the culture following the transfer from heavy to light medium (33). Denaturation of the mtDNA also yielded a single peak of a buoyant density intermediate between those expected for unsubstituted and fully substituted mtDNA strands. These results were ascribed to extensive and very rapid recombination between replicated and unreplicated mtDNA molecules. The differences between these observations and those described in the report may be explained by the different methods used in labeling and isolating DNA and perhaps by the use of different strains. Previous experiments using '5N as a label were performed in a defined medium in which nitrogen was in growth-limiting amounts. The yield of mtDNA from such suboptimaly grown cultures was quite small and, moreover, yielded a broad band in CsCl gradients. As a result, it was difficult to discem discrete peaks of different buoyant densitites even if they were present. On the other hand, cultures labeled with BrdUMP can be grown on complete medium with a nonferment-
J. BACTERIOL.
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DENSITY (g/cc) FIG. 7. Buoyant density profile of DNA from a culure of D273-IOB grown in the presence of (l5NH)2SO4 and transferred to light mediium. Samples were taken at 4, 8, and 12 h after the transfer. The reference was phage SP8 DNA. Nuclear DNA bands are at 1.698 g/ml (LL), 1.706 g/ml (HL), and 1.712 g/ml (HH). mtDNA bands are at 1.683 g/ml (LL), 1.689g/ml (HL), and 1.696g/ml (HH).
able carbon source, which greatly increases the yield of mtDNA (10). Consequently, in spite of unavoidable degradation during isolation, mtDNA is obtained in amounts that are sufficient to permit good resolution ofdifferent buoyant density species. Another difference between the two methods can be attributed to the nature of the label. In most cases, labeling with BrdU
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is the preferred method for studying DNA replication because it is specific for DNA and is rapidly incorporated via the salvage pathway. This is especially true for yeast stains auxotrophic for TMP that lack both thymidine kinase and thymidylate synthetase. Consequently, BrdUMP is incorporated as rapidly as 30 min after the addition of BrdUMP (0. Goldberg and J. G. Little, personal communication). However, in the experiments reported here (Fig. 7), as well as those reported by others (28, 33), incorporation of '4N into DNA during the downshift experiment shows a lag of variable duration due to the fact that inorganic "N has to be converted into deoxyribonucleotides before being incorporated into DNA. Moreover, presumably because of pool effects, heavy labeled nuclear DNA still persists after the third generation in light medium. It is also worth noting that BU-substituted DNA does not inhibit recombination. Little (18) has shown that BU actually promotes a sevenfold increase in recombination in A phage, and Vlachopoulou and Sadowski (30) have reported no inhibition of recombination of BU-substituted T7 DNA in vitro. The conclusion from the above data is that yeast mtDNA replicates semiconservatively with very little concomitant recombination. Some recombination cannot be excluded, and examination of the density tracings does show a skew of some of the mtDNA peaks at the second generation. This could be the result either of recombination or of some degradation of mtDNA. Further work is needed to distinguish between these altematives. However, if recombination does take place, it is most probably not as extensive and as rapid as was assumed previously. ACKNOWLEDGMENTIS We are grateful to D. Dubnau for gifts of ('5NH4)2SO4 and to M. Mandel for a generous supply of SP8 and Cellulomonas sp. DNA. The expert assistance of B. Buchferer is gratefully acknowledged. We also thank J. Marmur for critical readings of the manuscript. J.L. was supported by Public Health Service special fellowahip CA-05627 from the National Cancer Institute. This research was supported by Public Health Service grant CA12410 to J. Marmur from the National Cancer Institute.
LITERATURE CITED 1.
Bankcs, G. R. 1973. Mitochondrial DNA synthesis in
permeable cells. Nature (London) 245:196-199. 2. Bernardi, G. 1976. The mitochondrial genome of yeast: organization and recombination. In T. Bucher, W. Neupert, W. Sebald, and S. Werner (ed.), Genetics and biogenesis of chloroplasts and mitochondria. Elsevier/North Holland Biomedical Press, Amsterdam. 3. Bick, M., and R. Davidson. 1974. Total substitution of bromodeoxyuridine for thymidine in the DNA of a bromodeoxyuridine-dependent cell line. Proc. Natl.
