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Current Genetics 3, 229-233 (1981)

© Springer-Verlag 1981

Newly Synthesised DNA of High Molecular Weight in the Yeast Saccharomyces cerevisiae Leland H. Johnston Division of Microbiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, Great Britain

Summary. Two species of newly synthesised DNA larger than average replicons have been found in yeast. Their molecular weights are 60 million and 90 million daltons respectively. The exact nature of these molecules is not certain. They may represent entirely novel species of cellular DNA or they could be concatameric replication intermediates of some particular fraction of DNA, such as mitochondrial DNA or rDNA. Alternatively they could result from the fusion of adjacent completed replicons in a small cluster. K e y words: Yeast - Nascent DNA

Introduction We have previously used pulse-labelling of cells followed b y sedimentation in alkaline sucrose gradients to characterise newly sythesised DNA in the yeast S. cerevisiae. Broadly speaking, nascent chromosomal DNA fails into two size classes, small pieces of some 400 nucleotides in size (Johnston and Nasmyth 1978), presumably Okazaki fragments, and a heterogeneous collection o f molecules between 10 and 60 million daltons in size (Johnston and Williamson 1978). This latter class is in the size range of yeast replicons (Petes and Williamson 1975; Rivin and Fangman 1980) and a comparison of their behaviour in log phase and synchronous cultures supports the notion that they are indeed replicons. During the course o f these studies discrete classes of newly synthesised DNA larger than average replicons were noted and further observations on these molecules are described here.

Materials and Methods Strains and Cultural Conditions. The haploid strain D273-11a has been described previously (Johnston and Game 1978). The haploid cdc9 alleles are in an A364A genetic background (Hartwell et al. 1973). Strain g676 (a/~ cdc9-1/cdc9-1 canS/ canr +/hom3-10 hisl-1/hisl-7 +/lys2 tyrl/+ ade2/+ ura/Ura +/ leu2) was a gift from Dr John Game. Liquid cultures were incubated with shaking in YEPD medium (Johnston and Game 1978) at a temperature of 25 °C. For meiotic cultures g676 was grown in YEPA medium, YEP containing 1% potassium acetate, to 107 cells/ml at a temperature of 21 °C. The cells were then filtered, washed twice with 1% potassium acetate, resuspended at 107 cells/ml in 1% potassium acetate buffered with morpholinopropane sulphonic acid pH 6.5 (McCusker and Haber 1977) and incubated at 21° with vigorous shaking. Radioactive Labelling of Cells for Sedimentation. Cells were grown in YEPD containing 0.07 /~Ci (2.6 kBq)/ml [2-14C] uracil (Amersham; 62 mCi (2.3 GBq)/mmol) for approximately 20 h to 107 ceUs/ml. They were centrifuged, resuspended in 1 ml of fresh YEPD and [8-3H] adenine (Amersham; 22 Ci (0.8 TBq)/mmol) was then added and the cells were labelled for the required period. This was terminated by the addition of an equal volume of stop-mix (Johnston and Williamson 1978) and the cells were placed on ice. Labelling of meiotic ceils (see Fig. 4) was terminated by rapid filtration and placing the cells in 5 ml of ice-cold 0.5 M sodium thioglycollate in 0.1 M Tris-HC1 pH 8.9 (Jacobson et al. 1975). Alkaline Sucrose Gradient Sedimentation. Full details of these procedures have been described elsewhere (Johnston and Willlamson 1978; Johnston 1980) and only the salient points are mentioned here. The cells in stop-mix or thioglycoUate were washed and treated with zymolase to remove their cell wails. The resultant spheroplasts were lysed in alkaline sarkosyl and the lysate was heated to ensure complete denaturion of the DNA before loading onto 5-20% alkaline sucrose gradients with a 0.5 ml cushion of 80% sucrose. The gradients were centrifuged in a Beckman SW40 rotor at 19,000 rpm at 20 °C for 16 h. Fractions were collected by suction from the bottom of the gradient, dripped into tubes, made 0.5 N with NaOH and after O172-8083/81/0003/0229/$01.00

