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A Partially Single-stranded Intermediate Occuring during Synthesis of Nuclear DNA in Chlorella
Biochem. Physiol. Pflanzen 174, 215-222 (1979)
A Partially Single-stranded Intermediate Occuring during Synthesis of Nuclear DNA in Chlorella VOLKER
SSY~IANK
Pflanzenphysiologisches Institut, Universitat, D-3400 Giittingen, FRG Key Term Index: DNA synthesis, nuclear DNA, uridine, guanosine; Chiarella pyrenoidosa
Snmmary Pulse labelling of Chiarella DNA by incubation with uridine or guanosine resulted in labelled molecules with a higher buoyant density than the bulk of nuclear DNA. No incorporation into chloroplast D~A could be observed. The label could be chased into mature nuclear DNA indicating that the labelled molecules represent an intermediate in the synthesis of nuclear DNA. The labelled molecules have a sedimentation coefficient of less than 7 S, smaller than procaryotic Okazaki pieces. In contrast to these, they do not contain RNA segments. The observed density difference is not due to posterior methylation of the labelled intermediate molecules. Two factors contribute to their high density: The labelled intermediate molecules contain single-stranded regions, and the precursors nridine and guanosine preferentially enter (GC)-rich portions of the DNA.
Introduction
Uracil and uridine are incorporated by cells of the green alga Chlorella pyremoidosa not only into RKA, but also rather rapidly into DNA (WANKA et al. 1970; SsnIANK 1975). Presumably they enter DNA as a 2'-deoxycytidine nucleotide (PROllST et al. 1974). Pulse labelling experiments with uridine or with guanosine which is incorporated into DNA more readily always showed a buoyant density of the labelled molecules of 1. 721 g/cm 3 compared to the bulk of nuclear DNA with a density of 1. 710 g/cm3 ; chloroplast DXA (density: 1.689 g/cm 3 ) remained unlabelled up to 24 h incubation time (see e.g. SSY)fANK 1975, Fig. 1). The question now arises whether the labelled molecules arc intermediates in the course of the synthesis of nuclear DNA, and if they are, due to which reason they have a higher density. In the following investigations it is shown that uridine and guanosine are incorporated into nuclear DNA of Chlorella via an intermediate of higher buoyant density. Four possibilities to explain the high density are investigated: (i) RNA fragments might be connected with the intermediates of DNA synthesis like in procaryotic Okazaki pieces; (ii) intermediate molecules might become methylated and thus be shifted to a lower density; (iii) the intermediate molecules might contain single-stranded regions; (iv) uri dine or guanosine preferentially enter (GC)-rich portions of the DNA. Material and Methods Culture of algae The experiments described were carried out with the unicellular green alga Chiarella pyrenaidasa, strain 211-8 b from the algal collection of the Institute for Plant Physiology, Giittingen, FRG. The algae were grown at 30°C under sterile conditions as described by KUHL and LORENZEN (1964).
216
V. SSYMANK
Labelling procedures were described previously (SSYMANK 1972, 1973). The following radioactive precursors were used: [5-3H]uridine (30 Ci/mmol), [8-3H]guanosine (15 Ci/mmol), L-[methyPH]methionine (15 Ci/mmol) and [methyPH]thymidine (60 Ci/mmol), all obtained from the Radiochemical Centre, Amersham, U.K. Myxin (a gift from Dr. P. F. LURQUIN, Mol, Belgium, and Dr. R. M. BEHKI, Ottawa, Canada) was dissolved in methanol and added to the culture medium at 1pgl ml; the final concentration of methanol was less than 0.2 %. Preparation and fractionation of DNA
Nucleic acids were extracted with phenol as described (SSYMANK 1973) and from these preparations DNA was released by treatment with RNase and pronase. This treatment yielded DNA with a molecular weight of about 3 million daltons on the average. CsCI gradients had an average density of 1.695 g/cm3 in 0.2 X SSC (3 mmoi/lsodium citrate, pH 7.0, and 30 mmolll NaCI); they were centrifuged for at least 44 h at 20°C at 33,000 rev./min in a S52 swinging bucket rotor of a Christ Vacufuge ultracentrifuge. Five to 20% (w/w) sucrose gradients in 0.1 X SSC (1.5 mmol/l sodium citrate, pH 7.0, and 15 mmol/l NaCI) were centrifuged for 18 h at 4 °C at 35,000 rev./min in a SW56Ti rotor of a Beckman L5-65 ultracentrifuge. Alkali and nuclease 8 1 treatments of DNA
For hydrolysis of accompanying RNA, DNA preparations were incubated for 2 h at 37 °0 in 0.6 mol/l NaOH, neutralized and allowed to renature overnight. Incubations with nuclease S1 were carried out in 0.02 molll phosphate buffer, pH 6.9, containing 16 pg/ml each of denatured and native calf thymus DNA (Sigma, St. Louis, Mo.); one assay of 1 ml contained about 50 pg of labelled Ohlorella DNA (about 0.2pCi). Nuclease S1 was prepared from commercial IX-amylase (Sigma) by the following procedure (M. MELLI, personal communication): 1 g IX-amylase was stirred overnight at 4 °C in 40 ml 0.02 mol/l phosphate buffer, pH 6.9, containing 0.2 mol/l NaCI, heated to 70°C and immediately cooled. The supernatant after centrifugation for 10 min at 3,000 rev./min in a Christ Cryofuge centrifuge was stored under 50 % glycerol at -18°C. Per assay, 0.25 ml of this stock solution were used as the source of nuclease S1.
