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10 kb DNA replication intermediates are formed from Okazaki-fragments in human malignant melanoma cells
Cell Biology
International
Reports,
Vol. 6, No. 7, July
687
1982
10 kb DNA REPLICATION INTERMEDIATES ARE FORMED FROM OKAZAKIFF&MENTS IN HUMAN MALIGNANT MELANOMA CELLS
Ulf Department
L&n
of Histology, Karolinska Karolinska Hospital, S-104
Institutet, and Radiumhemmet, 01 Stockholm, Sweden.
ABSTRACT Human melanoma cells were synchronized with hydroxyurea. The formed .either during the hydroxyDNA replication intermediates urea-treatment or after the release of the hydroxyurea-block were The intermediates were released from the parental investigated. DNA by lysing the cells in dilute alkali and were then analyzed After the release of the hydroxyby agarose gel electrophoresis. urea block radioactivity was first detected in Okazaki-fragments and then later also in larger replication intermediates (ranging in size up to 10 kb). The results indicate that the 10 kb DNA replication intermediate is derived from the Okazaki-fragments. Thus during its maturation to high molecular weight DNA the Okazaki-fragments form a large, well defined DNA replication intermediate, which later forms the high molecular weight DNA. Howit is not possible to determine whether all or only a part ever, of the Okazaki-fragments give rise to the 10 kb DNA. INTRODUCTION DNA replication in human cells is a very complex process is probably formation of several DNA replication (1) - There intermediates which are ligated to form the stable high molecular weight DNA. So far, we have obtained little knowledge about the different steps. The best known replication intermediate is the short Okazaki-fragment. However, we do not know if this fragment during its maturation to high molecular weight DNA forms larger DNA replication intermediates with a discrete size and a different half-life or if there is a continuous increase in the size of the fragment up to high molecular weight DNA with no formation of larger discrete replication intermediates. 030~1651/82/070687-1
O/$03.00/0
@ 1982 Academic
Press
Inc. (London)
Ltd.
688
Cell Biology
International
Reports,
Vol. 6, No. 7, July 7982
In an earlier paper we have described a procedure where cells that are growing in cell culture as monolayers are treated with dilute alkali directly in the culture dish (2). This will lyse the cells and the alkaline milieu will start the uncoiling of the DNA. The uncoiling is initiated at points where there are single-stranded gaps in the DNA. Such gaps occur normally in during the uncoiling of the DNA active replicons. Therefore, there is a release from the parental DNA of single-stranded DNA replication intermediates which will appear free in the solution. In asynchronous cell populations one would expect the release of DNA replication intermediates that range in size from Okazaki-fragments up to replicon-size. However, when we analyzed an asynchronous population of human melanoma cells we detected the release of single-stranded DNA replication intermediates ranging in size from that of Okazaki-fragments up to 10 kilobases (kb) (2). We did not detect DNA molecules ranging in size from 10 kb up to replicon size. The 10 kb DNA replication intermediate represents a discrete population of DNA molecules that can be easily detected using agarose gel electrophoresis. In the asynchronous cell population which we analyzed earlier we were not able to show that the Okazaki-fragments were labeled with tritiated thymidine before the 10 kb DNA intermediTherefore, we have now performed experiments in melanoma ate. cells that have been synchronized with hydroxyurea. The action of It causes the depletion of the hydroxyurea is well understood. dNTPs required for DNA synthesis, thereby inhibiting DNA repThe effect is readily reversible. The results lication (3,4). described in this paper show that the 10 kb DNA is formed by the joining of Okazaki-fragments. MATERIALS AND METHODS cells synchronization with Culturing of melanoma and obtained from Flow Laboratohydroxyurea: Human melanoma cells, ries, were grown as monolayers as earlier described (2). To synchronize the cells hydroxyurea was added to the medium to give a final concentration of 1 mM. The cells were incubated for 18 h and were then washed free of the drug and cultivated in fresh media for 6 h after which a second block with hydroxyurea was delivered. The duration of the second block was likewise 18 h. When the cells were released from the second block they were highly synchronized with more than 90 96 of the cells in S-phase as judged by autoradiography according to Ashihara and Baserga (5) -
Cell Biology
International
Reports,
Vol. 6, No. 7, July 1982
