©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mimosine Arrests DNA Synthesis at Replication Forks by Inhibiting Deoxyribonucleotide Metabolism (*)

David M. Gilbert (2)(§), Ann Neilson (1), Hiroshi Miyazawa (2)(¶), Melvin L. DePamphilis (2), William C. Burhans (1)(**)

From the (1) Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263 and the (2) Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Mimosine has been reported to specifically prevent initiation of DNA replication in the chromosomes of mammalian nuclei. To test this hypothesis, the effects of mimosine were examined in several DNA replication systems and compared with the effects of aphidicolin, a specific inhibitor of replicative DNA polymerases. Our results demonstrated that mimosine inhibits DNA synthesis in mitochondrial, nuclear, and simian virus 40 (SV40) genomes to a similar extent. Furthermore, mimosine and aphidicolin were indistinguishable in their ability to arrest SV40 replication forks and mammalian nuclear chromosomal replication forks. In contrast to aphidicolin, mimosine did not inhibit DNA replication in lysates of mammalian cells supplied with exogenous deoxyribonucleotide triphosphate precursors for DNA synthesis. Mimosine also had no effect on initiation or elongation of DNA replication in Xenopus eggs or egg extracts containing high levels of deoxyribonucleotide triphosphates. In parallel with its inhibitory effect on DNA synthesis in mammalian cells, mimosine altered deoxyribonucleotide triphosphate pools in a manner similar to that reported for another DNA replication inhibitor that affects deoxyribonucleotide metabolism, hydroxyurea. Taken together, these results show that mimosine inhibits DNA synthesis at the level of elongation of nascent chains by altering deoxyribonucleotide metabolism.


INTRODUCTION

Chemical agents that reversibly inhibit DNA synthesis have proven to be effective tools for investigating DNA replication in mammalian cells. Until recently, however, all of these agents were believed to target cellular processes involved in the elongation phase of DNA replication. In fact, the lack of agents that specifically inhibit initiation of DNA replication is in part responsible for the paucity of information that exists on the initiation process.

A recent candidate for a specific inhibitor of initiation of DNA replication is the plant amino acid mimosine (- N(3-hydroxy-4-pyridone)--amino propionic acid). Fluorescence activated cell sorter analyses of exponentially proliferating mammalian cells arrested with mimosine suggested that they accumulated in the late Gphase of their cell division cycle (1, 2, 3, 4) . More detailed studies revealed that mimosine inhibited DNA replication throughout S phase but only after several hours of treatment (5, 6, 7) . Two observations in these studies supported the conclusion that mimosine specifically blocked initiation of new replicons. First, the length of time required for mimosine to inhibit DNA replication in cells that were synchronized at the beginning of S phase with aphidicolin was substantially longer than the time required to inhibit replication in exponentially proliferating cells. Since aphidicolin allows formation of replication bubbles but prevents subsequent expansion of these bubbles by inhibiting specifically DNA polymerases , , and (8) , mimosine appeared to inhibit replication only when new initiation events were required to sustain S phase, analogous to ``slow stop'' mutations in bacteria or yeast that specifically block initiation of DNA replication (9) . Second, replication fork structures were not detected by two-dimensional gel electrophoresis after prolonged treatment of cells with mimosine. This observation suggested that mimosine prevented formation of new replication forks, while allowing previously existing replication forks (present in replicons initiated prior to addition of mimosine) to complete replication. As a result of these studies, mimosine has recently been employed by a number of laboratories under the assumption that this drug synchronizes cells just prior to the onset of DNA replication (1, 3, 4, 10, 11, 12, 13, 14, 15, 16, 38) .

While mimosine may serve as a useful agent for synchronizing cells, accurate interpretation of experimental data depends on understanding the mechanism of mimosine action. The evidence that mimosine inhibits initiation of DNA replication is indirect, and alternative explanations exist for the effects described above. For example, mimosine could inhibit nucleotide metabolism, resulting in delayed arrest of DNA synthesis at replication forks followed by destabilization of replication fork structures. Therefore, it was important to determine whether or not mimosine was a specific inhibitor of initiation of DNA replication in cellular chromosomes or a general inhibitor of DNA synthesis at replication forks.

To this end, we compared the effects of mimosine on nuclear, mitochondrial, and simian virus 40 (SV40) DNA replication, as well as on DNA replication in Xenopus eggs. The mechanisms by which DNA replication occurs in these four systems differ substantially. For example, initiation of mitochondrial DNA replication occurs throughout the cell cycle, whereas initiation of nuclear DNA synthesis is limited to S phase. Moreover, mitochondria do not utilize any of the enzymes required at DNA replication forks in mammalian nuclei (17) . Although SV40 and nuclear DNA synthesis appear to share most of the enzymes required at replication forks, initiation of SV40 replication differs significantly in that it is controlled by the viral-encoded origin recognition protein large tumor antigen (T-ag), and it is not limited to one initiation event per cell cycle (18) . The mechanism by which nuclear DNA replication is initiated is as yet unknown, but it restricts cellular chromosomes to one initiation event per origin per S phase (19) . Xenopus eggs provide the most well characterized system that can initiate replication in non-viral DNA sequences as well as continue DNA synthesis at replication forks. Like mammalian cells, DNA replication in Xenopus eggs is restricted to one initiation event per cell cycle (20) . However, in contrast to mammalian cells, which initiate replication at specific chromosomal loci (21) , Xenopus eggs can initiate replication within most (perhaps all) DNA sequences that are presented to them (22, 23, 24) . Furthermore, whereas initiation of replication in mammalian chromosomes requires specific DNA sequences (25) , initiation of replication in Xenopus eggs does not (22, 26) .

The results of our study indicated that mimosine inhibited DNA synthesis in cellular, mitochondrial, and SV40 chromosomes to the same extent despite fundamental differences in the way these types of chromosomes are replicated. Furthermore, mimosine had the same inhibitory effect on elongation of nascent DNA chains in both cellular and SV40 replicating chromosomes as did aphidicolin, a specific inhibitor of replicative DNA polymerases. Finally, in parallel with its inhibitory effect on DNA synthesis, prolonged mimosine treatment altered intracellular levels of deoxyribonucleotide triphosphates (dNTPs) in a manner similar to that of another DNA replication inhibitor that affects deoxyribonucleotide metabolism, hydroxyurea.