Results and Discussion
Labelled DN.A as an intermediate in nuclear DN.A synthesis As can be seen from Fig. 1, incubation of Chlorella cells with radioactive guanosine for 15 min results in a labelled peak at a density of 1. 721 g/cm3 as it had been observed after pulse labelling with uri dine (SSYMANK 1975). An incubation period of 24 h, however, results in a main radioactivity peak at 1.710 g/cm3 , the density of the bulk nuclear DNA. Fig. 2 shows that the radioactivity of a guanosine pulse can be shifted by a chase experiment from the higher density peak to the density of the nuclear DNA. These results indicate a precursor/product relationship between the labelled molecules of 1. 721 g/cm3 and the mature nuclear DNA. Thus an intermediate molecule of higher density occurs during the course of synthesis of nuclear DNA. Furthermore, it can be seen from Fig. 1 that the antibiotic myxin (1-hydroxy-6-methoxyphenazine-5,10-dioxide; PETERSON et al. 1966) interferes with the processing of this intermediate to mature nuclear DNA. There is no explanation of this effect; myxin had been reported to cause breakdown of chloroplast DNA after prolonged incubations i'n vivo in Chlamydomonas reinhardii (BEHKI and LURQUIN 1974) or in Scenedesmus obliquus (SSYMANK et al. 1976) without having an effect on the amount of nuclear DNA in these algae.
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217
The size distribution of the DNA preparations is presented in Fig. 3. The distribution of the total DNA is rather inhomogeneous whereas the labelled molecules form a distinct peak smaller than 7S. Procaryotic Okazaki fragments sediment at about lOS (OKAZAKI et aI. 1968); thus the size of the labelled intermediates in Chlorella would be compatible with the assumption that they might correspond to Okazaki fragments.
Possible reasons for the high density of labelled Jj N A In order to test the possibility that the higher density of the labelled DNA might be due to RNA segments connected with the DNA molecules, the DNA preparation was denatured with alkali; this treatment hydrolyses accompanying RNA. A CsCl gradient of such a de- and renatured DNA preparation is shown in Fig. 4; the density pattern of the labelled DNA does not differ distinctly from that of un denatured DNA preparations indicating that RNA fragments connected with the DNA intermediate molecules
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Fig. 1. Short- and long-term incorporation of [8-3 HJguanosine into DNA of Chlorella and the effect of myxin. Chlorella mass cultures were incubated with 1 ,uCi/ml [8-3 HJguanosine for 15 min (a) or 24 h (b), respectively. DNA preparations were centrifuged in CsC! gradients. - - OD 26o (with or without myxin the optical densities were essentially identical); y - - y radioactivity without myxin; / ' : , - - 6. radioactivity after 24 h incubation with 1,ug/ml myxin.
,... V.
218
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are not responsible for the high density. This is in contrast to Okazaki fragments of procaryotes (SUGINO et al. 1972) and to some reports on mammalian DNA (Fox et al. 1973; REICHARD et al. 1974), but in accordp,nce with results of PROBST et al. (1974) with newly synthesized mammalian DNA. If post-synthetic methylation of DNA intermediates would occur, the DNA should be shifted to a lower density as could be observed in the chase experiment. In order to test this, [methyPH]methionine was offered as a source of labelled methyl groups. Table 1 shows that there is no difference in mcthyllabel between highcr and lower density DNA neither in the presence nor in the absence of myxin. A density shift of methyl label after prolonged incubation cannot be observed. It should be noted that the incorporation of methyl groups is very low compared with the much higher incorporation into RNA observed under the same labelling conditions (GALLING and SSYMANK 1970).
--
sedim.
5000
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1500
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1000
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500
3000
1000
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23
Fig. 2. Chase after pulse labelling of Chlorella DNA with [8- 3 H]guanosine. To Chlorella mass cultures iucubated for 15 min with 1 ,uCi/ml [8- 3 H]guanosine a 100fold excess of unlabelled guanosine was given and the DNA prepared after further incubation for the times indicated. The radioactivity profiles of parallel esCl gradients are shown. The optical density distribution was identical in both gradients. 6 - - 6 5 h chase; T - - T 23 h chase. Fig. 3. Sedimentation of pulse-labellerl Chlorelltt DNA. A Chlorella mass culture was incubated for 15 min with l,uCi/ml [8- 3 H]guanosine and its DNA sedimented in a sucrose gradient (5 % to 20 %). - - OD 26o ; 0 - - - 0 radioactivity.