Labeling with tritiated thymidine and cell lysis: Cells to be used in experiments were grown as monolayers in small petri the DNA 100 uCi dishes (35x10 mm) with 3 ml medium. To label activity 30 Ci/mmol) was tritiated thymidine (Amersham, specific the labeling the medium was added to the medium. To terminate sucked off and the petri dishes placed on ice and the cells washed with cold phosphate buffered-saline. Cell lysis was performed in the dark at O°C by the addition into the petri dish of 2.25 ml 0.03 M NaOH. After 30 min the solution was neutralized by the addition of 0.9 ml 0.067 M HCl concencontaining 0.02 M NaH2P04 and SDS was added to a final tration of 1 %. After 60 min at room temperature the labeled DNA was analyzed by gelelectrophoresis either in 0.75 % agarose or in The electrophoretic separations were 6 % acrylamide gels (2,6). standardized by the separation in a parallel lane of the same gel-slab of labeled DNA size markers obtained from NEN (see The markers were denatured immediately before Figure Legends). the run by heating to 80°C for 10 min in 90 % formamide. RESULTS Formation of replication intermediates in the presence of hydroxyurea In the human melanoma cell line used here hydroxyurea (1 mM) rapidly inhibits the incorporation of thymidine in cold TCA-precipitable material. More than 90 % of the precipitable material was sensitive to deoxyribonuclease. The rate of DNA synthesis in hydroxyurea-treated cells were measured by 5 min pulses of tritiated thymidine at different times after the addition of the drug. It was found that after 15 min of treatment the rate of DNA synthesis had decreased to 6 % of the control value. Moreover the inhibition of DNA synthesis is reversible. When the drug was removed after treatment for 18 h and the cells incubated in fresh media this resulted in rapidly resumed synthesis of DNA. The rate of DNA synthesis was 46 % of the control value after incubation. of the cells in fresh media for 15 min and 88 % after incubation for 7 h. To analyze the formation of DNA replication intermediates human melanoma cells were lysed in dilute alkali (0.03 M NaOH) in the dark at O°C for 30 min. This approach, originally used to measure the amount of single-stranded DNA breaks present in the cellular DNA (7,8), has been modified by us to allow analysis of the formation of single-stranded DNA replication intermediates (2). In the alkaline milieu the melanoma DNA starts to uncoil; the uncoiling being initiated at single-stranded gaps present in
Cell Biology
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Reports,
Vol. 6 No. 7, July 1982
active replicons. This will release single-stranded DNA replication intermediates into solution. When the solution is neutralized before the addition of detergent the high molecular weight DNA which is not completely denatured will renature and form double-stranded DNA whereas the released single-stranded DNA replication intermediates remain free in solution. When the total solution is then analyzed by agarose gel electrophoresis, the high molecular weight double-stranded DNA is located close to the trough (slices 3-6), whereas the single-stranded DNA replication intermediates enter the gel and are easily distinguished from the double-stranded DNA (2). The size of the single-stranded DNA range from that of Okazaki-fragments up to 10 kb and is divided into three populations ((2) cf. the control below in Fig. la). To establish the effect of hydroxyurea on the formation of DNA replication intermediates in human melanoma cells the following experiments were performed. Asynchronously growing cells were treated with the drug (1 mM) for 30 min and were then pulsed with tritiated thymidine during the last 45 seconds of the drug treatment. The cells were lysed in dilute alkali and the DNA then were always treated analyzed in 0.75 % agarose gels. The controls in parallel with the difference, however, that hydroxyurea was not present. The control DNA, when separated in the agarose gel (Fig. is located either close to the trough (the double-stranded la), high molecular weight DNA) or enters the gel (single-stranded DNA). The DNA entering the gel consists of three populations: the 10 kb DNA population (slices 19-25), the Okazaki-fragments (sliand the intermediate sized DNA population (slices ces 36-45), These three DNA populations have been found earlier to 26-35). have the kinetic behavior expected of DNA replication intermediates (2). After a pre-treatment with hydroxyurea for 30 min the main part of the radioactivity is located at slices 36-45 (OkazakiThe inhibition of labeling of the high fragments) (Fig. la). molecular weight DNA located close to the trough (slices 3-6) and the 10 kb DNA replication intermediate located at slices 19-25 is almost complete. This contrasts with the control described above where these two DNA populations are equally or more extensively labeled than the Okazaki-fragments. The size of the labeled Okazaki-fragments were established in 6 % acrylamide-gels (Fig. lb). The results show that the labeled DNA pieces are between LOO-200 bases, i.e. the same as in the control cells (2). Likewise control experiments in which the DNA was banded in CsCl showed that this DNA is a single-stranded DNA (not shown).