These results demonstrate that mimosine can block cell cycle progression within S phase at the level of elongation of nascent DNA chains by causing the depletion of intracellular pools of deoxyribonucleotides. This is consistent with our observation that, in contrast to aphidicolin, mimosine did not inhibit DNA replication in lysates of mammalian cells supplied with an exogenous source of deoxyribonucleotides or in extracts of Xenopus eggs or intact Xenopus eggs, both of which contain high levels of deoxyribonucleotides. Consequently, studies that have employed mimosine to block cell cycle progression in late Gmay require reinterpretation.


MATERIALS AND METHODS

Culture and Synchronization of Cells

CHO() C400 hamster and BSC-1 monkey cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and nonessential amino acids. Xenopus Xtc kidney cells were grown at 23 °C in amphibian medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and 25 m M HEPES (pH 7.0). For experiments requiring populations of cells synchronized with aphidicolin at their G/S boundary, cells were first collected in Gby isoleucine deprivation (27) followed by release into complete medium containing 5 µg/ml aphidicolin for 12 h. Mimosine (Aldrich) was stored at 4 °C as a 10 m M solution in phosphate-buffered saline, and aphidicolin (Boehringer-Mannheim) was stored as a 2 mg/ml solution in dimethyl sulfoxide at -20 °C.

Nuclear and Mitochondrial DNA Replication

Nascent nuclear and mitochondrial DNA (mtDNA) was radiolabeled for 20 min with 10 µCi/ml [H]thymidine (62 Ci/mmol) (Amersham Corp.) per 10-cm dish of cells. When mtDNA replication was to be quantified, culture medium included 5 µg/ml 1-(- D-arabinofuranosyl)cytosine to suppress nuclear DNA replication. Cells were lysed with 3 ml/dish of 50 m M Tris-HCl (pH 7.4), 10 m M EDTA, 400 m M NaCl, 0.6% sodium dodecyl sulfate, and 0.2 mg/ml Proteinase K (Boehringer Mannheim) by incubating them for 16 h at 37 °C. DNA was purified by extracting the lysate with phenol and chloroform-isoamyl alcohol and was then precipitated with ethanol and redissolved in 100 µl/dish of 10 m M Tris-HCl (pH 7.8) and 1 m M EDTA. Aliquots containing 8 µg of DNA were digested with various restriction enzymes according to the supplier's instructions and then fractionated by electrophoresis in 0.8% agarose gels in a Tris borate-EDTA buffer (28) . H-DNA was visualized by exposing dried gels to preflashed x-ray film for various lengths of time at -70 °C with an intensifying screen (29) after they had been processed for fluorography (30) . Total DNA replication was quantified by precipitation with trichloroacetic acid (28) . Mitochondrial DNA replication was quantified by scanning densitometry of autoradiograms that were exposed within the linear response range of the film.

SV40 DNA Replication

BSC-1 monkey cells (50% confluence) in 10-cm dishes were infected with SV40 virus (m.o.i. = 10). At 24 h post-infection, either 0.4 m M mimosine or 5 µg/ml aphidicolin was added to the medium, and 3 h later, nascent viral DNA was labeled with 50 µCi/ml [H]thymidine (80 Ci/mmol) (Amersham) for 15 min. The cells were then lysed with 3 ml/plate of 10 m M EDTA, 0.6% SDS (31) . After 5 min, 1 ml of 5 M NaCl was added dropwise to each plate, and lysates were transferred gently to Falcon 2059 tubes, chilled on ice overnight to precipitate cellular DNA, and then centrifuged in a Beckman JS13.1 rotor at 10,000 rpm for 30 min (4 °C) to remove cellular DNA. DNA in the supernatant was extracted with phenol and chloroform-isoamyl alcohol, precipitated with ethanol, dissolved in distilled water (100 µl/dish of cells), and then adjusted to 150 m M NaCl with a 10 buffer stock (New England BioLabs) appropriate for digesting DNA with BamHI.

DNA was analyzed by neutral-neutral two-dimensional gel electrophoresis (32) . Aliquots (10 µl) of purified SV40 DNA were digested with a 10-fold excess of BamHI for 2 h at 37 °C and then applied to a 0.6% agarose gel in a 40 m M Tris acetate and 1 m M EDTA (40) containing 3 µg/ml ethidium bromide and subjected to electrophoresis for 16 h at 1 V/cm (ambient temperature). DNA was visualized by 360 nm UV irradiation to excise each lane accurately. Each excised lane was laid across the top of a 1% agarose gel in 45 m M Tris borate, 1 m M EDTA, and 30 µg/ml ethidium bromide (28) and embedded with additional agar. Electrophoresis in the second dimension was carried out in buffer containing ethidium bromide at 4 °C for 20 h at 6 V/cm. DNA was then transferred to nylon membranes (GeneScreen Plus) and probed with P-labeled SV40 DNA, and replication intermediates were visualized by exposure to Phosphorimager screens (Molecular Dynamics). Alternatively, gels were prepared for fluorography to visualize nascent [H]thymidine-labeled DNA as described above.

