Periodic Expression of Nuclear and Mitochondrial DNA Replication Genes During the Trypanosomatid Cell Cycle

Journal of Cell Science 107, 3515-3520 (1994) 3515 Printed in Great Britain © The Company of Biologists Limited 1994

Periodic expression of nuclear and mitochondrial DNA replication genes during the trypanosomatid cell cycle

Sally G. Pasion, Grant W. Brown, Lisa M. Brown and Dan S. Ray* Molecular Biology Institute and Department of Biology, University of California, Los Angeles, California 90024, USA *Author for correspondence

SUMMARY

In trypanosomatids, DNA replication in the nucleus and in drial topoisomerase, was disrupted by the insertion of a the single mitochondrion (or kinetoplast) initiates nearly NEO drug-resistance cassette was found to express both a simultaneously, suggesting that the DNA synthesis (S) truncated TOP2 mRNA and a truncated topoisomerase phases of the nucleus and the mitochondrion are coordi- polypeptide. The truncated mRNA was also expressed peri- nately regulated. To investigate the basis for the temporal odically coordinate with the expression of the endogenous link between nuclear and mitochondrial DNA synthesis TOP2 mRNA indicating that cis elements necessary for phases the expression of the genes encoding DNA ligase I, periodic expression are contained within cloned sequences. the 51 and 28 kDa subunits of replication protein A, dihy- The expression of both TOP2 and nuclear DNA replication drofolate reductase and the mitochondrial type II topoiso- genes at the G1/S boundary suggests that regulated merase were analyzed during the cell cycle progression of expression of these genes may play a role in coordinating synchronous cultures of Crithidia fasciculata. These DNA nuclear and mitochondrial S phases in trypanosomatids. replication genes were all expressed periodically, with peak mRNA levels occurring just prior to or at the peak of DNA synthesis in the synchronized cultures. A plasmid clone Key words: cell cycle, replication, topoisomerase, kinetoplast, (pdN-1) in which TOP2, the gene encoding the mitochon- mitochondrion

INTRODUCTION 1993). Minicircle replication intermediates also have been shown by in situ hybridization methods to be present in two Trypanosomes contain a novel form of mitochondrial DNA or antipodal sites on the periphery of the kinetoplast disc in vivo kinetoplast DNA (kDNA), which consists of thousands of (Ferguson et al., 1992). The antipodal location of the newly copies of minicircles and a small number of maxicircles topo- synthesized minicircles coincides with that of two kinetoplast- logically interlocked to form a cup-shaped sheet of DNA associated replication proteins, a type II DNA topoisomerase termed a DNA network (Simpson, 1987). Maxicircles encode (Melendy and Ray, 1989; Melendy et al., 1988) and a DNA rRNAs and proteins essential for mitochondrial function. polymerase (Ferguson et al., 1992), and suggests that these Expression of maxicircle genes involves extensive editing of sites may represent multiprotein replication complexes. primary transcripts in which U residues are added and/or The temporal link between nuclear and kinetoplast DNA deleted to create functional mRNAs (Stuart, 1991). The speci- replication raises the possibility that nuclear and kinetoplast S ﬁcity of the editing process is determined by small ‘guide phases are coordinately regulated in trypanosomes. This RNAs’ encoded in both maxicircles and minicircles and which feature of kDNA replication differs from that observed for serve as templates for the editing process (Sturm and Simpson, other mitochondrial DNAs in which replication occurs 1990). throughout the cell cycle (Guttes et al., 1967). The coordina- Replication of kinetoplast DNA occurs in approximate tion of nuclear DNA synthesis with the cell cycle in eukary- synchrony with nuclear S phase (Cosgrove and Skeen, 1970) otic organisms appears to be under the control of a p34cdc2 and involves a mechanism in which individual minicircles are protein kinase that directly or indirectly modulates the released from the kDNA network prior to their duplication expression and/or activity of numerous proteins required for (Englund, 1979). Upon replication the daughter minicircles are DNA replication (Norbury and Nurse, 1992). In the budding rejoined to the periphery of the network while still containing yeast, one level of control involves the regulation of genes nicks and gaps in the newly synthesized strands (Kitchin et al., encoding DNA replication proteins. At least eighteen Saccha- 1984). Newly synthesized minicircles have been observed to romyces cerevisiae genes required for DNA synthesis are be concentrated in two sites on opposite sides of the network expressed under cell cycle control at, or near to, the G1/S (Simpson and Simpson, 1976; Perez-Morga and Englund, boundary (Johnston and Lowndes, 1992). All of these genes