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:'i"ueJear DXA Synthesis in ehloulla
219
Table 1. Incorporation of [methyl-3 H] methioll1:/Ie into Chlorella nuclear DNA. Chlorella mass cultures were incubated for the times indieated with 1,uCi/ml L-[methyPH] methionine and, if indicated, with 1,ug/1 myxin either simultaneously or 23 h in advance. DNA preparations were centrifuged in CsCI gradients and the specific activities of the heavier and lighter portions of the DNA gradient region determined Labelling time [h]
Myxin treatment [h]
)Iethyl groups incOl"porated [cpm/OD 26o ]
> 1.714 g/cm3
(!
1 1 24 24
4520 2610 4910 3110
24 24
(!
< 1. 714 g/cm3
4440 2160 4900 2390
These results indicate that methylation of DNA intermediates is not responsible for the shift of buoyant density. If methylation occurs at all, its rate must be extremely low. In order to test whether single-stranded regions within the labelled DNA molecules produce their higher densities, D~A preparations were treated with the single-strand specific nuclease S~. Fig. 5 shows that nuclease SL treatment results in a shift of labelled DNA from a density of 1. 721 g/cm 3 to a density of 1. 711 g/cm 3 • Denaturation of Escheri-
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35
Fig. 4. De- and renatured labelled DNA from Chlorella. A Chlorella mass culture was incubated for 1 h with 1,uCi/ml [8-3 H]guanosine and its DNA denatured with alkali. After renaturation it was centrifuged in a CsCI gradient. • - - - . OD 26o ; l:,---l:, radioactivity.
220
V.8SYMANK
ckia coli DNA which has the same density as nuclear DNA of Oklorella pyrenoidosa yields in a density shift from 1. 710 g/cm3 to 1. 725 g/cm3 (ERIKSON and SZYBALSKI 1964); comparison of these data with the data of Fig. 5 suggests that about two thirds of the labelled DNA intermediate molecules consist of single-stranded regions which become double-stranded during further processing of the intermediate molecules. Since uri dine is incorporated into DNA mainly as a 2'-deoxycytidine nucleotide {WANKA et al. 1970; PROBST et al. 1974), it might be possible that the two precursors used so far, guanosine and uridine, are incorporated preferentially into (GC)-rich portions of the DNA. During growth of the DNA chain the density of such (GC)-rich small molecules would become lower because their nucleotide composition would approach the average v£'Jue of Oklorella DNA. Fig. 6 shows that there is in fact a difference in the distribution of label versus density between incorporation of thymidine on one side and guanosine or uridine on the other. However, even with thymidine incorporation, the density of the labelled DNA is still higher than that of unlabelled nuclear DNA. In the case that the high density of guanosine or uridine labelled precursors were only due to the effect of the nucleotide composition of the labelled molecules, thymidine should cause a density shift into the other direction. By the experiments described above, it could be shown that small DNA molecules representing an intermediate in the course of nuclear DNA synthesis are rapidly labelled
30000
15000
10
20
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38
Fig. 5. Labelled Chlorella DNA treated with nuclease 8 1 . A Chlorella mas" culture wa3 incubated for 1 h with l,uCijml [8- 3 H]guanosine and its DNA prepared. Half of the DNA was treated with nuclease 8 1 , the other half incubated in the same assay, but without nuclease 81 . The radioactivity profiles of parallel CsCl gradients are shown. The optical density distribution was identical in both gradients .• - - - . control; 0 - - - 0 treated with nuclease 8 1 .
Nuelear DNA Synthesis in Chlorella
221
in Chlorella. They have a higher buoyant density than mature nuclear DNA; this effect is in part caused by preferential incorporation of labelled guanosine or uridine into (GC)-rich portions. Their size is similar to Okazaki pieces (OKAZAKI et al. 1968). In contrast to Okazaki pieces of procaryotes (SUGINO et al. 1972), they do not contain RNA segments connected with the DNA fragments. No extensive, if any, methylation occurs after synthesis of the DNA molecules. Like Okazaki pieces (OKAZAKI et al. 1968, OISHI 1968), the labelled DNA molecules in Chlorella consist of partially single-stranded DNA explaining in part their higher density. Taking into account the density shift portion due to the nucleotide composition, one can estimate that about half of the DNA molecules must be single-stranded. Such a single-stranded portion in newly synthesized DNA had been described by several authors for mammalian cells (Nuzzo et al. 1970; Fox 1.73
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Fig. 6. Labelling of Chiarella DNA with different precursors. Chiarella mass cultures were incubated for 24 h with l.uCi/ml of the radioactive precursors indicated. DNA preparations were centrifuged in CsCI gradients and the specific radioactivities of the single fractions determined. Labelling conditions: a [S-3H]guanosine; b [5-3H]uridine; c [methyl-3H]thymidine. 15 Biochem. Physioi. Pflanzen, Bd.174
222
V. SSYMANK, Nuclear DNA Synthesis in Chlorella
et al. 1973; WANKA 1973); it is supposed in these cases that it functions as a primer during DNA replication. Acknowledgements I wish to thank Mrs. M. BINNEWIES for excellent technical assistance and the Deutsche Forschungsgemeinschaft for financial support.