Early Labeled DNA Fragment Assay

CHO C400 cells were synchronized in Gby isoleucine deprivation and then subjected to one of three protocols. Gphase cells were prepared by releasing them into Dulbecco's modified Eagle's medium supplemented with nonessential amino acids and 10% fetal bovine serum (complete medium) for 2 h. Mimosine-arrested cells were prepared by releasing them into complete medium containing 0.4 m M mimosine for 12 h. Aphidicolin-arrested cells were prepared by releasing them into complete medium containing 5 µg/ml aphidicolin for 12 h and then replacing the aphidicolin-containing medium with prewarmed complete medium and incubating them for an additional 5 min. Cells were harvested by trypsinization, counted, and transferred to 1.5-ml microcentrifuge tubes (5 million cells/tube) on ice, washed with cold hypotonic buffer (33) , and finally swollen for 10 min on ice in hypotonic buffer. Cells were pelleted by centrifugation, supernatant completely removed, and then lysed in the same microcentrifuge tube by homogenization with 20 strokes of a Kontes pellet pestle. Nuclei were then isolated by centrifugation at 14,000 rpm for 3 min (4 °C) in an Eppendorf 5415C microcentrifuge and resuspended in 100 µl of S phase extract prepared from CHO K1 cells (33) . To this mixture, 25 µl of 5 replication mixture (33) was added; the mixture was then transferred to a 1.5-ml microcentrifuge tube containing 20 µl of lyophilized [-P]dATP (6000 Ci/mmol) (Amersham). Tubes were incubated at 37 °C, and 25-µl aliquots were taken at 10, 20, 30, 60, and 120 min. These samples were added to 300 µl of 0.5% SDS, 50 m M Tris-HCl (pH 8.0), and 10 m M EDTA to stop the reaction. Acid-precipitable radioactivity was measured from 50-µl aliquots of this cell lysate by addition of trichloroacetic acid (28) . DNA was isolated from the remaining sample by digesting it with 100 µg/ml Proteinase K for 2 h at 56 °C and then extracting once with phenol:chloroform and once with chloroform alone before adding 100 µl of 7.5 M ammonium acetate and adjusting the solution to 80% ethanol. DNA was precipitated, resuspended in EcoRI buffer (New England BioLabs), digested with 15 units EcoRI, and fractionated by electrophoresis in an 0.65% agarose gel containing 45 m M Tris borate and 1 m M EDTA (28) for 14 h at 2.3 V/cm and 21 °C. Gels were dried and exposed to Kodak XAR-5 film at room temperature.

DNA Replication in Xenopus Eggs and Egg Extracts

Xenopus egg extracts and Xenopus sperm chromatin were prepared as described by Blow and Laskey (20) . Sperm chromatin was added to 20 µl of extract supplemented with 0.5 µl of [-P]dATP (6000 Ci/mmol) (Amersham) to a final concentration of 3 ng of DNA/µl extract, and the indicated concentrations of mimosine or aphidicolin were added. Extracts were incubated at 21 °C, and at the indicated times, 2.5-µl aliquots were removed, and DNA was precipitated with trichloroacetic acid (28) . For analysis of plasmid replication, 1.6 ng of pneoS13 (34) was injected into unactivated Xenopus eggs that were activated 2 h later by addition of Caand Caionophore A24187 (35) . Ten eggs were collected at each of the indicated times and pooled, and plasmid DNA was isolated from each pool of eggs. One-tenth of the plasmid DNA was supplemented with 300 ng of bacteriophage DNA to provide an internal standard to monitor subsequent digestions and to ensure complete digestion with DpnI (36) . DNA was digested for 1 h with 5 units of DpnI (New England BioLabs) per ng of isolated plasmid DNA in buffer 4 (New England BioLabs) supplemented with 200 m M NaCl to prevent cleavage of hemimethylated DNA (36) . All forms of DpnI-resistant DNA as well as identical aliquots that were not digested with DpnI were subsequently converted to linear molecules with PvuI, and the products fractionated by electrophoresis in 0.65% agarose gels in 45 m M Tris borate and 1 m M EDTA for 15 h at 2.4 V/cm (28) . DNA was then transferred to a nylon membrane (Hybond-N+, Amersham) by capillary blotting using 0.4 M NaOH and 0.6 M NaCl. DNA on the membrane was hybridized with pUC19 P-DNA (37) , and the amount of linear monomeric plasmid DNA was determined by scanning the filters in a Betagen Betascope 603. Identical aliquots of plasmid DNA were digested with 5 units of MboI (New England BioLabs) in buffer 4 (New England Biolabs). DNA was hybridized with P-labeled neomycin gene (0.75-kb PvuII fragment of pSV2neo). The amount of the largest MboI DNA digestion product, a 0.56-kb P-DNA band, was determined by scanning filters in the Betascope.

Measurement of dNTP Pools

dNTPs were extracted according to the method of Skoog and Nordenskjold (48) . Briefly, 10-cm plates containing 10exponentially proliferating CHO C400 cells were washed once with phosphate-buffered saline and then scraped into ice-cold 60% methanol (1 ml/plate). Lysates were stored at -20 °C overnight and were then centrifuged to remove cell debris. The supernatant was evaporated to dryness, and dNTPs were redissolved in 100 µl/plate water. Amounts of dNTP in the extracts were determined by measuring incorporation by Klenow enzyme of complementary [H]dNTPs in DNA synthesis reactions using 10 µl of extract and poly(dAdT) or poly(dIdC) as a template (39) .


RESULTS

Mimosine Inhibits Both Nuclear and Mitochondrial DNA Synthesis

To determine whether the effects of mimosine were specific to nuclear DNA synthesis, the ability of mimosine to inhibit [H]thymidine incorporation into both mitochondrial and nuclear DNA was tested in CHO cells. One group of cells was treated with various concentrations of mimosine for 3 h and then, in the continued presence of mimosine, incubated with [H]thymidine for 20 min. Total cellular DNA was isolated, digested with EcoRI restriction endonuclease, and then fractionated by gel electrophoresis. A second group of cells was treated in the same manner except that 1-(- D-arabinofuranosyl)cytosine was included to specifically inhibit nuclear DNA synthesis and thus reveal the presence of mitochondrial DNA restriction fragments (30) . These data revealed that mimosine inhibited both mitochondrial and nuclear DNA synthesis (Fig. 1). Moreover, the sensitivities of mtDNA and nuclear DNA synthesis to mimosine were indistinguishable. Both mitochondrial and nuclear DNA synthesis were inhibited to the same extent at the same concentrations of mimosine (Fig. 2 A), and the rates of recovery for both mitochondrial and nuclear DNA synthesis upon removal of mimosine were the same (Fig. 2 B). These results suggested that mimosine acts on some metabolic pathway that is common to both nuclear and mitochondrial DNA replication. Furthermore, since mtDNA replication occurs throughout the cell cycle and the length of the mimosine treatment of exponentially proliferating cells in these experiments was a small fraction of the total time required for cell division (20 h), mimosine must inhibit mtDNA replication throughout the entire cell cycle, not just during S phase.