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Acad. Sci. U.SA. 71:2082-2086. 4. Bisson, L, and J. Thorner. 1977. Thymidine 5'-mono-
phosphate-requiring mutants of Saccharomyces cerevisiae are deficient in thymidylate synthetase. J. Bacteriol. 132:44-50. 5. Brendel, L, W. W. Fath, and L Laskowsi. 1975. Isolation and characterization of mutants of Saccharomyces cerevisiae able to grow after inhibition of dTMP synthesis. Methods Cell Biol 11:287-294. 6. Brendel ML, and R. H. Haynes. 1972. Kinetics and genetic control of the incorporation of thymidine monophosphate in yeast DNA. Mol. Gen. Genet. 117:39-44. 7. Chun, E. H. L, and J. W. L{ttlefield. 1961. The separation of the light and heavy strands of bromouracilsubstituted mammalian DNA. J. Mol. Biol. 3:668-673. 8. Cryer, D. R., C. D. Goldthwaite, S. Zinker, K.-B. Lam, E. Storm, R. Hirschberg, J. Blamire, D. B. Finkelstein, and J. Marmur. 1974. Studies on nuclear and mitochondrial DNA of Saccharomyces cerevisiae. Cold Spring Harbor Symp. Quant. Biol. 38:17-29. 9. Flory, P. J., Jr., and J. Vinograd. 1973. 5-Bromodeoxyuridine labeling of monomeric and catenated circular mitochondrial DNA in HeLa cells. J. Mol. Biol. 74:81-94. 10. Goldthwaite, C. D., D. R. Cryer, and J. Marmur. 1974. Effect of carbon source on the replication and transmissions of yeast mitochondrial genomes. Mol. Gen. Genet. 133:87-104. 11. Grigg, G. W. 1977. Selective breakage of DNA alongside 5-bromodeoxyuridine nucleotide residues by high temperature hydrolysis. Nucleic Acids Res. 4:969-987. 12. Grivell, A. R., and J. F. Jackson. 1968. Thymidine kinase: evidence for its absence from Neurospora crassa and some other micro-organims, and the relevance of this to the specific labeling of deoxyribonucleic acid. J. Gen. Microbiol. 54:307-317. 13. Gross, N. J., and ML Rabimowitz. 1969. Synthesis of new strands of mitochondrial and nuclear deoxyribonucleic acid by semiconservative replication. J. Biol. Chem. 244:1563-1566. 14. Hanawalt, P. C. 1967. Preparation of 5-bromouracil-labeled DNA. Methods Enzymol. 12A:702-708. 15. Hewitt, R. R., and K. Marburger. 1975. The photolability of DNA containing 5-bromouracil-1. Single-strand breaks and alkali-labile bands. Photochem. Photobiol. 21:413-417. 16. Hutchinson, F. 1973. The lesions produced by ultraviolet light in DNA containing 5-bromouracil. Q. Rev. Biophys. 6:201-246. 17. Leff, J., and K.-B. Lam. 1976. Bromodeoxyuridine 5'monophosphate incorporation into yeast nuclear and mitochondrial deoxyribonucleic acid. J. Bacteriol. 127:354-361. 18. Little, J. W. 1976. The effect of 5-bromouracil on recombination of phage lambda. Virology 72:530-535. 19. LAu, D. C., and L. 0. Bick. 1977. Determination of 5'bromodeoxyuridine in DNA by buoyant density. Anal. Biochem. 77:346-349. 20. Mattick, J. S., and R. M. Hall. 1977. Replicative deoxyribonucleic acid synthesis in isolated mitochondria from Saccharomyces cerevisiae. J. Bacteriol. 130:973-982. 21. Meselson, M., and F. W. Stahl. 1968. The replication of DNA in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 44:671-682. 22. Michelson, A. J., J. Dondon, and M. Grunberg-Manago. 1962. The action of polynucleotide phosphorylase on 5-halogenouridine-5' pyrophosphate. Biochimn. Biophys. Acta 55:529-548. 23. Pietrzykowska, I., and M. Krych. 1977. Lethal and mutagenic BU-induced lesions in DNA and their repair. Studia Biophys. 61:17-22. 24. Prunell, A., F. G. Goutorbe, F. Strauss, and G. Bernardi. 1977. Yield of restriction fragments from yeast
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mitochondrial DNA. J. Mol. BioL 110:47-52. 25. Reich, EL, and D. J. L Luck. 1966. Replication and inheritance of mitochondrial DNA. Proc. Natl. Acad. Sci. U.S.A. 55:1600-1608. 26. Richards, 0. C., and R. S. Ryan. 1974. Synthesis and turnover of Eugkena graciles mitochondrial DNA. J. Mol. Biol. 82:57-75. 27. Rownd, R. 1969. Replication of a bacterial episome under relaxed controL J. Mol. Biol. 44:387-402. 28. Sena, E., J. Welch, and 8. Fogel. 1976. Nuclear and mitochondrial DNA replication during zygote formation and maturation in yeast. Science 194:433-435. 29. Sena, E. P., J. W. Welch, H. 0. Halvorson, and S. Fogel. 1975. Nuclear and mitochondrial deoxyribonucleic acid replication during mitosis in Saccharomyces
J. BACTERIOL. cerevisiae. J. Bacteriol. 123:497-504. 30. Vlachopoulou, P. J., and P. D. Sadowkik 1977. Genetic recombination of bacteriophage T7 DNA in vitro. m. A physical assay for recombinant DNA. Virology
78:203-215. 31. Wenls, J. R. 1974. Mitochondrial DNA synthesis during the cell cycle of Saccharomyces cerevisiae. Exp. Cell Res. 85:278-286. 32. Wickner, R. B. 1975. Mutants of Saccharomyces cerevisiae that incorporate deoxythymidine-5'-monophosphate into DNA in vivo. Methods Cell Biol. 11:295-302. 33. Wilfanison, D. H., and D. J. FenneL 1974. Apparent dispersive replication of yeast mitochondrial DNA as revealed by density labeling experiments. Mol. Gen. Genet. 131:193-207.