References BEHKI, R. M., and LURQUIN, P. F.: Effect of myxin on the biosynthesis and degradation of chloroplast and nuclear DNA of Chlamydomonas reinhardi. Plant Sci. Letters 3, 419-429 (1974). ERIKSON, R. L., and SZYBALSKI, W.: The CS 2S04 equilibrium density gradient and its application for the study of T-even phage DNA: Glucosylation and replication. Virology 22, 111-124 (1964). Fox, R. M., MENDELSOHN, J., BARBOSA, E., and GOULIAN, M.: RNA in nascent DNA from cultured human lymphocytes. Nature (London) New BioI. 24a, 234-237 (1973). GALLING, G., and SSYMANK, V.: Bevorzugter Einbau markierten Uridins in die Vorlaufer von Chloroplasten-Ribosomen in Algenzellen. Planta 94, 203-212 (1970). KUHL, A., and LORENZEN, H.: Handling and CUlturing of Chlorella. Methods of Cell Physiology (Edit. PRESCOTT, D. M.). Vol. 1, pp. 159-187. Academic Press, New York 1964. Nuzzo, F., BREGA, A., and FALASCHI, A.: DNA replication in mammalian cells, 1. The size of newly synthesized helices. Proc. Natl. Acad. Sci. (Wash.) 6a, 1017-1024 (1970). OISHI, M.: Studies on DNA replication in vivo, 1. Isolation of the first intermediate of DNA replication in bacteria as single-stranded DNA. Proc. Nat!. Acad. Sci. (Wash.) 60,329-336 (1968). OKAZAKI, R., OKAZAKI, T., SAKABE, K., SUGIMOTO, K., and SUGINO, A.: Mechanism of DNA chain growth, 1. Possible discontinuity and unusual secondary structure of newly synthesized chains. Proc. Nat!. Acad. Sci. (Wash.) a9, 598-605 (1968). PETERSON, E. A., GILLESPIE, D. C., and COOK, F. D.: A wide-spectrum antibiotic produced by a species of Sorangium. Can. J. Microbiol. 12, 221-230 (1966). PROBST, H., GENTNER, P. R., HOFSTATTER, T., and JENKE, S.: Newly synthesized mammalian cell DNA: Evidence for effects simUlating the presence of RNA in the nascent DNA fraction isolated by nitrocellulose column chromatography. Biochim. Biophys. Acta 340, 361-373 (1974). REICHARD, P., ELIASSON, R., and SODERMAN, G.: Initiator RNA in discontinuous polyoma DNA synthesis. Proc. Nat!. Acad. Sci. (Wash.) 71, 4901-4905 (1974). SSYMANK, V.: Influence of nitrogen deficiency on uridine incorporation into ribosomes in the green alga Chlorella. Arch. Mikrobio!. 82, 311-324 (1972). Incorporation of different labelled precursors into chloroplast RNA of Chlorella. Planta 111, 157 to 166 (1973). - Hybridization of chloroplast RNA from Chlorella to DNA fractions from Chlorella. Les cycles cellulaires et leur blocage chez plusieurs protistes (Edit. LEFORT-TRAN, M., and VALENCIA, R.). pp. 199-202. CNRS, Paris 1975. STEUP, M., and SENGER, H.: Studies on nucleic acids in plastids of the pigment mutant C-2A' of Scenedesmus obliquus. Plant Cell Physio!. 17, 787-798 (1976). SUGINO, A., HIROSE, S., and OKAZAKI, R.: RNA-linked nascent DNA fragments in Escherichia coli. Proc. Natl. Acad. Sci. (Wash.) 69, 1863-1867 (1972). WANKA, F.: Separation of rapidly labeled intermediates of DNA synthesis in mammalian cells. Biochern. Biophys. Res. Commun. 54, 1410-1417 (1973). - JOOSTEN, H. F. P., and DE GRIP, W. J.: Composition and synthesis of DNA in synchronously growing cells of Chlorella pyrenoidosa. Arch. Mikrobio!. 7a, 25-36 (1970). Received October 14, 1978.