Figure 2: Characteristics of mimosine inhibition of mitochondrial and nuclear DNA replication were the same. A, exponentially proliferating cultures of CHO C400 cells were treated for 3 h with mimosine at the indicated concentration. [H]Thymidine was added for 20 min. Nuclear DNA synthesis () was quantified in cells not treated with araC by acid precipitation of total cellular DNA, while mtDNA synthesis () was quantified in cells treated with araC by densitometry of fluorographs as described in Fig. 1. AraC suppressed nuclear DNA synthesis and thus revealed mtDNA synthesis (Fig. 1). Relative DNA synthesis was expressed as a percentage of the amount detected in untreated cultures or just prior to addition of mimosine (time = 0). B, exponentially proliferating cultures of CHO C400 cells were treated with 0.4 m M mimosine for 3 h to completely arrest DNA synthesis and then released from mimosine arrest by washing them twice with fresh medium without mimosine and culturing them in the absence of mimosine for the times indicated. [H]Thymidine was added for 20 min, and both nuclear and mitochondrial DNA synthesis was measured as in panel A. C, cells were synchronized at their G/S boundary with aphidicolin (see ``Materials and Methods'') and then released from the aphidicolin block in the presence of 0.4 m M mimosine ( solid symbols). Replicating mtDNA was monitored as in previous experiments except that nuclear DNA replication was suppressed by not withdrawing aphidicolin from the culture medium (rather than the addition of araC). Exponentially proliferating cells were also treated with 0.4 m M mimosine and analyzed in parallel as in panel A.



In previous studies, a crucial observation was that mimosine took substantially longer to arrest DNA synthesis in cells that had been synchronized at the beginning of their S phase with aphidicolin than in unsynchronized cells that were proliferating exponentially (7) . Aphidicolin does not prevent synthesis of short RNA-p-DNA chains that precede formation of Okazaki fragments (40) . Thus, aphidicolin allows both SV40 (41) and cellular (5) chromosomes to initiate DNA replication by forming replication forks but limits subsequent DNA synthesis according to the dose and time of exposure. Therefore, the delayed response to mimosine suggested that mimosine inhibition did not occur until new initiation events took place to continue S phase, analogous to the ``slow-stop'' phenotype that characterizes mutations in genes required specifically to initiate replication in bacteria and yeast (9) .

We were able to confirm this observation with mimosine inhibition of nuclear DNA synthesis. When CHO C400 cells were synchronized with aphidicolin near the G/S boundary (shortly after entry into S phase) and released from the aphidicolin block into medium containing mimosine, inhibition of DNA replication did not begin until 2-3 h and was complete after a 4-5-h incubation (Fig. 2 C). In contrast, inhibition of DNA synthesis in exponentially proliferating cells began 30-60 min after mimosine addition and was completed after 2-3 h (Fig. 2 C). These data on mimosine inhibition of nuclear DNA synthesis are in excellent agreement with those of Mosca et al. (7) . However, we observed the same phenomenon with mtDNA synthesis. Since mtDNA replication responded in an identical manner even though mtDNA replication is not affected by aphidicolin, the delayed response phenotype in G/S cells cannot be related to synchronization of chromosomal replicons by aphidicolin. The fact that mimosine inhibition of nuclear and mtDNA synthesis were indistinguishable under all conditions strongly suggests that mimosine does not specifically inhibit some step in the initiation of cellular chromosome replication.

Mimosine Inhibits DNA Synthesis at SV40 Replication Forks

To examine directly the effects of mimosine on DNA replication forks, replication of SV40 DNA was analyzed in mimosine-treated virus-infected monkey cells. At 24 h post-infection, BSC-1 cells were cultured for 3 h with different amounts of mimosine. Uninfected cultures were treated in parallel. Cells were then incubated with [H]thymidine for 20 min before DNA was extracted, digested with EcoRI, and fractionated by gel electrophoresis. Ethidium bromide staining of the gels indicated that, prior to addition of mimosine, SV40 DNA had replicated to the same extent in both treated and untreated cells (data not shown). Fluorographic detection of H-DNA in the same gels revealed that both nuclear and SV40 DNA synthesis were inhibited strongly by 200 µ M mimosine (Fig. 3).

If mimosine prevents initiation of SV40 DNA replication, then incorporation of [H]thymidine into SV40 replicating intermediates will cease only when all of the viral replicating intermediates present prior to mimosine treatment complete their replication cycle (18, 42) . At that time, no SV40 replicating intermediates will be present. However, if mimosine blocked DNA synthesis at replication forks, then replicating intermediates will be present but inactive. To distinguish between these two possibilities, virus-infected BSC-1 cells were treated with 0.4 m M mimosine for either 3 or 6 h and then incubated with [H]thymidine for 20 min to radiolabel replicating intermediates. Viral DNA was isolated and cut at the single BamHI site located in its termination region for replication to produce a collection of replication bubble structures (``-structures'') of various sizes in the absence of replication fork structures (``Y-structures'') (Fig. 4 A). The products of this digestion were then fractionated by the neutral-neutral two-dimensional gel electrophoresis technique of Brewer and Fangman (32) to identify bubble and fork structures (Fig. 4 A). Newly synthesized SV40 H-DNA was detected by fluorography (Fig. 4 B). Total SV40 DNA was detected by transferring the DNA from the gel to a nylon filter and then hybridizing it with SV40 P-DNA (Fig. 4, C and D).


Figure 4: Mimosine arrests SV40 replication forks. Replicating SV40 DNA was radiolabeled in three parallel groups of virus-infected BSC-1 cells. At 24 h post-infection, one group of cells was treated with 0.4 m M mimosine, one group with 5 µg/ml aphidicolin, and one group left untreated for either a 3- or 6-h period. SV40-replicating DNA was then radiolabeled for 20 min with [H]thymidine, and SV40-replicating intermediates were purified, digested at the single BamHI site located in the termination region to produce a population of replication bubbles, and then analyzed by neutral-neutral two-dimensional gel electrophoresis ( A). The migration pattern of unreplicated SV40 DNA, unreplicated cellular DNA remaining in the Hirt supernatant, replication bubbles ( solid line), and replication forks ( dotted line) are indicated. B, H-DNA was visualized by fluorography. C and D, DNA in duplicate gels was transferred to a membrane, hybridized with SV40 P-DNA, and detected by phosphorimage analysis.