230

L.H. Johnston: Newly Synthesised DNA in Yeast

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Fraction No. Fig. 1. Alkaline sucrose gradient analysis of pulse-labelled D273-11a to show the DNA sedimenting to fraction 11. Mid-log phase ceils prelabelled with 14C-uracil were resuspended in 1 ml of fresh YEPD at 8 x 107 cells/rot. 50 ~zCiof 3H-adenine was added and pulses were terminated at the times shown. The DNA was extracted and sedimented on alkaline sucrose gradients as described in Methods. Sedimentation is from right to left. The total 14C-label recovered per gradient was 618 cpm, 2,217 cpm and 2,483 cpm respectively, while the 3H-label recovered was 1,528 cpm, 17,827 cpm and 29,251 cpm. o 14C-label; • 3H-label

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incubation at 37 °C for 24 h the alkali-stable, acid-precipitable radioactivity was determined. Molecular weights were determined using the equation of Levin and Hutchinson (1973) with a coefficient c~ of 0.36 (Johnston and Williamson 1978). Phage ~ DNA was used as the marker.

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edc9-7. Procedure as in the legend to Fig. 1 b u t after resuspension in 1 ml of YEPD the cells were incubated at 37 °C for 10 min and t h e n labelled with 25 #Ci of 3H-adenine for 30 min at this temperature. Sedimentation is f r o m right to left. The total 14C4abel was 1857 cpm while the 3H4abel was 5,241 cpm.Inset: shows t h e 3H-label f r o m the b o t t o m 19 fractions plotted as absolute counts per rain. o 14C-label; • 3H-label

In the experiments reported here the DNA of the cells was labelled with ~4C for many generation before sedimentation. This prelabel was used to monitor the recovery of DNA from the gradients and also to indicate the size of the parental strands. The synthesis of new DNA was then followed by the use of shorter periods of labelling with 3H (pulse-label) and then observing the increase in size of this pulse-labelled DNA to that of the prelabel. In the course of such experiments a small peak of 3H-labelled DNA has frequently been observed in fraction 11 of the alkaline sucrose gradients (Fig. 1). It was a minor species o f newly-synthesised DNA, never accounting for more than a small percentage of the total pulse-label and has never been apparent as a peak in the prelabelled DNA. It was not specifically associated with any particular length of pulse but was more clearly evident in short pulses (Fig. 1A), and tended to be less apparent in longer pulses. Single-stranded DNA o f phage T4 sediments to fraction 11 in these gradients so that DNA in this fraction has a molecular weight of some 60 million daltons. The conditional cell cycle mutant cdc9 is defective in DNA ligase (Johnston and Nasmyth 1978). Since it was possible that this ligase could be involved in the me-

L. H. Johnston: Newly Synthesised DNA in Yeast

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10 Fraction No. Fig. 5. Accumulation of pulse-labelled DNA in fraction 11 in edc9 during premeiotic DNA synthesis. A meiotic culture of g676 was incubated at 21 °C for 5 h, approximately mid-way through premeiotic DNA synthesis, then transferred to 34 °C for 45 min and returned to 21 °C. The culture was then divided into 4 sub-cultures of 5 ml each and pulse-labeUed with 20 ~Ci/ml 3H-adenine for 30 min, (A) immediately, (B) after 30 rain, (C) after 60 min and (D) after 90 min further incubation. The DNA was extracted as described in Methods and sedimented on alkaline sucrose gradients, Sedimentation is from right to left and only the label in the botton 16 fractions is shown. Inset: shows the proportionate increase in label in fractions 9 - 1 3 as a percentage of the total label in all fractions of each gradient