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synchronized with hydroxyurea Hydroxyurea is often used to synchronize cell cultures at Cl/S (5). To synchronize the human melanoma cells used in this study the following procedure was used. The cells were treated with 1 mM hydroxyurea for 18 h, washed free of the drug and then cultivated further for 6 h in fresh media. Then a second block with hydroxyurea was delivered for another 18 h. To evaluate the degree of synchronization the cells were labeled with tritiated thymidine after the release of the second block and then examined by autoradiography (5). It was found that there is good synchrony of the cells with more than 90 % in S-phase immediately after the second hydroxyurea block was removed. In order to establish whether it is possible to label the Okazaki-fragments before the 10 kb DNA molecules we investigated the synthesis of the DNA replication intermediates after the release of the second hydroxyurea block. Thus, the drug was thus removed and the cells were allowed to grow in fresh media and were pulsed with tritiated thymidine for 45 seconds at different times after the removal of the drug. In the first set of experiments the formation of DNA replication intermediates were analyzed immediately after the removal of hydroxyurea (Fig. Za). Analysis of the labeled material shows the preferential labeling of Okazaki-fragments as described earlier in Fig. la. There was almost no label in the position of the 10 kb DNA replication intermediate or in the high molecular weight DNA. However, 15 min after the release of the block there was label, apart from the Okazaki-fragment, also in the 10 kb DNA replication intermediate, the DNA located at slices 26-35 and the high molecular weight DNA located at slices 3-6 (Fig. 2b). Still the Okazaki-fragments dominated over the 10 kb DNA fragments. Seven hours after the release of the hydroxyurea block the electrophoretic separation is similar to the controls with roughly equal amounts of radioactivity in the 10 kb DNA fragments and the Okazaki-fragments (Fig. 2c, compare Fig. la). To demonstrate that the labeled DNA intermediates formed after the release of the block are incorporated into the high molecular weight DNA the following experiment was performed. Cells were labeled for 45 seconds immediately after the release of the second block and were then cultivated further for 24 h. The electrophoretic analysis of the labeled DNA thus obtained showed a stable high molecular weight DNA with no indication of the release of labeled DNA intermediates (Fig. 2d). This is the same result as in the parallel control cells not treated with hydroxyurea (see also (2)). Thus, during the recovery of DKA synthesis one first detects the
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Vol. 6, No. 7, July 7982
Okazaki-fragments and later also 10 kb DNA intermediates, the intermediate located at slices 26-35, and the high molecular that the 10 kb DNA intermediate is weight DNA. This indicates formed by the joining of Okazaki-fragments. DISCUSSION Using the procedure to lyse asynchronously growing melanoma cells in dilute alkali we were previously able to detect three different single-stranded DNA replication intermediates, i.e. Okazaki-fragments, a 10 kb DNA fragment and a population of molecules ranging in size from Okazaki-fragments up to 10 kb (slices 26-35 in the agarose gel) (2). In this paper we have analyzed the formation of these DNA replication intermediates in cells synchronized with hydroxyurea. The function of hydroxyurea is well known. It inhibits the DNA synthesis by inhibiting the enzyme ribonucleotide reductase and thereby depleting the pools for dGTP and dATP (3,4). This frequently results in the accumulation of Okazaki-fragments which are not joined together to form high molecular weight DNA (9). As expected, also in human melanoma cells there is an accumulation of Okazaki-fragments after a pretreatment with hydroxyurea. Neither the other single-stranded DNA replication intermediates nor the high molecular weight DNA as with tritiated thymidine. labeled When the cells are released from the second hydroxyureablock more than 90 % of the cells are in S-phase. When pulsed with tritiated thymidine for 45 seconds at different times after the release of the block, at first there is radioactivity only in the Okazaki-fragments. 15 min after the release of the block there is also radioactivity in the intermediate DNA population at slices 26-35, the 10 kb DNA intermediate, and the high molecular weight DNA. Finally, when the cells were pulsed 7 h after the removal of the hydroxyurea one can detect a distribution of radioactivity among the different DNA populations which is similar to that in the control cells which were growing asynchronously. Moreover, experiments in which the DNA was labeled for 45 seconds immediately after the release from the block and then analyzed 24 hours later showed no release of labeled DNA replication intermediates. Thus the labeled intermediates formed after the hydroxyurea synchronization will after a time-lag form a stable high molecular weight DNA, as in the control cells. It is not possible to distinguish between the appearance of radioactivity in the 10 kb DNA fragments and the intermediate DNA population (slices 26-35). However, it is obvious that both populations are formed later than the Okazaki-fragments and therefore
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in all probability are derived from the Okazaki-fragments. Thus during the maturation to high molecular weight DNA the Okazakifragments form a large, well defined DNA replication intermediate, which later form the high molecular weight DNA. However, it is not possible from our work to determine whether all or only a part of the Okazaki-fragments give rise to the 10 kb DNA. A prerequisite for the release of the 10 kb DNA from the parental DNA during our cell lysis condition is that there exist gaps in the continuity of the newly synthesized DNA spaced roughThese gaps would serve as initialy 10 kb away from each other. tion points for the uncoiling of the DNA when the cells are lysed in dilute alkali and thereby produce a discrete population of 10 kb DNA molecules. However, these gaps should represent a transient state since in the steady-state labeled DNA one can not detect the release of these DNA fragments. Hence, it is probable that the presence of these gaps should represent a defined step during the maturation of the newly synthesized DNA.