The results showed clearly that mimosine arrested DNA synthesis at replication forks. Cells treated with mimosine for 3 h no longer synthesized SV40 DNA, as determined by incorporation of [H]thymidine (Fig. 4 B), but they still contained at least 60% as many SV40 replicating intermediates as untreated cells (Fig. 4 C), as determined by Phosphorimager analysis of the two-dimensional gels. The slow disappearance of SV40 replicating intermediates observed at 3 and 6 h after addition of mimosine (Fig. 4 C) resulted from inhibition of DNA synthesis at replication forks because experiments carried out in parallel with aphidicolin instead of mimosine gave identical results (Fig. 4 D). Furthermore, ``Y-arcs'' (Fig. 4 A) produced by molecules containing a single fork also were observed in DNA recovered from mimosine or aphidicolin treated cells but not from untreated cells (Fig. 4, C and D). These molecules result from a break at one of the two forks in a single replicating intermediate; breaks at both forks would produce simple linear molecules. These results are consistent with previous studies showing that prolonged exposure to aphidicolin produces breaks at SV40 DNA replication forks (43, 44) . Thus, the effects of mimosine and aphidicolin on SV40 DNA replication were indistinguishable; inhibition of viral DNA synthesis at replication forks followed by destabilization of replication forks and slow disappearance of replicating intermediates.

Mimosine Does Not Block Entry of Mammalian Cells into S Phase

To determine whether mimosine blocks cells before or after they have initiated DNA replication ( i.e. entered S phase), nuclei from mimosine-arrested mammalian cells were tested for their ability to carry out DNA replication in an extract prepared from S phase CHO cells. These extracts, when supplemented with the four deoxyribonucleotide substrates for DNA synthesis, the four ribonucleotide substrates for RNA primer synthesis, and an ATP regenerating system, can support DNA synthesis in nuclei from S phase cells but cannot initiate replication in nuclei from Gphase cells (22, 33) . CHO C400 cells were synchronized in their Gphase by first arresting their proliferation through isoleucine deprivation and then releasing them into complete medium for 2 h. Other cells were synchronized at the beginning of S phase by first arresting them through isoleucine deprivation and then releasing them into complete medium containing 5 µg/ml aphidicolin for 12 h. A third group of cells was released into complete medium containing 0.4 m M mimosine for 12 h. Nuclei were then isolated from each population and incubated in CHO cell extract supplemented with a reaction mixture that included [-P]dATP. As previously reported (22, 33) , nuclei from cells that had entered S phase (arrested with aphidicolin) continued to synthesize DNA when placed in these cell extracts, while nuclei from cells in Gphase did not (Fig. 5 A). Nuclei from cells arrested in mimosine behaved like nuclei from cells arrested in S phase (Fig. 5 A), revealing that mimosine did not arrest cells prior to initiation of DNA replication.


Figure 5: Mimosine arrests nuclear DNA synthesis after formation of replication forks. CHO C400 cells were synchronized in their Gphase, arrested with aphidicolin ( Aph), or arrested with mimosine ( Mim) and subjected to early labeled fragment analysis as described under ``Materials and Methods.'' Isolated nuclei were allowed to synthesize DNA in the presence of [-P]dATP, and the amount of acid-precipitable P-DNA was measured at the indicated times ( A). The number of nuclei per microliter of extract was counted, and the DNA synthesis was expressed as a percentage of input DNA (6.6 pg of DNA/nucleus). B, alternatively, DNA was extracted from nuclei radiolabeled for 20 min as in A, digested with EcoRI, and then fractionated by gel electrophoresis. Nuclei were also isolated from exponentially proliferating CHO C400 cells and radiolabeled as in panel A. The gel was dried and exposed to film. Indicated are the positions of mtDNA fragments ( mt), DNA fragments from the amplified dihydrofolate reductase locus, and the DNA fragment containing the origin of bidirectional replication ( OBR-1) 17 kb downstream from the dihydrofolate reductase gene. The schematic representation at the bottom represents the 273-kb amplified dihydrofolate reductase gene region that includes another structural gene, 2BE2121. Horizontal arrows indicate directions of transcription.



The pattern of early labeled DNA restriction fragments from the dihydrofolate reductase gene locus was examined to determine whether mimosine-arrested cells could be distinguished from aphidicolin-arrested cells. The dihydrofolate reductase locus in CHO C400 cells has been amplified about 500-fold by stepwise selection in methotrexate and comprises approximately 3% of the total DNA (45) . As a result, restriction fragments from this locus can be visualized above a background smear of genomic DNA. An origin of bidirectional DNA replication (OBR-1) has been mapped by a number of different methods to a site about 17 kb downstream of the dihydrofolate reductase gene (21) . The restriction fragment containing this origin of bidirectional DNA replication constitutes the earliest labeled DNA in this region of the genome and can be identified as such by synchronizing CHO C400 cells at the beginning of their S phase in aphidicolin, isolating nuclei from these cells, and then briefly radiolabeling these nuclei in CHO cell extracts as described above. The restriction fragment containing OBR-1 appears as a prominent P-DNA band above a smear of P-DNA fragments generated from throughout the CHO C400 genome (22, 33) . The same pattern of early labeled P-DNA restriction fragments was observed with nuclei isolated from either aphidicolin-arrested cells (Fig. 5 B, APH) or from mimosine-arrested cells (Fig. 5 B, MIM). In contrast, nuclei isolated from Gphase cells revealed only P-DNA bands from the mitochondrial genome (Fig. 5 B, G), and nuclei from non-synchronized, exponentially proliferating cells (Fig. 5 B, EXP) incorporated radiolabel equally well throughout their genome. Thus, nuclei from cells synchronized with mimosine continue DNA synthesis in vitro in a manner indistinguishable from cells synchronized with aphidicolin, demonstrating that mimosine, like aphidicolin, arrests proliferating mammalian cells after they have initiated DNA replication.