03

10 Fraction No. Fig. 4. Average 3H-cpm from the first 16 fractions of 10 gradients

prepared from cells treated with HU. Legend as in Fig. 3

tabolism of the DNA in fraction 11, alleles of cdc9 were examined for any effects on this species of DNA. There was a small peak in fraction 11 in gradients of cdc9 cells pulse-labelled at the restrictive temperature (Fig. 2; see also Fig. 1A in Johnston and Nasmyth, 1978). Although it rarely exceeded 2% of the total 3H-label it was very clearly evident when the absolute aH-cpm were plotted (Fig. 2, inset). Moreover, it was a reproducible feature of all the alleles of cdc9 pulselabelled at 37 °C. Out of 15 such gradients from various

experiments, eleven had a peak in fraction 11 and three in fraction 12 and only one showed no peak in this region of the gradient. When the data from all fifteen of these gradients was pooled and the average 3H-cpm in each of the bottom 16 fractions calculated, a peak in fraction 11 was clearly evident (Fig. 3). Other treatments wich reduce the amount of pulselabel sedimenting to the bottom of the gradient also revealed the DNA in fraction 11. For instance, after treatment of cells with relatively high concentrations of hydroxyurea (HU) these molecules were clearly evident (Johnston 1980) and in Fig. 4 pooled data from 10 such gradients are shown. The relative prominence of this DNA in these HU treated cells, and in cdc9 labelled at 37°C, does not necessarily represent an abnormal quantity of this DNA. The normal complement in the cells may simply have been revealed by the reduced label in adjacent fractions. Attempts to increase the proportion of all-label in fraction 11 by treatment of cells with different concentrations of HU or by varying the temperature stress of cdc9 cells before or during pulse-labelling were not fruitful. The only slight success was with a cdc9 homozygous

232 diploid undergoing meiosis. If the ligase was inactivated by transferring meiotic cells of cdc9 to the restrictive temperature, on return to the permissive there was only a very slow regeneration of the enzyme activity (manuscript in preparation). If the culture was pulse-labelled at successive intervals during this regeneration, there was a slight accumulation of 3H-label in fraction 11 (Fig. 5). Incidentally this experiment confirmed that the molecules sedimenting to this fraction were completed entitites since the peak of pulse-label remained in the same fraction throughout the two hour period of the experiment. As well as the DNA in fraction 11, another minor species was frequently detected in fraction 9. This was evident as a shoulder or as a distinct peak in gradients where there was little label in adjacent fractions, such as after short pulses or in cdc9 labelled at 37 ° or in cells treated with HU (Fig. 1A, 2, 3, 4), although it was not always apparent. For example, in the fifteen gradients of cdc9 referred to above, nine had either a peak or shoulder in fraction 9, while of the ten gradients from HU-treated cells in Fig. 4, eight had a peak or shoulder in this fraction. Nevertheless, this species was evident when the pooled data from the bottom sixteen fractions of these gradients was considered (Figs. 3 and 4). This suggests it may be a normal, if very minor, component of nascent cellular DNA which tends to be obscured in gradients. The molecular weight of DNA sedimenting to fraction 9 is 90 million daltons.

Discussion This paper demonstrates the existence of previously underscribed classes of newly synthesised single-stranded DNA molecules, one of 60 million daltons and possibly a second of 90 million daltons. Although not detectable in every gradient, they are fairly reproducible under defined conditions. For example, out of 25 gradients of cdc9 and HU-treated cells, 24 showed a peak corresponding to 60 million daltons and 17 had a peak or shoulder in fraction 9, corresponding to 90 million daltons. It therefore seems reasonable to conclude that these molecules are significant species of newly synthesised DNA. Their exact nature, however, is uncertain. They are not simply a pathological consequence of incubating cdc9 at 37 °C or of treating cells with HU as they were present in untreated wild type cells (Fig. 1). They may be previously undetected molecules, such as plasmids, although then they might be expected to be evident in the 14C-prelabelled parental DNA and this was not the case. There was a relatively high proportion of 14C-labelled DNA in this part of the gradient, however, which might obscure a discrete minor species. The possibility that they may be novel species of DNA cannot