JOURNAL OF BACTERIOLOGY, Aug. 1978, p. 436-444 0021-9193/78/0135-0436$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Replication of Bromodeoxyuridylate-Substituted Mitochondrial DNA in Yeast JUDITH LEFFt* AND T. R. ECCLESHALL Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 Received for publication 14 March 1978
The DNA of several strains of Saccharomyces cerevisiae was labeled by growing the culture in medium supplemented with thymidylate and bromodeoxyuridylate. It was thus possible to follow the course of mitochondrial DNA replication in density shift experiments by determining the buoyant density distribution of unreplicated and replicated DNAs in analytical CsCl gradients. DNA replication was followed for three generations after transfer of cultures from light medium to heavy medium and heavy medium to light medium. Under both conditions, the density shifts observed for mitochondrial DNA were those expected for semiconservative, nondispersive replication. This was further confirmed by analysis of the buoyant density of alkali-denatured hybrid mitochondrial DNA. With this method, no significant recombination between replicated and unreplicated DNA was detected after three generations of growth. It is generally believed that all duplex DNA replication is semiconservative. However, this mode of replication can be obscured in density transfer experiments if hybrid DNA molecules arise which do not have the predicted buoyant density. When this occurs, the DNA is said to replicate dispersively. This phenomenon could be due either to the presence of large precursor nucleotide pools or to extensive recombination between replicated and unreplicated DNA. Previous workers studying the replication of mitochondrial DNA (mtDNA) have obtained buoyant density patterns for newly replicated DNA which seem dependent on the organism studied or on the method of labeling used. In mammalian cells such as rat liver (13) and HeLa cells (9), mtDNA buoyant densities were those expected for semiconservative replication of mtDNA. In Euglena, however, only dispersive patterns were observed; this was attributed to recombination between hybrid DNA strands, possibly accompanied by turnover of the mtDNA (26). The results for Neurospora crassa were interpreted to indicate a semiconservative pattern of replication of mtDNA (25), but the density labeling experiments using 15N showed a large pool of nucleotides in the cells. The presence of this pool made the interpretation of the results more difficult. In cells of Ustilago maydis made permeable to deoxyribonucleotides, no dispersive replication of mtDNA was observed (1). t Present address: 5829 Liebig Avenue, Riverdale, NY 10471.