Author's address: Dr. VOLKER SSYMANK, Pflanzenphysiologisches Institut, Universitat, Untere Karspiile 2, D-3400 Gottingen.
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A Partially Single-stranded Intermediate Occuring during Synthesis of Nuclear DNA in Chlorella VOLKER
SSY~IANK
Pflanzenphysiologisches Institut, Universitat, D-3400 Giittingen, FRG Key Term Index: DNA synthesis, nuclear DNA, uridine, guanosine; Chiarella pyrenoidosa
Snmmary Pulse labelling of Chiarella DNA by incubation with uridine or guanosine resulted in labelled molecules with a higher buoyant density than the bulk of nuclear DNA. No incorporation into chloroplast D~A could be observed. The label could be chased into mature nuclear DNA indicating that the labelled molecules represent an intermediate in the synthesis of nuclear DNA. The labelled molecules have a sedimentation coefficient of less than 7 S, smaller than procaryotic Okazaki pieces. In contrast to these, they do not contain RNA segments. The observed density difference is not due to posterior methylation of the labelled intermediate molecules. Two factors contribute to their high density: The labelled intermediate molecules contain single-stranded regions, and the precursors nridine and guanosine preferentially enter (GC)-rich portions of the DNA.
Introduction
Uracil and uridine are incorporated by cells of the green alga Chlorella pyremoidosa not only into RKA, but also rather rapidly into DNA (WANKA et al. 1970; SsnIANK 1975). Presumably they enter DNA as a 2'-deoxycytidine nucleotide (PROllST et al. 1974). Pulse labelling experiments with uridine or with guanosine which is incorporated into DNA more readily always showed a buoyant density of the labelled molecules of 1. 721 g/cm 3 compared to the bulk of nuclear DNA with a density of 1. 710 g/cm3 ; chloroplast DXA (density: 1.689 g/cm 3 ) remained unlabelled up to 24 h incubation time (see e.g. SSY)fANK 1975, Fig. 1). The question now arises whether the labelled molecules arc intermediates in the course of the synthesis of nuclear DNA, and if they are, due to which reason they have a higher density. In the following investigations it is shown that uridine and guanosine are incorporated into nuclear DNA of Chlorella via an intermediate of higher buoyant density. Four possibilities to explain the high density are investigated: (i) RNA fragments might be connected with the intermediates of DNA synthesis like in procaryotic Okazaki pieces; (ii) intermediate molecules might become methylated and thus be shifted to a lower density; (iii) the intermediate molecules might contain single-stranded regions; (iv) uri dine or guanosine preferentially enter (GC)-rich portions of the DNA. Material and Methods Culture of algae The experiments described were carried out with the unicellular green alga Chiarella pyrenaidasa, strain 211-8 b from the algal collection of the Institute for Plant Physiology, Giittingen, FRG. The algae were grown at 30°C under sterile conditions as described by KUHL and LORENZEN (1964).
216
V. SSYMANK
Labelling procedures were described previously (SSYMANK 1972, 1973). The following radioactive precursors were used: [5-3H]uridine (30 Ci/mmol), [8-3H]guanosine (15 Ci/mmol), L-[methyPH]methionine (15 Ci/mmol) and [methyPH]thymidine (60 Ci/mmol), all obtained from the Radiochemical Centre, Amersham, U.K. Myxin (a gift from Dr. P. F. LURQUIN, Mol, Belgium, and Dr. R. M. BEHKI, Ottawa, Canada) was dissolved in methanol and added to the culture medium at 1pgl ml; the final concentration of methanol was less than 0.2 %. Preparation and fractionation of DNA
Nucleic acids were extracted with phenol as described (SSYMANK 1973) and from these preparations DNA was released by treatment with RNase and pronase. This treatment yielded DNA with a molecular weight of about 3 million daltons on the average. CsCI gradients had an average density of 1.695 g/cm3 in 0.2 X SSC (3 mmoi/lsodium citrate, pH 7.0, and 30 mmolll NaCI); they were centrifuged for at least 44 h at 20°C at 33,000 rev./min in a S52 swinging bucket rotor of a Christ Vacufuge ultracentrifuge. Five to 20% (w/w) sucrose gradients in 0.1 X SSC (1.5 mmol/l sodium citrate, pH 7.0, and 15 mmol/l NaCI) were centrifuged for 18 h at 4 °C at 35,000 rev./min in a SW56Ti rotor of a Beckman L5-65 ultracentrifuge. Alkali and nuclease 8 1 treatments of DNA
For hydrolysis of accompanying RNA, DNA preparations were incubated for 2 h at 37 °0 in 0.6 mol/l NaOH, neutralized and allowed to renature overnight. Incubations with nuclease S1 were carried out in 0.02 molll phosphate buffer, pH 6.9, containing 16 pg/ml each of denatured and native calf thymus DNA (Sigma, St. Louis, Mo.); one assay of 1 ml contained about 50 pg of labelled Ohlorella DNA (about 0.2pCi). Nuclease S1 was prepared from commercial IX-amylase (Sigma) by the following procedure (M. MELLI, personal communication): 1 g IX-amylase was stirred overnight at 4 °C in 40 ml 0.02 mol/l phosphate buffer, pH 6.9, containing 0.2 mol/l NaCI, heated to 70°C and immediately cooled. The supernatant after centrifugation for 10 min at 3,000 rev./min in a Christ Cryofuge centrifuge was stored under 50 % glycerol at -18°C. Per assay, 0.25 ml of this stock solution were used as the source of nuclease S1.