L.H. Johnston: Newly Synthesised DNA in Yeast therefore be excluded. In fact a number of non-Mendelian elements have been detected genetically in yeast for which no nucleic acid determinant is known (Cox 1965; Aigle and Lacroute 1975; Schamhart et al. 1975; Wickner 1980) and one or other of these could be the DNA observed in fractions 11 or 9 In general, however, plasmids are considerably smaller than 90 or even 60 million daltons (equal to 180 and 120 million daltons double-stranded) but these molecules could be concatameric replication intermediates of a smaller DNA species. Conceivably, yeast mitochondrial DNA might replicate in this way or rDNA may be amplified by this means, as occurs with rDNA in certain higher cells (Long and Dawid 1980). Whether yeast rDNA is amplified at all is not known. Indeed little is known about replication of rDNA in yeast and these molecules could be replicons of unique size derived from this DNA, but 60 and 90 million daltons are considerably larger than average yeast replicons (Petes and Williamson 1975 ; Rivin and F angman 1980; Newlon and Burke 1980). These molecules could, however, result from the joining together of adjacent completed replicons in a small cluster, as occurs in higher cells (for a review see Hand 1978). Since average yeast repllcons are 30 million daltons, clusters of two or three would neatly account for the DNA in fractions 9 and 11. Against this is the fact that the DNA in these fractions is of discrete sizes and replicons have a relatively wide size range (Petes and Williamson 1975; Johnston and Williamson 1978) but possibly 60 and 90 million daltons define the size of a cluster, and the number of replicons in one is variable. A more telling criticism of this interpretation, however, is the re-producible occurrence of this DNA in situations where their putative precursors, the replicons, are absent (Figs. 2, 3, 4). In summary, the existence of single-stranded molecules of 60 and 90 million daltons in the newly synthesised DNA of yeast seems quite clear. Attempts to investigate the nature of these molecules are complicated by the occurrence of somewhat large amounts of parental, non-specific, DNA in this region of the gradient, so for the time being their precise nature remains uncertain. Acknowledgements. I am grateful to John Game for providing strain g676 and to Don Williamson for critical reading of the manuscript.

References Aigle M, Lacroute F (1975) Mol Gen Genet 136:327-335 Cox BS (1965) Heredity 20:505-521 Hand R (1978) Cell 15:317-325

L. H. Johnston: Newly Synthesised DNA in Yeast HartweU LH, Mortimer RK, Culotti J, Culotti M (1973) Genetics 74:267-286 Jaeobson GK, Pinon R, Esposito RE, Esposito MS (1975) Proc Natl Aead Sci USA 72:1887-1891 Johnston LH (1980) Curr Genet 2:175-180 Johnston LH, Game JC (1978) Mol Gen Genet 161:205-214 Johnston LH, Nasmyth KA (1978) Nature 274:891-893 Johnston LH, Williamson DH (1978) Mol Gen Genet 164:217225 Levin D, Hutchinson F (1973) J Mol Bio175 :495-502 Long EO, Dawid IB (1980) Ann Rev Biochem 49:727-764 McCusker JH, Haber JE (1977) J Bacteriol 132:180-185 Newlon CS, Burke W (1980) In: Alberts B, Fow CF (eds) Meehamistic studies of DNA replication and genetic recombina-

233 tion. ICN-UCLA Symposium on Molecular and Cellular Biology, Vol 19. Academic Press, New York, pp 399-409 Petes TD, Williamson DH (1975) Exp Cell Res 95:103-110 Rivin CJ, Fangman WL (1980) J Cell Biol 85:108-115 Schamhart DHJ, Ten Berge AMA, Van De Poll KW (1975) J Bacteriol 121:747-752 Wickner RB (1980) Cell 21:217-226

Communicated by B. S. Cox Received April 1, 1981

Newly synthesised DNA of high molecular weight in the yeast Saccharomyces cerevisiae.

Two species of newly synthesised DNA larger than average replicons have been found in yeast. Their molecular weights are 60 million and 90 million dal...
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