ACKNOWLEDGEMENTS This Stockholm,
work was supported by grants from the Cancer and the E. Welander Foundation.
Society
in
REFERENCES 1. Hand, R (1978) Eukaryotic DNA: Organization of the genome for replication. Cell 15, 317-325 2. Liinn, U (1982) Detection of a 10 kb DNA replication intermediate in human melanoma cells. Chromosoma (in press) 3. Krakoff, IHN, Brown, C and Reichard, P (1968) Inhibition of ribonucleoside diphosphate reductase by hydroxyurea. Cancer Res. 28, 1559-1565 4. Skoog, L and Nordenskjcld, B (1971) Effects of hydroxyurea and 1-B-arabinofuranosylcytosine on deoxyribonucleotide pools in mouse embryo cells. Eur. J. Biochem. 19, 81-89 5. Ashihara, T and Baserga, R (1979) Cell Synchronization, in "Methods in Enzymology", vol. LVIII 248-262 6. L&n, U (1977) Exclusive nuclear location of precursor 4 S RNA. J. Mol. Biol. 112, 661-666 7. AhnstrGm, G and Erixon, K (1973) Radiation induced strand breakage in DNA from mammalian cells. Strand separation in alkaline solution. Int. J. Radiat. Biology 23, 285-289
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8. Rydberg, G (1975) The rate of strand separation in alkali DNA of irradiated mammalian cells. Radiation Res.
of 61,
274-287 9. Magnusson, G (1973) Hydroxyurea-induced accumulation of short fragments during polyoma DNA replication. 1. Characterization of fragments. J. Virol. 12, 600-608
Received:
22nd December 2981
Modified version accepted: 16th Apm-2 1982
Fig. 1 Formation of DNA replication intermediates in the presence of hydroxyurea. The human melanoma cells were treated with hydroxyurea (1 mM) for 30 min and during the last 45 seconds tritiated were lysed as described and the thymidine was added. The cells DNA then subjected to electrophoresis in either (a) 0.75 % agaroControls were always treated se gels or (b) 6 % acrylamide gels. in parallel with the difference that hydroxyurea was not present. the size (in kb) and location of In (a) 25, 10 and 2 denotes DNA populations are single-stranded marker DNA. Four labeled visible. The high molecular weight double-stranded DNA located at slices 3-6 and three populations of single-stranded DNA located at slices 19-25, 26-35 (hatched area) and 36-45, respectively. In (b) 194, 118 and 72 denotes the size (in bases) and location of single-stranded DNA markers. -e- hydroxyurea-treated cells, -ocontrol cells. Fig. 2 Cells synchronized with hydroxyurea. The cells were released from the second block with hydroxyurea and were then pulsed with tritiated thymidine for 45 seconds either (a) immediately after the release, (b) after 15 min of cultivation in fresh media, (c) after 7 hours of cultivation in fresh media or (d) cells labeled for 45 seconds immediately after the release of the block and then cultivated for 24 hours in fresh media. The cells were lysed in dilute alkali and the labeled DNA then separated in 0.75 % agarose gels. A total of four DNA populations are formed in the The high molecular weight double-stranded DNA different panels. Three populations of single-stranded is located at slices 3-6. DNA is located at slices 19-25 (10 kb), 26-35 (hatched area) and 36-45 (Okazaki-fragments). 25, 10 and 2 denotes the size (in kb) and location of single-stranded DNA markers.