3516 S. G. Pasion and others are coordinately regulated through a common cis-acting supernatant was decanted and the cells were resuspended in the sequence recognized by a specific transcription factor residual media and plated onto 1% agar plates containing BHI plus (Johnston et al., 1991). Since all kinetoplast DNA replication 10 µg hemin/ml, 100 µg streptomycin sulfate/ml and 80 µg G418/ml. proteins are likely to be nuclear encoded, their expression may The plates were wrapped in parafilm and incubated at 28¡C. G418- be under the same controls as those of genes encoding nuclear resistant colonies appeared within 4 to 5 days. replication proteins. To investigate this possibility, we have Construction of the pdN-1 plasmid examined the cell cycle expression of genes encoding both The vector pX.2 was derived from pX (LeBowitz et al., 1990), which nuclear and kinetoplast replication proteins in the trypanoso- contains a mutant neomycin phosphotransferase (NPT) gene by matid Crithidia fasciculata. replacing the 350 bp BalI-NcoI fragment contained within the NPT gene with the corresponding 350 bp BalI-NcoI fragment from the wild-type NPT gene from pSV2neo (Yenofsky et al., 1990). ′ MATERIALS AND METHODS Two oligonucleotides (Kpn 1: 5 -CGCGGATCCTCTAGAG- GTAC-3′ and Kpn 2: 5′-CTCTAGAGGATCCGCGGTAC-3′) were synthesized, annealed, and subcloned into the unique KpnI site of Cell synchronization pX.2 introducing BamHI, SacII and XbaI sites flanked by KpnI sites C. fasciculata was synchronized using a modification of the method and generating the vector pX.2-KO. This construct allows the described for synchronizing Leishmania tarentolae (Simpson and movement of the 3.3 kb NEO cassette (the wild-type NPT gene with Braly, 1970). Cells were grown to late log phase (~108 cells/ml) at flanking Leishmania major DHFR-TS sequences) as a BamHI, SacII 28¡C in Difco brain heart infusion medium (BHI) supplemented with or XbaI fragment. 10 µg hemin/ml and 100 µg streptomycin sulfate/ml prior to the To create a disruption of the plasmid-encoded CfaTOP2 gene, the addition of 200 µg hydroxyurea/ml for 6 hours. Growth was continued NEO cassette was subcloned as a SacII fragment from pX.2-KO into at 28¡C in fresh medium lacking hydroxyurea, and samples were the pt2-1 plasmid (Pasion et al., 1992), which contains the CfaTOP2 removed every 30 minutes. coding region and 883 bp 5′ flanking and 1.5 kb 3′ flanking sequences. The pt2-1 plasmid was constructed by inserting a 6.1 kb genomic Measurement of DNA synthesis fragment into the bacterial cloning vector pGEM-7Zf(+), which Total DNA synthesis was measured by labeling 65 µl of culture with contains no other trypanosomatid sequences. The inserted NEO 10 µl of [3H]thymidine (1 mCi/ml; 79 Ci/mmol) at 28¡C for 10 cassette replaces a 1.4 kb SacII fragment within the CfaTOP2 coding minutes. Cells were lysed and incorporation of [3H]thymidine into sequence to generate pdN-1 in which the CfaTOP2 gene and the NPT trichloroacetic acid-precipitable form was measured. Mitochondrial gene are in the same orientation. DNA synthesis was measured by scintillation counting of [3H]thymidine label in minicircle DNA bands on Southern blots Western blot analysis following digestion with XhoI and agarose gel electrophoresis. Cell lysates prepared from C. fasciculata strains were analyzed on 7% acrylamide-SDS gels as described previously (Pasion et al., 1992). Northern blots Each lane contained lysate from 2.5×106 cells. Blots were probed with RNA extracted from 109 cells by guanidinium isothiocyanate lysis polyclonal antisera (1:5000) raised against purified topoIImt was used for northern blotting and hybridization as described (Pasion (Melendy and Ray, 1989) and detected with alkaline phosphatase con- et al., 1992). Either 50 µg (for CfRPA1) or 25 µg (all others) of RNA jugated to goat anti-rabbit IgG. was fractionated. Probes for analysis of C. fasciculata RNA were a 980 bp EcoRI-MluI fragment of the CfaRPA1 gene (EMBL