Mimosine Does Not Inhibit DNA Synthesis in Cell Extracts

To determine whether or not mimosine inhibited directly any of the proteins required for DNA synthesis at replication forks, exponentially proliferating CHO C400 cells were treated with mimosine for 3 h to arrest DNA synthesis, and then the cells were lysed and incubated further in the presence of unlabeled dGTP, dCTP, dTTP, and [-P]dATP under conditions that support DNA synthesis in S phase nuclei (46) . Whereas incorporation of radiolabeled DNA precursors in vivo was strongly inhibited by this treatment (Figs. 1 and 2), incorporation of [-P]dATP in vitro was not inhibited either by pretreatment of cells with mimosine or by pretreatment of cells with mimosine and addition of mimosine to the cell lysate (Fig. 6). In contrast, aphidicolin effectively inhibited DNA synthesis in vivo or in vitro (Fig. 6). These results demonstrated that mimosine does not act directly on some protein at replication forks but indirectly through cellular metabolism. In fact, the observation that DNA synthesis was reproducibly greater in lysates from cells pretreated with mimosine relative to untreated cells suggested that mimosine had reduced the intracellular dATP pool size, thereby increasing the specific radioactivity of [-P]dATP in the lysate.


Figure 6: Mimosine does not inhibit DNA synthesis in cell lysates. Exponentially proliferating CHO C400 cells were cultured either in the presence (+) or absence (-) of 0.4 m M mimosine for 3 h ( Mimosine Cells). Cells were then lysed in the presence of 0.4% Nonidet P-40, supplemented with the four deoxyribonucleotide substrates for DNA synthesis, the four ribonucleotide substrates for RNA primer synthesis, and [-P]dATP (6000 Ci/mmol, Amersham) (46). Lysates were then incubated for 1 h at 32 °C in the presence (+) or absence (-) of either 0.4 m M mimosine ( Mimosine Lysate) or 5 µg/ml aphidicolin ( Aphidicolin Lysate). The amount of acid-precipitable P-DNA present at the end of the reaction was expressed relative to untreated lysates from cells that had not been treated with mimosine.



Mimosine Does Not Inhibit DNA Synthesis in Xenopus Eggs

In contrast to mammalian cells, Xenopus eggs and egg lysates can efficiently initiate de novo replication of exogenously introduced nonviral DNA templates. Replication is independent of DNA sequence but dependent on the presence of a chromatin substrate that is organized into a nuclear structure (22, 47) . Thus, Xenopus provided an opportunity to test the effects of mimosine on a process of initiation of DNA replication that is directed entirely by cellular proteins.

After incubation of Xenopus sperm nuclei in Xenopus egg extracts supplemented with either mimosine or aphidicolin, the time course for incorporation of [-P]dATP into Xenopus sperm DNA was monitored by acid precipitation of radioactively labeled DNA. As was observed with mammalian cell lysates supplemented with deoxyribonucleotide precursors of DNA synthesis, mimosine had no effect on DNA synthesis in Xenopus egg extracts, although aphidicolin completely arrested DNA synthesis under the same conditions (Fig. 7 A).

Analogous experiments were carried out with intact Xenopus eggs. Plasmid DNA was injected into unfertilized Xenopus eggs that were then allowed to convert the injected DNA into chromatin before activating the eggs in the presence of Caand a Caionophore (35) . This procedure increased the efficiency of plasmid DNA replication at least 10-fold during the first S phase (35, 36) . At various times after activation, plasmid DNA was purified, linearized, and then digested with either DpnI (Fig. 7 B) or MboI (Fig. 7 C). Plasmid DNA that was resistant to DpnI had undergone at least one round of replication, whereas plasmid DNA that was sensitive to MboI had undergone at least two rounds of DNA replication (36) . DNA products were fractionated by gel electrophoresis and identified by blotting-hybridization using appropriate P-labeled DNA probes.


Figure 7: Mimosine does not inhibit DNA replication in Xenopus eggs. A, replication of Xenopus sperm chromatin was measured at the indicated times after addition of sperm chromatin to Xenopus egg extracts in the presence () or absence () of 2 m M mimosine or to Xenopus egg extracts supplemented with 8 µg/ml aphidicolin (). Incorporation of [-P]dATP into acid-precipitable DNA was expressed as a percentage of the total sperm DNA added to the extract. B, Xenopus eggs were injected with an 18-kb plasmid DNA (pneoS13) and incubated in the presence of either 2 m M mimosine () or 40 µg/ml aphidicolin () or in the absence of either inhibitor (). The fraction of plasmid DNA that had replicated at least once was determined by measuring the fraction of DpnI resistant, full-length plasmid DNA monomers ( DpnI resistant full-length plasmid)/(total full-length plasmid). C, samples of plasmid DNA isolated from the same injected eggs shown in panel B also were digested PvuI and MboI to determine the fraction of plasmid DNA that had replicated at least twice. The amount of the largest MboI DNA digestion product was measured. D, the sensitivities of exponentially proliferating CHO C400 cells () and Xenopus Xtc kidney cells () to 0.4 m M mimosine were compared. The amount of [H]thymidine incorporated into cellular H-DNA was expressed as a percentage of the incorporation observed in untreated cultures (time = 0). Since Xtc cells divide once every 40 h while CHO C400 cells divide once every 20 h, the time scale for Xtc cells was divided by 2 and plotted on the same time scale as CHO C400 cells to compensate for the difference in their cell cycle times.



Incubation of Xenopus eggs with mimosine had no effect on either the first or subsequent rounds of replication of plasmid DNA, whereas incubation with aphidicolin completely inhibited replication under all conditions. In fact, newly fertilized embryos continued to divide and develop to the tadpole stage when cultured in the presence of high levels of mimosine or when mimosine was injected into the eggs. This result was remarkably similar to the previous observation that the deoxyribonucleotide synthesis inhibitor hydroxyurea also has no effect on DNA replication in Xenopus embryos, presumably because they contain large stores of deoxyribonucleotide precursors of DNA synthesis (39) . The inability of mimosine to inhibit DNA replication was not species specific because Xenopus cells in culture were as sensitive to mimosine as CHO C400 cells (Fig. 7 D). Therefore, both the initiation and elongation phases of DNA replication in Xenopus eggs or egg lysates were resistant to mimosine at concentrations that were 5-10-fold greater than required to inhibit DNA synthesis in either mammalian or Xenopus somatic cells in culture. Similar results were obtained with mouse preimplantation embryos. Mouse 2-cell embryos cultured in the presence of 0.4 m M mimosine developed up to the blastula stage at the same rate and with the same efficiency as did 2-cell embryos that were cultured in the absence of mimosine. In contrast, aphidicolin rapidly arrested embyronic development as expected.