Cell Biology
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Vol. 6, No. 7, July 1982
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International
Reports,
Vol. 6, No. 7, July
687
1982
10 kb DNA REPLICATION INTERMEDIATES ARE FORMED FROM OKAZAKIFF&MENTS IN HUMAN MALIGNANT MELANOMA CELLS
Ulf Department
L&n
of Histology, Karolinska Karolinska Hospital, S-104
Institutet, and Radiumhemmet, 01 Stockholm, Sweden.
ABSTRACT Human melanoma cells were synchronized with hydroxyurea. The formed .either during the hydroxyDNA replication intermediates urea-treatment or after the release of the hydroxyurea-block were The intermediates were released from the parental investigated. DNA by lysing the cells in dilute alkali and were then analyzed After the release of the hydroxyby agarose gel electrophoresis. urea block radioactivity was first detected in Okazaki-fragments and then later also in larger replication intermediates (ranging in size up to 10 kb). The results indicate that the 10 kb DNA replication intermediate is derived from the Okazaki-fragments. Thus during its maturation to high molecular weight DNA the Okazaki-fragments form a large, well defined DNA replication intermediate, which later forms the high molecular weight DNA. Howit is not possible to determine whether all or only a part ever, of the Okazaki-fragments give rise to the 10 kb DNA. INTRODUCTION DNA replication in human cells is a very complex process is probably formation of several DNA replication (1) - There intermediates which are ligated to form the stable high molecular weight DNA. So far, we have obtained little knowledge about the different steps. The best known replication intermediate is the short Okazaki-fragment. However, we do not know if this fragment during its maturation to high molecular weight DNA forms larger DNA replication intermediates with a discrete size and a different half-life or if there is a continuous increase in the size of the fragment up to high molecular weight DNA with no formation of larger discrete replication intermediates. 030~1651/82/070687-1
O/$03.00/0
@ 1982 Academic
Press
Inc. (London)
Ltd.
688
Cell Biology
International
Reports,
Vol. 6, No. 7, July 7982
In an earlier paper we have described a procedure where cells that are growing in cell culture as monolayers are treated with dilute alkali directly in the culture dish (2). This will lyse the cells and the alkaline milieu will start the uncoiling of the DNA. The uncoiling is initiated at points where there are single-stranded gaps in the DNA. Such gaps occur normally in during the uncoiling of the DNA active replicons. Therefore, there is a release from the parental DNA of single-stranded DNA replication intermediates which will appear free in the solution. In asynchronous cell populations one would expect the release of DNA replication intermediates that range in size from Okazaki-fragments up to replicon-size. However, when we analyzed an asynchronous population of human melanoma cells we detected the release of single-stranded DNA replication intermediates ranging in size from that of Okazaki-fragments up to 10 kilobases (kb) (2). We did not detect DNA molecules ranging in size from 10 kb up to replicon size. The 10 kb DNA replication intermediate represents a discrete population of DNA molecules that can be easily detected using agarose gel electrophoresis. In the asynchronous cell population which we analyzed earlier we were not able to show that the Okazaki-fragments were labeled with tritiated thymidine before the 10 kb DNA intermediTherefore, we have now performed experiments in melanoma ate. cells that have been synchronized with hydroxyurea. The action of It causes the depletion of the hydroxyurea is well understood. dNTPs required for DNA synthesis, thereby inhibiting DNA repThe effect is readily reversible. The results lication (3,4). described in this paper show that the 10 kb DNA is formed by the joining of Okazaki-fragments. MATERIALS AND METHODS cells synchronization with Culturing of melanoma and obtained from Flow Laboratohydroxyurea: Human melanoma cells, ries, were grown as monolayers as earlier described (2). To synchronize the cells hydroxyurea was added to the medium to give a final concentration of 1 mM. The cells were incubated for 18 h and were then washed free of the drug and cultivated in fresh media for 6 h after which a second block with hydroxyurea was delivered. The duration of the second block was likewise 18 h. When the cells were released from the second block they were highly synchronized with more than 90 96 of the cells in S-phase as judged by autoradiography according to Ashihara and Baserga (5) -
Cell Biology
International
Reports,
Vol. 6, No. 7, July 1982