accession number Z23163), a 606 bp HaeII fragment of the CfaRPA2 gene RESULTS (EMBL accession number Z23164), a 630 bp EcoRI-AvaI fragment of the CfaLIG1 gene (EMBL accession number Z23078), a 2000 bp XhoI fragment of the CfaTOP2 gene encompassing amino acid Expression of DNA replication genes in residues 311 to 1007 (Pasion et al., 1992) and a 260 bp fragment of synchronized cells a C. fasciculata gene designated CaBP due to its sequence similarity C. fasciculata cultures undergoing synchronous progress to trypanosome calcium-binding proteins (Lee et al., 1990). The probe through the cell cycle were prepared by a hydroxyurea arrest for the analysis of Cf (pdN-1) RNA was a 2.1 kb HindIII-SacII method (Simpson and Braly, 1970). Cells were released from ′ fragment of pdN-1 designated 5 TOP2 US and CfaTOP2 (1-1186) in the hydroxyurea block and sampled every 30 minutes to Fig. 2. Northern blots were quantitated using a Molecular Dynamics determine the cell number, the percentage of dividing cells and PhosphorImager. the incorporation of pulse label into DNA. Cultures released Transformation of Crithidia fasciculata from hydroxyurea treatment entered S phase immediately, as C. fasciculata cells were cultured in BHI media supplemented with indicated by the high level of DNA synthesis at 0 and 30 10 µg hemin/ml and 100 µg streptomycin sulfate/ml to a density of minutes (Fig. 1A). Following this peak of DNA synthesis, the 1×108 cells/ml. Cells were harvested in a clinical centrifuge at 4¡C percent of cells undergoing division (cells with two nuclei) for 10 minutes at 1,500 rpm. The supernatant was discarded and the increased from <1% to 30% at 120 minutes post-release cells were washed in ice-cold BHI media. Cells were collected again followed by an approximate doubling of the cell number by and finally resuspended in ice-cold BHI media at a density of 108 150 minutes post-release. The culture then entered another µ cells/ml and placed on ice. Next, 0.4 ml of cells and 1 g of super- round of DNA synthesis, with peak incorporation at 210 coiled plasmid DNA in a 0.2 cm Bio-Rad electroporation cuvette were µ minutes, followed by another round of division at about 300 subjected to the discharge of a 500 F capacitor charged at 500 volts. 3 The cells were allowed to sit on ice for 10-15 minutes and were then minutes. Incorporation of [ H]thymidine into mitochondrial transferred to 10 ml M199 media supplemented with 20 µg hemin/ml, minicircle DNA was also measured by quantitation of radioac- 2 µg folic acid/ml, and 0.4 mM adenine and incubated at 28¡C in an tivity in linear minicircle DNA following digestion of a sample air shaker for 4 hours. After recovery, the cells were harvested at room of the pulse-labeled DNA with XhoI, an enzyme that cuts all temperature in a clinical centrifuge for 10 minutes at 1,500 rpm. The minicircles once, and gel electrophoresis and blotting to a Trypanosomatid DNA replication genes 3517 nitrocellulose membrane. Minicircle DNA synthesis occurred as evidenced by a broadening of the peaks of DNA synthesis in synchrony with total DNA synthesis during the second S and percentage dividing cells in the second cell cycle. The phase, with peak synthesis at 180 minutes, but was delayed gradual loss of synchrony can be attributed, at least in part, to relative to total DNA synthesis immediately following release natural variation in individual cell cycle times. In Try- from the hydroxyurea block in the experiment shown (Fig. 1c). panosoma brucei, for example, cell cycle kinetic studies on This delay was an anomaly and was not reproducible in sub- asynchronous populations of cells indicate that individual cells sequent synchronizations. The culture gradually became can have a G2 phase ranging from 1.44 hours to 2.5 hours increasingly asynchronous during the course of the experiment, (Woodward and Gull, 1990). In the first division cycle the cell number does not exactly double, probably due to the presence of