Results and Discussion
Labelled DN.A as an intermediate in nuclear DN.A synthesis As can be seen from Fig. 1, incubation of Chlorella cells with radioactive guanosine for 15 min results in a labelled peak at a density of 1. 721 g/cm3 as it had been observed after pulse labelling with uri dine (SSYMANK 1975). An incubation period of 24 h, however, results in a main radioactivity peak at 1.710 g/cm3 , the density of the bulk nuclear DNA. Fig. 2 shows that the radioactivity of a guanosine pulse can be shifted by a chase experiment from the higher density peak to the density of the nuclear DNA. These results indicate a precursor/product relationship between the labelled molecules of 1. 721 g/cm3 and the mature nuclear DNA. Thus an intermediate molecule of higher density occurs during the course of synthesis of nuclear DNA. Furthermore, it can be seen from Fig. 1 that the antibiotic myxin (1-hydroxy-6-methoxyphenazine-5,10-dioxide; PETERSON et al. 1966) interferes with the processing of this intermediate to mature nuclear DNA. There is no explanation of this effect; myxin had been reported to cause breakdown of chloroplast DNA after prolonged incubations i'n vivo in Chlamydomonas reinhardii (BEHKI and LURQUIN 1974) or in Scenedesmus obliquus (SSYMANK et al. 1976) without having an effect on the amount of nuclear DNA in these algae.
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:'\ tldear D~ A Synthesis in Chiarella
217
The size distribution of the DNA preparations is presented in Fig. 3. The distribution of the total DNA is rather inhomogeneous whereas the labelled molecules form a distinct peak smaller than 7S. Procaryotic Okazaki fragments sediment at about lOS (OKAZAKI et aI. 1968); thus the size of the labelled intermediates in Chlorella would be compatible with the assumption that they might correspond to Okazaki fragments.
Possible reasons for the high density of labelled Jj N A In order to test the possibility that the higher density of the labelled DNA might be due to RNA segments connected with the DNA molecules, the DNA preparation was denatured with alkali; this treatment hydrolyses accompanying RNA. A CsCl gradient of such a de- and renatured DNA preparation is shown in Fig. 4; the density pattern of the labelled DNA does not differ distinctly from that of un denatured DNA preparations indicating that RNA fragments connected with the DNA intermediate molecules
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Fig. 1. Short- and long-term incorporation of [8-3 HJguanosine into DNA of Chlorella and the effect of myxin. Chlorella mass cultures were incubated with 1 ,uCi/ml [8-3 HJguanosine for 15 min (a) or 24 h (b), respectively. DNA preparations were centrifuged in CsC! gradients. - - OD 26o (with or without myxin the optical densities were essentially identical); y - - y radioactivity without myxin; / ' : , - - 6. radioactivity after 24 h incubation with 1,ug/ml myxin.
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are not responsible for the high density. This is in contrast to Okazaki fragments of procaryotes (SUGINO et al. 1972) and to some reports on mammalian DNA (Fox et al. 1973; REICHARD et al. 1974), but in accordp,nce with results of PROBST et al. (1974) with newly synthesized mammalian DNA. If post-synthetic methylation of DNA intermediates would occur, the DNA should be shifted to a lower density as could be observed in the chase experiment. In order to test this, [methyPH]methionine was offered as a source of labelled methyl groups. Table 1 shows that there is no difference in mcthyllabel between highcr and lower density DNA neither in the presence nor in the absence of myxin. A density shift of methyl label after prolonged incubation cannot be observed. It should be noted that the incorporation of methyl groups is very low compared with the much higher incorporation into RNA observed under the same labelling conditions (GALLING and SSYMANK 1970).
--
sedim.
5000
.3
1500
.2
1000
.1
500
3000
1000
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10
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23
Fig. 2. Chase after pulse labelling of Chlorella DNA with [8- 3 H]guanosine. To Chlorella mass cultures iucubated for 15 min with 1 ,uCi/ml [8- 3 H]guanosine a 100fold excess of unlabelled guanosine was given and the DNA prepared after further incubation for the times indicated. The radioactivity profiles of parallel esCl gradients are shown. The optical density distribution was identical in both gradients. 6 - - 6 5 h chase; T - - T 23 h chase. Fig. 3. Sedimentation of pulse-labellerl Chlorelltt DNA. A Chlorella mass culture was incubated for 15 min with l,uCi/ml [8- 3 H]guanosine and its DNA sedimented in a sucrose gradient (5 % to 20 %). - - OD 26o ; 0 - - - 0 radioactivity.