Labeling with tritiated thymidine and cell lysis: Cells to be used in experiments were grown as monolayers in small petri the DNA 100 uCi dishes (35x10 mm) with 3 ml medium. To label activity 30 Ci/mmol) was tritiated thymidine (Amersham, specific the labeling the medium was added to the medium. To terminate sucked off and the petri dishes placed on ice and the cells washed with cold phosphate buffered-saline. Cell lysis was performed in the dark at O°C by the addition into the petri dish of 2.25 ml 0.03 M NaOH. After 30 min the solution was neutralized by the addition of 0.9 ml 0.067 M HCl concencontaining 0.02 M NaH2P04 and SDS was added to a final tration of 1 %. After 60 min at room temperature the labeled DNA was analyzed by gelelectrophoresis either in 0.75 % agarose or in The electrophoretic separations were 6 % acrylamide gels (2,6). standardized by the separation in a parallel lane of the same gel-slab of labeled DNA size markers obtained from NEN (see The markers were denatured immediately before Figure Legends). the run by heating to 80°C for 10 min in 90 % formamide. RESULTS Formation of replication intermediates in the presence of hydroxyurea In the human melanoma cell line used here hydroxyurea (1 mM) rapidly inhibits the incorporation of thymidine in cold TCA-precipitable material. More than 90 % of the precipitable material was sensitive to deoxyribonuclease. The rate of DNA synthesis in hydroxyurea-treated cells were measured by 5 min pulses of tritiated thymidine at different times after the addition of the drug. It was found that after 15 min of treatment the rate of DNA synthesis had decreased to 6 % of the control value. Moreover the inhibition of DNA synthesis is reversible. When the drug was removed after treatment for 18 h and the cells incubated in fresh media this resulted in rapidly resumed synthesis of DNA. The rate of DNA synthesis was 46 % of the control value after incubation. of the cells in fresh media for 15 min and 88 % after incubation for 7 h. To analyze the formation of DNA replication intermediates human melanoma cells were lysed in dilute alkali (0.03 M NaOH) in the dark at O°C for 30 min. This approach, originally used to measure the amount of single-stranded DNA breaks present in the cellular DNA (7,8), has been modified by us to allow analysis of the formation of single-stranded DNA replication intermediates (2). In the alkaline milieu the melanoma DNA starts to uncoil; the uncoiling being initiated at single-stranded gaps present in
Cell Biology
international
Reports,
Vol. 6 No. 7, July 1982
active replicons. This will release single-stranded DNA replication intermediates into solution. When the solution is neutralized before the addition of detergent the high molecular weight DNA which is not completely denatured will renature and form double-stranded DNA whereas the released single-stranded DNA replication intermediates remain free in solution. When the total solution is then analyzed by agarose gel electrophoresis, the high molecular weight double-stranded DNA is located close to the trough (slices 3-6), whereas the single-stranded DNA replication intermediates enter the gel and are easily distinguished from the double-stranded DNA (2). The size of the single-stranded DNA range from that of Okazaki-fragments up to 10 kb and is divided into three populations ((2) cf. the control below in Fig. la). To establish the effect of hydroxyurea on the formation of DNA replication intermediates in human melanoma cells the following experiments were performed. Asynchronously growing cells were treated with the drug (1 mM) for 30 min and were then pulsed with tritiated thymidine during the last 45 seconds of the drug treatment. The cells were lysed in dilute alkali and the DNA then were always treated analyzed in 0.75 % agarose gels. The controls in parallel with the difference, however, that hydroxyurea was not present. The control DNA, when separated in the agarose gel (Fig. is located either close to the trough (the double-stranded la), high molecular weight DNA) or enters the gel (single-stranded DNA). The DNA entering the gel consists of three populations: the 10 kb DNA population (slices 19-25), the Okazaki-fragments (sliand the intermediate sized DNA population (slices ces 36-45), These three DNA populations have been found earlier to 26-35). have the kinetic behavior expected of DNA replication intermediates (2). After a pre-treatment with hydroxyurea for 30 min the main part of the radioactivity is located at slices 36-45 (OkazakiThe inhibition of labeling of the high fragments) (Fig. la). molecular weight DNA located close to the trough (slices 3-6) and the 10 kb DNA replication intermediate located at slices 19-25 is almost complete. This contrasts with the control described above where these two DNA populations are equally or more extensively labeled than the Okazaki-fragments. The size of the labeled Okazaki-fragments were established in 6 % acrylamide-gels (Fig. lb). The results show that the labeled DNA pieces are between LOO-200 bases, i.e. the same as in the control cells (2). Likewise control experiments in which the DNA was banded in CsCl showed that this DNA is a single-stranded DNA (not shown).