436
Williamson and Fennel (33), as well as Sena et al. (29), reported that the replication of yeast (Saccharomyces cerevisiae) mtDNA is apparently dispersive as a consequence of extensive and rapid recombination of the mtDNA molecules after replication. However, when mtDNA replication was studied in isolated S. cerevisiae
mitochondria, using bromodeoxyuridine triphosphate, no evidence for recombination was observed (20). These differences may reflect the inherent difficulties in studying the replication of yeast mtDNA. One problem is that yeast mtDNA is unusually susceptible to enzymatic degradation by specific nucleases associated with an mtDNA membrane complex (24). Another difficulty, until recently, was the inability to use radioactive thymidine or its heavy analog, 5-bromodeoxyuridine (BrdU), in studying yeast DNA replication. This is because yeast lacks the enzyme thymidine kinase to phosphorylate the nucleoside for use in DNA synthesis (12). As a result, previous studies in vivo have been carried out using 15N as a density label. The drawback of this method is that the presence of significant pools of nucleotide precursors in the cell may obscure the pattern of mtDNA replication. In recent years several workers (5, 32) have isolated yeast mutants that could incorporate thymidylate (TMP), either because they were auxotrophic for TMP or because de novo synthesis of TMP was inhibited by drugs. These mutants permit the specific labeling of yeast DNA with radioactive TMP or with its heavy analog, bromodeoxyuridylate (BrdUMP). In a previous study (17), the incorporation of BrdUMP into
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yeast nuclear DNA and mtDNA was investigated. In this report, further work is descibed using this method for the study of mtDNA rep-
lication. MATERIALS AND METHODS Yeast strains. A strain auxotrophic for TMP, tnpl (tmpl-6a hisl,7 ilvl-92 lys tiyp [p+J), was obtained from J. Iittle. The wild-type strain D273-1OB was obtained from F. Sherman. Strain cdc7-2, a cell division cycle mutant of A364A (a adel ade2 ural his7 1l lys2gall), wa obtained from the Yeast Genetic Stock Center at Berkeley. Colonies that could incorporate TMP when grown in the presence of drug were selected from strain cdc7 and D273-10B as decribed by Leff and Lam (17). Mi1ds. Strain tpl was grown on complete medium containing 1% Difco yeast extract, 2% Difco peptone, 1.5 mg of KH2P04 per mL 2% ethanoL and 3% glycerol (YPPEG). The pH was adjusted to 5.7. This medium was supplemented before use with TMP (30 pg/mI) (Sigma Chemical Co.) and variable amounts of BrdUMP (7 to 20 pg/ml), synthesized by the method of Michelson et aL (22). Strains D273-10B and cdc7 were grown on a medium described by Brendel and Haynes (6) containing 0.25% Difco yeast extract, 1% Difco peptone, sulfanilamide (Sigma Chemical Co.) (6 mg/ml), opterin (K and K Laboratories) (50 pg/mI), and a carbon source which was 3% glycerol and 2% ethanol for strain D273-10B (SATEG medium) and 2% dextrose for strin cdc7 (SATD medium). The medium used to label DNA with "'N contained 0.117 g of yeast carbon base (Difco) per mL 3% glyceroL and 2% ethanol (CBGE medium), supplemented with 300 pg of ("NH)2S04 (Merck, Sharpe and Dohme, Canada; 96% isotope substitution) per ml. Denity transfer experiments. Transfer of cultures from light medium to heavy medium (upshift experiments) was carred out by growing the cultures in the presence of TMP alone until early exponential phase and then adding BrdUMP. Samples for analysis were withdrawn at various times. Transfer of the cultures from heavy medium to light medium (downshift experiments) were carried out by growing the culture in the presence of BrdUMP and TMP until either midexponentia or statioy phase, washing the ceUls twice with YPPEG medium, and resupendim contining TMP. The ing them in YPPEG labeling of strain D273-1OB with "N was performed by growng a culture to stationary phase in CBGE medium having ("NHO4SO4 as the only nitrogen source. A portion (0.5 ml) of that culture was then used to inoculate a new 10-ml culture. The procedure was repeated three times to ensure that the DNA was fully substituted with 15N. The downshift experiment consisted of tansferring the cells, after washing them with CBGE medium, to fresh medium supplemented with 0.4 mg of (NH4)2S04 per mL This concentration of (NHJ2SO permitted optimal growth of the culture with a doubling time of 90 min at 300C. All cultures were grown at 300C unless specified otherwise. Growth was monitored with a Klett-Summerson photometer equipped with a red filter. DNA isolaon and analysis. DNA was isolated
437
as described by Goldthwaite et al. (10), using zymolyase (Kirin Brewery Co.) to prepare spheroplasts. Since BrdUMP-containing DNA is very susceptible to degradation by 330-nm light (16), the isolation procedure was carried out exercising great care to avoid exposure to fluorescent light. In addition, the DNA was analyzed by analytical centrifugation as soon as possible after isolation because BrdUMP-containing DNA is unstable and because yeast mtDNA degrades on storage. It was more appropriate to prepare DNA from whole cells than from isolated mitochondria because it is impossible to prepare intact yeast mitochondria and to free mitochondria of nuclear DNA without extensive DNase treatment. Preparation of bromouracil (BU)-substituted mtDNA from mitochondrial preparations yielded mtDNA of variable quality. Analytical density gradient centrifugation was carried out in a Spinco model E ultracentrifuge at 44,770 rpm for at least 16 h at 250C as described by Meselson and Stahl (21). UV photographs were scanned with a Joyce-Loebl microdensitometer. Bacillus subtilis phage SP8 DNA (p = 1.742 g/ml) and Celulomonas sp. DNA (p = 1.734 g/ml) were used as density markers. Single-standed DNA was obtained by alkali denaturation and neutralization of native DNA; 0.01 ml of 10 N NaOH was added to 0.35 ml of DNA solution and neutrlized after 2 min with 0.05 ml of 2 N HCl in 1 M tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.5). The DNA was then examined in a neutal CsCl grdient uXsin phage SP8 DNA as a density marker.