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219
Table 1. Incorporation of [methyl-3 H] methioll1:/Ie into Chlorella nuclear DNA. Chlorella mass cultures were incubated for the times indieated with 1,uCi/ml L-[methyPH] methionine and, if indicated, with 1,ug/1 myxin either simultaneously or 23 h in advance. DNA preparations were centrifuged in CsCI gradients and the specific activities of the heavier and lighter portions of the DNA gradient region determined Labelling time [h]
Myxin treatment [h]
)Iethyl groups incOl"porated [cpm/OD 26o ]
> 1.714 g/cm3
(!
1 1 24 24
4520 2610 4910 3110
24 24
(!
< 1. 714 g/cm3
4440 2160 4900 2390
These results indicate that methylation of DNA intermediates is not responsible for the shift of buoyant density. If methylation occurs at all, its rate must be extremely low. In order to test whether single-stranded regions within the labelled DNA molecules produce their higher densities, D~A preparations were treated with the single-strand specific nuclease S~. Fig. 5 shows that nuclease SL treatment results in a shift of labelled DNA from a density of 1. 721 g/cm 3 to a density of 1. 711 g/cm 3 • Denaturation of Escheri-
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Fig. 4. De- and renatured labelled DNA from Chlorella. A Chlorella mass culture was incubated for 1 h with 1,uCi/ml [8-3 H]guanosine and its DNA denatured with alkali. After renaturation it was centrifuged in a CsCI gradient. • - - - . OD 26o ; l:,---l:, radioactivity.
220
V.8SYMANK
ckia coli DNA which has the same density as nuclear DNA of Oklorella pyrenoidosa yields in a density shift from 1. 710 g/cm3 to 1. 725 g/cm3 (ERIKSON and SZYBALSKI 1964); comparison of these data with the data of Fig. 5 suggests that about two thirds of the labelled DNA intermediate molecules consist of single-stranded regions which become double-stranded during further processing of the intermediate molecules. Since uri dine is incorporated into DNA mainly as a 2'-deoxycytidine nucleotide {WANKA et al. 1970; PROBST et al. 1974), it might be possible that the two precursors used so far, guanosine and uridine, are incorporated preferentially into (GC)-rich portions of the DNA. During growth of the DNA chain the density of such (GC)-rich small molecules would become lower because their nucleotide composition would approach the average v£'Jue of Oklorella DNA. Fig. 6 shows that there is in fact a difference in the distribution of label versus density between incorporation of thymidine on one side and guanosine or uridine on the other. However, even with thymidine incorporation, the density of the labelled DNA is still higher than that of unlabelled nuclear DNA. In the case that the high density of guanosine or uridine labelled precursors were only due to the effect of the nucleotide composition of the labelled molecules, thymidine should cause a density shift into the other direction. By the experiments described above, it could be shown that small DNA molecules representing an intermediate in the course of nuclear DNA synthesis are rapidly labelled
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Fig. 5. Labelled Chlorella DNA treated with nuclease 8 1 . A Chlorella mas" culture wa3 incubated for 1 h with l,uCijml [8- 3 H]guanosine and its DNA prepared. Half of the DNA was treated with nuclease 8 1 , the other half incubated in the same assay, but without nuclease 81 . The radioactivity profiles of parallel CsCl gradients are shown. The optical density distribution was identical in both gradients .• - - - . control; 0 - - - 0 treated with nuclease 8 1 .
Nuelear DNA Synthesis in Chlorella
221
in Chlorella. They have a higher buoyant density than mature nuclear DNA; this effect is in part caused by preferential incorporation of labelled guanosine or uridine into (GC)-rich portions. Their size is similar to Okazaki pieces (OKAZAKI et al. 1968). In contrast to Okazaki pieces of procaryotes (SUGINO et al. 1972), they do not contain RNA segments connected with the DNA fragments. No extensive, if any, methylation occurs after synthesis of the DNA molecules. Like Okazaki pieces (OKAZAKI et al. 1968, OISHI 1968), the labelled DNA molecules in Chlorella consist of partially single-stranded DNA explaining in part their higher density. Taking into account the density shift portion due to the nucleotide composition, one can estimate that about half of the DNA molecules must be single-stranded. Such a single-stranded portion in newly synthesized DNA had been described by several authors for mammalian cells (Nuzzo et al. 1970; Fox 1.73
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Fig. 6. Labelling of Chiarella DNA with different precursors. Chiarella mass cultures were incubated for 24 h with l.uCi/ml of the radioactive precursors indicated. DNA preparations were centrifuged in CsCI gradients and the specific radioactivities of the single fractions determined. Labelling conditions: a [S-3H]guanosine; b [5-3H]uridine; c [methyl-3H]thymidine. 15 Biochem. Physioi. Pflanzen, Bd.174
222
V. SSYMANK, Nuclear DNA Synthesis in Chlorella
et al. 1973; WANKA 1973); it is supposed in these cases that it functions as a primer during DNA replication. Acknowledgements I wish to thank Mrs. M. BINNEWIES for excellent technical assistance and the Deutsche Forschungsgemeinschaft for financial support.