Mimosine Alters Deoxyribonucleotide Metabolism

Many of the results described above suggested that mimosine inhibits DNA replication by altering a metabolic process related to deoxyribonucleotide metabolism. We directly tested this possibility by measuring the size of intracellular pools of dNTPs in control and mimosine-treated CHO C400 hamster cells. dNTPs were extracted from cells and assayed for their ability to support DNA synthesis in reactions containing Klenow enzyme, complementary [H]dNTPs, and poly(dAdT) or poly(dIdC) templates (48) . Inhibition of cellular DNA synthesis by mimosine was assayed in parallel cell cultures by measuring incorporation of [H]thymidine into acid-precipitable DNA.

dCTP pools remained unaffected by mimosine treatment. However, dTTP pools increased, and dATP and dGTP pools decreased to less than 15% of levels observed in untreated cells (Fig. 8). The decrease in dATP and dGTP pools occurred in parallel with a gradual decrease in DNA synthesis (Fig. 8). In fact, the relative amounts of all four dNTPs observed after 3 h of mimosine treatment were very similar to those previously observed in mouse cells treated with hydroxyurea (48) . Since the effect of hydroxyurea on deoxyribonucleotide metabolism is believed to be responsible for its inhibitory effect on DNA replication, we concluded that the similar alterations introduced by mimosine treatment were responsible for the inhibitory effect of mimosine on DNA replication.


Figure 8: Mimosine alters intracellular levels of deoxyribonucleotide triphosphates. dNTPs were extracted from control cells and cells treated with 0.4 m M mimosine for various lengths of time, and the relative amounts of all four dNTP pools were determined for each extract (--, dNTP pools relative to controls). DNA synthesis was measured in parallel cultures by pulse labeling DNA with 5 µCi/ml [H]thymidine for an additional 20 min after extracts were made (- - -, % DNA replication). All values are normalized to the values obtained in control experiments using untreated cultures (100%). Values represent the averages of at least two independent experiments.




DISCUSSION

Mimosine has proven useful as an agent for synchronizing cells near the G/S border. However, whether or not this drug targets some specific step required for initiation of DNA replication in cellular chromosomes or generally inhibits DNA synthesis at replication forks after they have formed remained an open question (see Introduction). In the first case, mimosine would arrest cells in their Gperiod prior to the onset of DNA synthesis, while in the latter case, mimosine would arrest cells at the beginning of their S phase after the onset of DNA synthesis. If mimosine prevented initiation of DNA replication, it would provide a useful tool for dissecting the initiation process.

Results presented here show that mimosine does not act by specifically inhibiting initiation of DNA replication. Several lines of evidence support this conclusion. First, the dose response curve for inhibition of DNA synthesis by mimosine and the kinetics of this inhibition were the same for mitochondrial DNA as for cellular chromosomes (Figs. 1 and 2). These observations strongly suggest that mimosine is affecting a metabolic pathway shared by nuclear and mitochondrial DNA replication and is not targeting a specific step in the initiation of cellular DNA replication. Furthermore, mitochondrial DNA replication was efficiently inhibited throughout the cell cycle. Therefore, the inhibitory effect of mimosine cannot be confined to late Gor the S phase of the cell cycle.

The second line of evidence is that the same concentration of mimosine that inhibited cellular DNA synthesis also inhibited SV40 DNA synthesis (Fig. 3), a genome whose replication depends on the same proteins required to replicate cellular chromosomes but whose initiation process differs sharply from that of the cell in that it requires a unique viral-encoded protein and undergoes reinitiation multiple times within a single S phase (18) . Thus, it is unlikely that mimosine would specifically block initiation of both cellular and viral DNA replication. In fact, analysis of SV40 replicating intermediates by two-dimensional gel electrophoresis (Fig. 4) demonstrated directly that mimosine arrested DNA synthesis at SV40 replication forks. Moreover, the effects of mimosine on viral replicating intermediates were indistinguishable from those of aphidicolin, an established inhibitor of DNA synthesis at replication forks (8) . Both drugs produced the same pattern of arrested SV40 DNA replication intermediates, and both drugs induced a slow disappearance of replicating intermediates accompanied by conversion of replication bubble structures containing two replication forks into structures containing single replication forks. This phenomenon was strikingly similar to previous reports that aphidicolin induces destabilization of SV40 DNA replication forks (43, 44) . Destabilization of replication forks in cellular chromosomes could also account for the absence of detectable chromosomal replication forks in cells treated with mimosine (6, 7) as well as the increased fraction of breaks observed at fragile sites in human chromosomes in the presence of aphidicolin (51) and the stimulation of gene amplification in the presence of various DNA synthesis inhibitors (52) .


Figure 3: Mimosine inhibits SV40 as well as nuclear DNA replication. SV40-infected BSC-1 monkey cells were treated with various concentrations of mimosine for 3 h beginning at 24 h post-infection. Replicating DNA was then labeled with [H]thymidine for 30 min. Viral DNA was recovered by Hirt extraction and restricted with EcoRI; equivalent amounts of sample were fractionated by agarose gel electrophoresis, and H-DNA was detected by fluorography. Uninfected cells were treated similarly.



The third line of evidence supporting the hypothesis that mimosine arrests DNA synthesis at replication forks stems from the ability of cell extracts to support DNA synthesis in nuclei isolated from S phase cells but not in nuclei isolated from Gphase cells (22, 33) . The fact that nuclei isolated from either mimosine- or aphidicolin-arrested cells carried out DNA synthesis in these extracts (Fig. 5 A) demonstrated that these nuclei had already entered S phase. Furthermore, the pattern of DNA fragments that were rapidly labeled in nuclei from mimosine-arrested cells was indistinguishable from the pattern produced in nuclei from aphidicolin-arrested cells (Fig. 5 B). Therefore, both mimosine- and aphidicolin-arrested chromosomal DNA replication in S phase just after DNA synthesis had been initiated in vivo at specific sites within these fragments.