RESULTS Labeling with BrdUMP. To achieve maximum separation between replicated and unreplicated DNA in the presence of BrdUMP, complete substitution of thymine by BU would be desirable, as has been achieved with mammalian cells (3). However, strain tmpl reverted to prototrophy after several generations of growth in YPPEG medium supplemented only with BrdUMP. However, for at least two generations BrdUMP was incorporated into newly synthesized DNA as shown in Fig. 1 for an ethidium bromide-treated derivative of strain tmpl, ie., a derivative lacking mtDNA. After 4 h, a time corewponding to one generation, the majority of the nuclear DNA banded at a density of 1.755 g/ml. This value is that expected for hybrid DNA (HL) in which one strand is fully substituted with BU (19). (The substituted DNA
strand is designated H and the unsubstituted strand is designated L.) Unsubstituted nuclear DNA (LL), having a buoyant density of 1.698 g/ml, was still present at this time. Growth of cells in the presence of BrdUMP and in the absence of TMP normally leads to a lag in DNA replication. After 6 h of growth, nuclear DNA banded at densities of 1.755 and 1.812 g/ml, corresponding to hybrid (HL) and fully substi-
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ments. Strain tmpl was grown in YPPEG medium supplemented with TMP, and then BrdUMP (7 ,ug/ml) was added at early logarithmic phase. Samples were taken at 0, 1, 2, 4, 8, and 12 h following the addition of BrdUMP. Analytical density gradient centriftgation of the isolated DNA gave the CsCl density gradient profiles shown in Fig. 2 and 3. The yield of mtDNA relative to nuclear DNA is unusually high using the growth conditions and the isola4 tion method described in Materials and Methcr ~~~~~~~6ods. This is important because it permits an 0 improved resolution of the DNA species. After growth for only 1 h in the presence of BrdUMP, BrdUMP substitution in the DNA was clearly visible. Both nuclear and mtDNA showed distinct bands at the densities corresponding to hybrid DNA (HL). This rapid incorporation of the nucleotide analoges indicated an extremely small pool of TMP precursors, and it was especially evident in strain tmpl, which lacks both thymidine kinase and thymidine synthetase (4). After 2 h of growth following the addition of 1.811 734 1.755 1.698 BrdUMP, the hybrid bands were well separated from the corresponding unsubstituted DNA speDENSITY (g/cc) cies. The sharpness of the peaks and their resoFIG. 1. Buoyant density profiles of the DNA of an lution were such as to permit the conclusion that ethidium bromide derivative of strain tmpl grown in for both DNA species there was very little, if YPPD medium in the presence of 30 pg of TMP per any, DNA of intermediate buoyant density beml and transferred to a medium containing 20 jg of tween the light (LL) and hybrid (HL) species. BrdUMP per ml. Samples were taken at 4 and 6 h Figure 3 shows the incorporation of BrdUMP after the transfer. The reference is Celulomonas sp. into yeast DNA at various times up to three DNA, which has a buoyant density of 1. 734 g/ml. generations. It is interesting to note, as reported by Wells (31), that mtDNA replication was completed sooner than that of nuclear DNA. After tuted (HH) nuclear DNA, respectively. Unfortunately, in the case of mtDNA, which 8 h of growth (corresponding to two generahas long adenine-thymine-rich regions (2), ex- tions), the mtDNA peak was at the heavy dentensive BrdUMP substitution causes extensive sity (1.696 g/ml), with a slight skewing towards degradation of the genome. Moreover, since the hybrid density. Nuclear DNA was still mtDNA has a higher content of adenine-thy- mostly at hybrid buoyant density (1.705 g/ml). mine than nuclear DNA, growth of cells in in- The presence of a small peak of light unreplicreasing concentrations of BrdUMP causes the cated