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Vol. 6, No. 7, July 7982
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synchronized with hydroxyurea Hydroxyurea is often used to synchronize cell cultures at Cl/S (5). To synchronize the human melanoma cells used in this study the following procedure was used. The cells were treated with 1 mM hydroxyurea for 18 h, washed free of the drug and then cultivated further for 6 h in fresh media. Then a second block with hydroxyurea was delivered for another 18 h. To evaluate the degree of synchronization the cells were labeled with tritiated thymidine after the release of the second block and then examined by autoradiography (5). It was found that there is good synchrony of the cells with more than 90 % in S-phase immediately after the second hydroxyurea block was removed. In order to establish whether it is possible to label the Okazaki-fragments before the 10 kb DNA molecules we investigated the synthesis of the DNA replication intermediates after the release of the second hydroxyurea block. Thus, the drug was thus removed and the cells were allowed to grow in fresh media and were pulsed with tritiated thymidine for 45 seconds at different times after the removal of the drug. In the first set of experiments the formation of DNA replication intermediates were analyzed immediately after the removal of hydroxyurea (Fig. Za). Analysis of the labeled material shows the preferential labeling of Okazaki-fragments as described earlier in Fig. la. There was almost no label in the position of the 10 kb DNA replication intermediate or in the high molecular weight DNA. However, 15 min after the release of the block there was label, apart from the Okazaki-fragment, also in the 10 kb DNA replication intermediate, the DNA located at slices 26-35 and the high molecular weight DNA located at slices 3-6 (Fig. 2b). Still the Okazaki-fragments dominated over the 10 kb DNA fragments. Seven hours after the release of the hydroxyurea block the electrophoretic separation is similar to the controls with roughly equal amounts of radioactivity in the 10 kb DNA fragments and the Okazaki-fragments (Fig. 2c, compare Fig. la). To demonstrate that the labeled DNA intermediates formed after the release of the block are incorporated into the high molecular weight DNA the following experiment was performed. Cells were labeled for 45 seconds immediately after the release of the second block and were then cultivated further for 24 h. The electrophoretic analysis of the labeled DNA thus obtained showed a stable high molecular weight DNA with no indication of the release of labeled DNA intermediates (Fig. 2d). This is the same result as in the parallel control cells not treated with hydroxyurea (see also (2)). Thus, during the recovery of DKA synthesis one first detects the
Cell Biology
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Reports,
Vol. 6, No. 7, July 7982
Okazaki-fragments and later also 10 kb DNA intermediates, the intermediate located at slices 26-35, and the high molecular that the 10 kb DNA intermediate is weight DNA. This indicates formed by the joining of Okazaki-fragments. DISCUSSION Using the procedure to lyse asynchronously growing melanoma cells in dilute alkali we were previously able to detect three different single-stranded DNA replication intermediates, i.e. Okazaki-fragments, a 10 kb DNA fragment and a population of molecules ranging in size from Okazaki-fragments up to 10 kb (slices 26-35 in the agarose gel) (2). In this paper we have analyzed the formation of these DNA replication intermediates in cells synchronized with hydroxyurea. The function of hydroxyurea is well known. It inhibits the DNA synthesis by inhibiting the enzyme ribonucleotide reductase and thereby depleting the pools for dGTP and dATP (3,4). This frequently results in the accumulation of Okazaki-fragments which are not joined together to form high molecular weight DNA (9). As expected, also in human melanoma cells there is an accumulation of Okazaki-fragments after a pretreatment with hydroxyurea. Neither the other single-stranded DNA replication intermediates nor the high molecular weight DNA as with tritiated thymidine. labeled When the cells are released from the second hydroxyureablock more than 90 % of the cells are in S-phase. When pulsed with tritiated thymidine for 45 seconds at different times after the release of the block, at first there is radioactivity only in the Okazaki-fragments. 15 min after the release of the block there is also radioactivity in the intermediate DNA population at slices 26-35, the 10 kb DNA intermediate, and the high molecular weight DNA. Finally, when the cells were pulsed 7 h after the removal of the hydroxyurea one can detect a distribution of radioactivity among the different DNA populations which is similar to that in the control cells which were growing asynchronously. Moreover, experiments in which the DNA was labeled for 45 seconds immediately after the release from the block and then analyzed 24 hours later showed no release of labeled DNA replication intermediates. Thus the labeled intermediates formed after the hydroxyurea synchronization will after a time-lag form a stable high molecular weight DNA, as in the control cells. It is not possible to distinguish between the appearance of radioactivity in the 10 kb DNA fragments and the intermediate DNA population (slices 26-35). However, it is obvious that both populations are formed later than the Okazaki-fragments and therefore
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693
Vol. 6, No. 7, July 1982
in all probability are derived from the Okazaki-fragments. Thus during the maturation to high molecular weight DNA the Okazakifragments form a large, well defined DNA replication intermediate, which later form the high molecular weight DNA. However, it is not possible from our work to determine whether all or only a part of the Okazaki-fragments give rise to the 10 kb DNA. A prerequisite for the release of the 10 kb DNA from the parental DNA during our cell lysis condition is that there exist gaps in the continuity of the newly synthesized DNA spaced roughThese gaps would serve as initialy 10 kb away from each other. tion points for the uncoiling of the DNA when the cells are lysed in dilute alkali and thereby produce a discrete population of 10 kb DNA molecules. However, these gaps should represent a transient state since in the steady-state labeled DNA one can not detect the release of these DNA fragments. Hence, it is probable that the presence of these gaps should represent a defined step during the maturation of the newly synthesized DNA.