Several characteristics of the effects of mimosine suggest that it inhibits DNA replication by altering deoxyribonucleotide metabolism. First, DNA synthesis in nuclear and viral chromosomes share the same metabolic pathways for synthesis of deoxyribonucleotides and, although differences in deoxyribonucleotide metabolism occur in mitochondria compared to nuclei (54, 55) , mitochondrial DNA synthesis may share some of these pathways as well. An effect by mimosine on a shared metabolic pathway involved in dNTP metabolism would provide a simple explanation for the similarity of the effect of mimosine on all three DNA replication systems. Second, mimosine did not inhibit DNA synthesis in lysed cells supplemented with the deoxyribonucleotide substrates required for DNA synthesis (Fig. 6). Third, similar to the previous observation that Xenopus embryos are insensitive to the deoxyribonucleotide synthesis inhibitor hydroxyurea for at least the first 12 rounds of DNA replication after fertilization (39) , mimosine did not inhibit replication of DNA templates injected into Xenopus embryos or incubated in extracts of Xenopus eggs (Fig. 7 A). The failure of mimosine and hydroxyurea to inhibit DNA replication in Xenopus embryos may be related to the presence in these embryos of dNTP stores sufficient to support complete replication of the DNA in at least 2,500 nuclei (60) .

Measurement of the relative amounts of dNTPs in mimosine-treated cells compared with untreated cells demonstrated directly that mimosine alters intracellular dNTP pools in cultured cells (Fig. 8). A similar effect on dNTP metabolism by mimosine was reported recently by Dai et al. (53) . In fact, in our experiments and in those of Dai et al., mimosine treatment produced changes in amounts of individual dNTPs relative to levels in untreated cells that were very similar to those previously reported to occur in mouse cells treated with hydroxyurea (48) . In particular, cells treated with either compound are depleted of dATP and dGTP to similar levels relative to controls. Since hydroxyurea's inhibitory effect on DNA replication is believed to be mediated by the specific depletion of dATP and dGTP pools (48, 58) , mimosine very likely inhibits the elongation phase of DNA replication via the similar reduction in dATP and dGTP pools observed in our experiments and by Dai et al. (53) .

How mimosine effects these changes in dNTP metabolism is not clear. The similarity of the effects of mimosine and hydroxyurea on dNTP metabolism and DNA replication in cultured cells and of the lack of effect by either compound on DNA replication in early Xenopus embryos suggests that, similar to hydroxyurea (49) , mimosine inhibits the enzyme ribonucleotide reductase. Mimosine is a strong iron chelator (56) , and ribonucleotide reductase has an essential requirement for iron for proper function (50) . Thus, mimosine might disrupt ribonucleotide reductase function through its ability to chelate iron. In fact, Dai et al. (53) recently demonstrated a direct inhibitory effect of mimosine on ribonucleotide reductase, and this effect could be reversed by iron. Similarly, the addition of excess iron to mimosine-treated cell cultures restores DNA synthesis and dATP levels to those of untreated controls.()

The effect of mimosine on dNTP metabolism could reflect a direct interaction of mimosine with ribonucleotide reductase, similar to hydroxyurea. However, mimosine depletion of dGTP and dATP pools occurs slowly compared to depletion by hydroxyurea, and as reported here and in Mosca et al. (7) , the kinetics with which mimosine inhibits DNA replication also are slow compared to those of hydroxyurea, even when the longer time required to transport mimosine into the cell is taken into account. These observations and the fact that the subunit of ribonucleotide reductase which contains iron turns over fairly rapidly in cells (57) suggest an alternative hypothesis: mimosine sequesters iron required for the function of newly synthesized ribonucleotide reductase as previously synthesized enzyme is depleted.

The apparent Garrest observed in some mimosine-treated cells is correlated in some cases with altered post-translational modifications of proteins implicated in cell cycle regulation (38, 59) . However, the apparent lack of an S phase arrest in these cells may simply reflect levels of functional ribonucleotide reductase that are sufficient to support completion of one S phase after mimosine treatment begins but not sufficient to support significant amounts of DNA synthesis when a second S phase begins many hours later. Consequently, cells would accumulate with a Gcontent of DNA, which could be misinterpreted as a block in late G(2, 3) . This potential misinterpretation could affect the conclusions reached by a number of studies that employed mimosine to block cell cycle progression in late G(1, 3, 4, 10, 11, 12, 13, 14, 15, 16, 38, 59) .

Regardless of how mimosine alters dNTP metabolism, these alterations are expected to block DNA replication at the level of elongation. This mechanism is consistent with previously published data on mimosine when two aspects of mimosine inhibition become apparent. First, mimosine, like other DNA synthesis inhibitors, may induce breaks at replication forks that eventually cause them to disappear. Second, the action of mimosine on DNA replication is indirect through alterations in deoxyribonucleotide pools. The slow kinetics of this effect are very likely responsible for the ``slow-stop'' phenotype previously attributed to inhibition of initiation of DNA replication (7) . Thus, while mimosine remains a useful agent for efficiently synchronizing cultured cells, it may not be useful as a tool for probing late Gcell cycle regulatory events related to the onset of the S phase of the cell cycle.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant CA16056 and by Roswell Alliance. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Biochemistry and Molecular Biology, SUNY Health Science Ctr., Syracuse, NY 13210.

On leave from the Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-01, Japan and was supported in part by a grant from the Biodesign Research Program sponsored by RIKEN.

**
To whom correspondence should be addressed. Tel.: 716-845-7691; Fax: 716-845-8169.

The abbreviations used are: CHO, Chinese hamster ovary; kb, kilobase(s).

W. Burhans, unpublished results.


ACKNOWLEDGEMENTS

We thank Solon Rhode (University of Nebraska Medical Center) and Robert Kalejto and Joyce Hamlin (University of Virginia) for sharing results prior to the publication of their articles, Miriam Miranda for determining the effects of mimosine on development of mouse preimplantation embryos, Martin Weinberger, Christine Brun, and Joel Huberman for critical reading of the manuscript, and Joyce Hamlin for the gift of pneoS13.


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