mtDNA after the first doubling was not buoyant-density mtDNA to increase at a faster the result of a nonreplicating fraction the rate than that of nuclear DNA (17). As a result, mtDNA molecules, as will be shown below. Unthe difference in density between the two DNA replicated DNA has been reported in other studspecies becomes smaller until it becomes diffi- ies involving incorporation of BrdUMP into cult to distnguish them in CsCl gradients. Con- DNA (7, 14). The most likely explanation is that sequently, the percentage of BrdUMP substitu- a small fraction of the mitochondria did not tion (in the range of 11 to 12%) used in the utilize BrdUMP. following e'xperiments was the most convenient mtDNA replication in downshift experifor our present studies. This degree of substitu- ments. In the downshift experiments, cells were tion was obtained by growing the celLs in YP- first grown to stationary phase in YPPEG mePEG medium in the presence of 30 ig of TMP dium containing both TMP and BrdUMP. and 7 Ag of BrdUMP per ml. This concentration These conditions were previously shown to inof BrdUMP causes very little growth inhibition, crease the proportion of mtDNA relative to nusince the culture can grow to a cell density of 2 clear DNA (8). The culture was then transferred x 108 cells per ml with a doubling time of 4 h at to YPPEG supplemented only with TMP. The 300C. tracings in Fig. 4 summarize the results of sammtDNA replication in upshift experi- ples taken up to 6 h after the transfer. Up to 2
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REPLICATION OF YEAST MITOCHONDRIAL DNA
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cept that some unreplicated mtDNA was still present. After an additional 2 h, most of the mtDNA banded at its hybrid density. There was no evidence of any remaining heavy mtDNA. This lends support to the conclusion that all the mtDNA molecules replicate (33), so that the persistence of the light peak in the upshift experiments must be because some mitochondria cannot utilize BrdUMP. This set of experiments
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DENSITY (g/cc) FIG. 2. Buoyant density profiles of the DNA of a culure ofbnpl grown in the presence of 3Opg of TMP and 7 pg of Brd UMP per ml. Samples were taken at 0,1, and 2 h after the addition of Brd UMP. The shoulder on the hcavy side of the nuckar DNA at zero time represents the y sateUite band. Nuclear DNA bands are at 1.698 glml (LL) and 1.705 g/ml (HL). mtDNA bands are at 1.682 g/ml (LL) and 1.689
g/ml (HL). The reference is B. subtiis phage SP8 DNA, which has a buoyant density of 1.742 g/ml
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h after the transfer to light medium, there was FIG. 3. Buoyant density tracings of DNA isolated no change in the DNA buoyant densities befrom a culture of tnpl grown for 4, 8, and 12 h in cause cells emerging from, the stationary phase medium containing TMP and BrdUMP. The referexperience a time lag in their growth. After 4 h, ence DNA was from phage SP8. Nuclear DNA bands most of the nuclear DNA banded at a hybrid are at 1.698 glml (LL), 1.705 g/ml (HL), and 1.711 density but having a shoulder at heavier buoyant g/ml (HI). mtDNA bands are at 1.683 glml (LL), density. mtDNA showed a similar pattern, ex- 1.689 g/li (HL), and 1.696 glml (H11.
440
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LEFF AND ECCLESHALL
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DENSITY ( g/cc) FIG. 4. Buoyant density profiles of DNA from a culture of tmpl grown in the presence of TMP and BrdUMP for six generations and transferred to light medium. Samples were taken at 2,4, and 6 h after the transfer. The reference was phage SP8 DNA. Nuclear DNA bands are at 1.698g/ml (LL), 1.705g/ml (HL), and 1.711 g/ml (HH). mtDNA bands are at 1.683g/ml (LL), 1.689 g/ml (HL), and 1.696 g/ml (HI).