ACKNOWLEDGEMENTS This Stockholm,
work was supported by grants from the Cancer and the E. Welander Foundation.
Society
in
REFERENCES 1. Hand, R (1978) Eukaryotic DNA: Organization of the genome for replication. Cell 15, 317-325 2. Liinn, U (1982) Detection of a 10 kb DNA replication intermediate in human melanoma cells. Chromosoma (in press) 3. Krakoff, IHN, Brown, C and Reichard, P (1968) Inhibition of ribonucleoside diphosphate reductase by hydroxyurea. Cancer Res. 28, 1559-1565 4. Skoog, L and Nordenskjcld, B (1971) Effects of hydroxyurea and 1-B-arabinofuranosylcytosine on deoxyribonucleotide pools in mouse embryo cells. Eur. J. Biochem. 19, 81-89 5. Ashihara, T and Baserga, R (1979) Cell Synchronization, in "Methods in Enzymology", vol. LVIII 248-262 6. L&n, U (1977) Exclusive nuclear location of precursor 4 S RNA. J. Mol. Biol. 112, 661-666 7. AhnstrGm, G and Erixon, K (1973) Radiation induced strand breakage in DNA from mammalian cells. Strand separation in alkaline solution. Int. J. Radiat. Biology 23, 285-289
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8. Rydberg, G (1975) The rate of strand separation in alkali DNA of irradiated mammalian cells. Radiation Res.
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Received:
22nd December 2981
Modified version accepted: 16th Apm-2 1982
Fig. 1 Formation of DNA replication intermediates in the presence of hydroxyurea. The human melanoma cells were treated with hydroxyurea (1 mM) for 30 min and during the last 45 seconds tritiated were lysed as described and the thymidine was added. The cells DNA then subjected to electrophoresis in either (a) 0.75 % agaroControls were always treated se gels or (b) 6 % acrylamide gels. in parallel with the difference that hydroxyurea was not present. the size (in kb) and location of In (a) 25, 10 and 2 denotes DNA populations are single-stranded marker DNA. Four labeled visible. The high molecular weight double-stranded DNA located at slices 3-6 and three populations of single-stranded DNA located at slices 19-25, 26-35 (hatched area) and 36-45, respectively. In (b) 194, 118 and 72 denotes the size (in bases) and location of single-stranded DNA markers. -e- hydroxyurea-treated cells, -ocontrol cells. Fig. 2 Cells synchronized with hydroxyurea. The cells were released from the second block with hydroxyurea and were then pulsed with tritiated thymidine for 45 seconds either (a) immediately after the release, (b) after 15 min of cultivation in fresh media, (c) after 7 hours of cultivation in fresh media or (d) cells labeled for 45 seconds immediately after the release of the block and then cultivated for 24 hours in fresh media. The cells were lysed in dilute alkali and the labeled DNA then separated in 0.75 % agarose gels. A total of four DNA populations are formed in the The high molecular weight double-stranded DNA different panels. Three populations of single-stranded is located at slices 3-6. DNA is located at slices 19-25 (10 kb), 26-35 (hatched area) and 36-45 (Okazaki-fragments). 25, 10 and 2 denotes the size (in kb) and location of single-stranded DNA markers.
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