References BEHKI, R. M., and LURQUIN, P. F.: Effect of myxin on the biosynthesis and degradation of chloroplast and nuclear DNA of Chlamydomonas reinhardi. Plant Sci. Letters 3, 419-429 (1974). ERIKSON, R. L., and SZYBALSKI, W.: The CS 2S04 equilibrium density gradient and its application for the study of T-even phage DNA: Glucosylation and replication. Virology 22, 111-124 (1964). Fox, R. M., MENDELSOHN, J., BARBOSA, E., and GOULIAN, M.: RNA in nascent DNA from cultured human lymphocytes. Nature (London) New BioI. 24a, 234-237 (1973). GALLING, G., and SSYMANK, V.: Bevorzugter Einbau markierten Uridins in die Vorlaufer von Chloroplasten-Ribosomen in Algenzellen. Planta 94, 203-212 (1970). KUHL, A., and LORENZEN, H.: Handling and CUlturing of Chlorella. Methods of Cell Physiology (Edit. PRESCOTT, D. M.). Vol. 1, pp. 159-187. Academic Press, New York 1964. Nuzzo, F., BREGA, A., and FALASCHI, A.: DNA replication in mammalian cells, 1. The size of newly synthesized helices. Proc. Natl. Acad. Sci. (Wash.) 6a, 1017-1024 (1970). OISHI, M.: Studies on DNA replication in vivo, 1. Isolation of the first intermediate of DNA replication in bacteria as single-stranded DNA. Proc. Nat!. Acad. Sci. (Wash.) 60,329-336 (1968). OKAZAKI, R., OKAZAKI, T., SAKABE, K., SUGIMOTO, K., and SUGINO, A.: Mechanism of DNA chain growth, 1. Possible discontinuity and unusual secondary structure of newly synthesized chains. Proc. Nat!. Acad. Sci. (Wash.) a9, 598-605 (1968). PETERSON, E. A., GILLESPIE, D. C., and COOK, F. D.: A wide-spectrum antibiotic produced by a species of Sorangium. Can. J. Microbiol. 12, 221-230 (1966). PROBST, H., GENTNER, P. R., HOFSTATTER, T., and JENKE, S.: Newly synthesized mammalian cell DNA: Evidence for effects simUlating the presence of RNA in the nascent DNA fraction isolated by nitrocellulose column chromatography. Biochim. Biophys. Acta 340, 361-373 (1974). REICHARD, P., ELIASSON, R., and SODERMAN, G.: Initiator RNA in discontinuous polyoma DNA synthesis. Proc. Nat!. Acad. Sci. (Wash.) 71, 4901-4905 (1974). SSYMANK, V.: Influence of nitrogen deficiency on uridine incorporation into ribosomes in the green alga Chlorella. Arch. Mikrobio!. 82, 311-324 (1972). Incorporation of different labelled precursors into chloroplast RNA of Chlorella. Planta 111, 157 to 166 (1973). - Hybridization of chloroplast RNA from Chlorella to DNA fractions from Chlorella. Les cycles cellulaires et leur blocage chez plusieurs protistes (Edit. LEFORT-TRAN, M., and VALENCIA, R.). pp. 199-202. CNRS, Paris 1975. STEUP, M., and SENGER, H.: Studies on nucleic acids in plastids of the pigment mutant C-2A' of Scenedesmus obliquus. Plant Cell Physio!. 17, 787-798 (1976). SUGINO, A., HIROSE, S., and OKAZAKI, R.: RNA-linked nascent DNA fragments in Escherichia coli. Proc. Natl. Acad. Sci. (Wash.) 69, 1863-1867 (1972). WANKA, F.: Separation of rapidly labeled intermediates of DNA synthesis in mammalian cells. Biochern. Biophys. Res. Commun. 54, 1410-1417 (1973). - JOOSTEN, H. F. P., and DE GRIP, W. J.: Composition and synthesis of DNA in synchronously growing cells of Chlorella pyrenoidosa. Arch. Mikrobio!. 7a, 25-36 (1970). Received October 14, 1978.
Author's address: Dr. VOLKER SSYMANK, Pflanzenphysiologisches Institut, Universitat, Untere Karspiile 2, D-3400 Gottingen.
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