again points out the parallels between nuclear and mtDNA replication. In both cases, whether in upshift or in downshift experiments, the buoyant densities of the DNA are distributed among bands corresponding in density to light hybrid and heavy DNA, in accordance with a mode of replication that is semiconservative. The lack of dispersion indicates that the use of BrdUMP
overcomes any posiible pool problem and that little detectable recombination occurs between replicated and unreplicated mtDNA genomes. Density of denatured replicated DNA. Separation of the strands of DNA of hybrid density (HL) provided another means to detect the extent of recombination after semiconservative replication. DNA samples from the upshift experiments described in Fig. 2 and 3 (0 and 2 h and 8 h) were denatured as described in Materials and Methods. Figure 5 shows the DNA profiles after analytical centrifugation in neutral CsCl gradients. Two hours after the addition of BrdUMP to the culture, mtDNA gave rise to two peaks, the light peak banding at 1.696 g/ml, corresponding to the buoyant density expected for denatured light DNA, and the heavier peak banding at 1.703 g/ml. After 3 h of growth in the presence of BrdUMP, the resolution of the heavy mtDNA strand from the light mtDNA strand was very clear. The buoyant density of the heavy strand of mtDNA (1.703 g/ml) was significantly lower than that expected for mtDNA with 11% BrdUMP substitution (1.709 g/ml). However, in some experiments in which cultures were grown for five generations or more in the presence of BrdUMP, the buoyant density of single-stranded mtDNA was 1.709 g/ml. The lower buoyant density of the substituted mtDNA strands encountered in most experiments might be due to the presence of a region of neighboring BU bases which were preferentially degraded during alkali denaturation as the result of BU-induced single-strand breaks. Moreover, these observations, as well as those of others (15, 23), suggest that BrdUMP substitution in DNA increases its lability to alkaline treatment. It could also be argued that the density of 1.703 g/ml represents an intermediate density between the light and heavy strands due to recombinational events. This is not likely, however, for the following reasons. First, native mtDNA had buoyant densities expected for DNA replicating semiconservatively. If some recombination had occurred, the amount of the recombined DNA was too small to be detected in the buoyant density tracings of undernatured DNA. Consequently, the large peaks observed after denaturation of the hybrid mtDNA (HL) were unlikely to represent recombined strands of DNA. Moreover, extensive recombination between stands of hybrid DNA (HL) would result in DNA molecules in which both strands would be BU substituted. Denaturation of this DNA would yield two strands with very similar buoyant densities which would probably yield a single band in CsCl density gradients. The buoyant density of this band would have a value intermediate between those expected for unsubsti-
REPLICATION OF YEAST MITOCHONDRIAL DNA
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BrdUMP, with derivatives of the strains D273lOB and cdc7-2 selected for TMP uptake (17). The strains were grown as described in Materials and Methods. The results with both strains were similar to those observed with strain tmpl. Figure 6 shows the density profile of D273-1OB 3 and 6 h after the addition of BrdUMP. The generation time for strain D273-1OB under these conditions was 8 h. mtDNA banded at discrete values corresponding to unsubstituted DNA and to hybrid DNA. Denaturation of the DNA yielded one light and one heavy band as described for strain tmpl. Density labeling with 1N. It is usually assumed that incorporation of reasonable amounts of BrdUMP into DNA does not affect replication
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DENSITY (g/cc) FIG. 5. Buoyant density profies of analytical centrifiugation in neutral CsCI of alkali-denatured DNA. DNA was isolated from samples taken from tmpl cultures grown for 0, 2, and 8 h in the presence of BrdUMP and TMP. The reference was phage SP8 DNA. Nuclear DNA bands are at 1.711 g/ml (L) and 1.727 g/mi (H). mtDNA bands are at 1.696 g/ml (L) and 1.703 g/ml (H).
tuted (L) and BU-substituted (H) singlestranded DNA molecules. In our experiments, however, the light and heavy mtDNA strands were well separated, and their buoyant density remained essentially invariant (± 0.002 g/ml) throughout the course of replication of mtDNA. mtDNA replication in other strains. We have also studied DNA replication for one generation in upshift experiments in the presence of
DENSITY (g/cc) FIG. 6. Buoyant density profile of DNA from a culture of strain D273-IOB grown in SATEG medium and containing 25 pg of TMP and 7 pg of BrdUMP per ml. Samples were taken at 3 and 6 h after the addition of BrdUMP. The reference was phage SP8 DNA. Nuclear DNA bands are at 1.698 g/ml (LL) and 1.704g/ml (HL). mtDNA bands are at 1.683g/ml (LL) and 1.689g/ml (HL).
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(11). Nevertheless, we compared the results of BrdUMP and '5N labeling by growing strain D273-1OB in the presence of ("5NH4)2SO4. This experiment could not be performed with tmpl because it would have required '5N-substituted TMP, which was not available. The results of the downshift experiment with 16N-labeled DNA are shown in Fig. 7. After 1.5 h (equivalent to one generation) in light medium, half of the nuclear and most of the mtDNA still banded at the heavy density. After 4.5 h in the light medium, a significant proportion of the nuclear DNA was still at the heavy density, whereas most of the mtDNA was unsubstituted. Only a small fraction of mtDNA remained at hybrid density (1.689 g/ml). Nevertheless, it is apparent that mtDNA banded at buoyant density values corresponding successively to heavy, hybrid, and light DNA. The separation of the peaks was not as distinct as with BrdUMP-labeled DNA, but there was clearly no single band with progressively decreasing buoyant density, as would be expected if extensive recombination had occurred. Denaturation of 15N-labeled mtDNA yielded two bands with the expected buoyant densities for light (1.696 g/ml) and heavy (1.710 g/ml) strands. No detectable DNA of intermediate buoyant density was observed. In addition, this experiment also confirmed that all the mtDNA molecules replicate. DISCUSSION In previous experiments with 15N-labeled DNA, replication mtDNA appeared as a single unimodal peak whose buoyant density decreased continuously during growth of the culture following the transfer from heavy to light medium (33). Denaturation of the mtDNA also yielded a single peak of a buoyant density intermediate between those expected for unsubstituted and fully substituted mtDNA strands. These results were ascribed to extensive and very rapid recombination between replicated and unreplicated mtDNA molecules. The differences between these observations and those described in the report may be explained by the different methods used in labeling and isolating DNA and perhaps by the use of different strains. Previous experiments using '5N as a label were performed in a defined medium in which nitrogen was in growth-limiting amounts. The yield of mtDNA from such suboptimaly grown cultures was quite small and, moreover, yielded a broad band in CsCl gradients. As a result, it was difficult to discem discrete peaks of different buoyant densitites even if they were present. On the other hand, cultures labeled with BrdUMP can be grown on complete medium with a nonferment-
J. BACTERIOL.
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DENSITY (g/cc) FIG. 7. Buoyant density profile of DNA from a culure of D273-IOB grown in the presence of (l5NH)2SO4 and transferred to light mediium. Samples were taken at 4, 8, and 12 h after the transfer. The reference was phage SP8 DNA. Nuclear DNA bands are at 1.698 g/ml (LL), 1.706 g/ml (HL), and 1.712 g/ml (HH). mtDNA bands are at 1.683 g/ml (LL), 1.689g/ml (HL), and 1.696g/ml (HH).
able carbon source, which greatly increases the yield of mtDNA (10). Consequently, in spite of unavoidable degradation during isolation, mtDNA is obtained in amounts that are sufficient to permit good resolution ofdifferent buoyant density species. Another difference between the two methods can be attributed to the nature of the label. In most cases, labeling with BrdU
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REPLICATION OF YEAST MITOCHONDRIAL DNA
is the preferred method for studying DNA replication because it is specific for DNA and is rapidly incorporated via the salvage pathway. This is especially true for yeast stains auxotrophic for TMP that lack both thymidine kinase and thymidylate synthetase. Consequently, BrdUMP is incorporated as rapidly as 30 min after the addition of BrdUMP (0. Goldberg and J. G. Little, personal communication). However, in the experiments reported here (Fig. 7), as well as those reported by others (28, 33), incorporation of '4N into DNA during the downshift experiment shows a lag of variable duration due to the fact that inorganic "N has to be converted into deoxyribonucleotides before being incorporated into DNA. Moreover, presumably because of pool effects, heavy labeled nuclear DNA still persists after the third generation in light medium. It is also worth noting that BU-substituted DNA does not inhibit recombination. Little (18) has shown that BU actually promotes a sevenfold increase in recombination in A phage, and Vlachopoulou and Sadowski (30) have reported no inhibition of recombination of BU-substituted T7 DNA in vitro. The conclusion from the above data is that yeast mtDNA replicates semiconservatively with very little concomitant recombination. Some recombination cannot be excluded, and examination of the density tracings does show a skew of some of the mtDNA peaks at the second generation. This could be the result either of recombination or of some degradation of mtDNA. Further work is needed to distinguish between these altematives. However, if recombination does take place, it is most probably not as extensive and as rapid as was assumed previously. ACKNOWLEDGMENTIS We are grateful to D. Dubnau for gifts of ('5NH4)2SO4 and to M. Mandel for a generous supply of SP8 and Cellulomonas sp. DNA. The expert assistance of B. Buchferer is gratefully acknowledged. We also thank J. Marmur for critical readings of the manuscript. J.L. was supported by Public Health Service special fellowahip CA-05627 from the National Cancer Institute. This research was supported by Public Health Service grant CA12410 to J. Marmur from the National Cancer Institute.
LITERATURE CITED 1.
Bankcs, G. R. 1973. Mitochondrial DNA synthesis in
permeable cells. Nature (London) 245:196-199. 2. Bernardi, G. 1976. The mitochondrial genome of yeast: organization and recombination. In T. Bucher, W. Neupert, W. Sebald, and S. Werner (ed.), Genetics and biogenesis of chloroplasts and mitochondria. Elsevier/North Holland Biomedical Press, Amsterdam. 3. Bick, M., and R. Davidson. 1974. Total substitution of bromodeoxyuridine for thymidine in the DNA of a bromodeoxyuridine-dependent cell line. Proc. Natl.
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