inviable cells, as hydroxyurea treatment is selectively lethal to cells in S phase (Simpson and Braly, 1970). Cell cycle expression of genes encoding topoIImt (the kinetoplast DNA topoisomerase) (Pasion et al., 1992) and those of DNA ligase I (Brown and Ray, 1992), the 51 and 28 kDa subunits of replication protein A (Brown et al., 1994) and dihydrofolate reductase (Hughes et al., 1989) were examined on northern blots of RNA prepared from synchronized cultures (Fig. 1B). The Crithidia RP-A protein is the trypanosome homolog of the human RP-A protein, which has been shown to be required for SV40 DNA replication in vitro. Crithidia RP-A substitutes for human RP-A in the T-antigen-dependent unwinding of the SV40 origin of replication and stimulates both DNA synthesis and DNA priming by human DNA polymerase α/primase (Brown et al., 1992). CfaRPA1 and CfaRPA2 encode the 51 kDa and 28 kDa subunits of the het- erotrimeric C. fasciculata RP-A protein (Brown et al., 1994). CfaLIG1 encodes a DNA ligase with properties similar to other type I nuclear DNA ligases (Brown and Ray, 1992) and the C. fasciculata DHFR-TS gene encodes dihydrofolate reductase- thymidylate synthetase (Hughes et al., 1989), which is required for the synthesis of DNA precursors. The analysis of the CaBP transcript, which was found to be expressed at a constant level during cell cycle progression, is included as an internal loading control. The steady state levels of the CfaRPA1, CfaRPA2, and DHFR-TS transcripts varied during cell cycle progression, with peak mRNA levels at 0-30 minutes and 150-210 minutes, both during late G1 and S phases, with little expression between these peaks, during G2 and M phases. All three transcripts were most abundant at 180 minutes, just before the peak of DNA synthesis, indicating that maximal expression is at the transi- tion from G1 phase to S phase. The three transcripts were induced 5- to 7-fold between 90 and 180 minutes following release from hydroxyurea. The CfaLIG1 transcript is expressed periodically, but reached maximum levels at 210 minutes, at the peak of S phase. The approximately 3-fold induction of CfaLIG1 expression as cells progress into S phase is not as great as that seen for the CfaRPA1 and CfaRPA2 genes and the DHFR-TS gene. These results suggest that the mechanism reg- Fig. 1. Expression of RP-A p51 and p28, DHFR-TS, DNA ligase I, ulating expression of CfaLIG1 may be distinct from that reg- topoIImt, and calcium-binding protein genes during progression ulating expression of the DHFR-TS and CfaRPA1 and through the cell cycle in C. fasciculata synchronized by hydroxyurea CfaRPA2 genes. treatment. (A) Cell number (squares), percent of cells with two The CfaTOP2 gene, which is also encoded in the nucleus, 3 nuclei (triangles), and incorporation of a [ H]thymidine pulse label is also under cell cycle control. The periodic expression pattern (circles), measured every 30 minutes following release from the of the CfaTOP2 transcript is similar to that of the CfaRPA1 hydroxyurea block to monitor synchrony and cell cycle position. (B) Northern blot analysis of RNA carried out on synchronized C. and CfaRPA2 transcripts and the DHFR-TS transcript, with the fasciculata cells. The transcript levels of CfaRPA1, CfaRPA2, peak of expression occurring just prior to the peak of total DHFR-TS, CfaLIG1 and CfaTOP2 are shown together with that of DNA synthesis (Fig. 1A) and mitochondrial minicircle DNA the calcium-binding protein gene (CaBP), which served as a control synthesis (Fig. 1C). A 5-fold difference in CfaTOP2 transcript for gel loading. (C) Incorporation of [3H]thymidine into levels occurred between the 90 and 180 minute time points. mitochondrial minicircle DNA in synchronized cells. The CfaTOP2 gene is the first example of a mitochondrial

3518 S. G. Pasion and others

EcoRI HindIII pdN-1 (Fig. 2). The inserted NEO cassette replaces approxi- AAAAAAA TOP2 5'US AAAAAAA mately 1.4 kb of TOP2 coding sequence and confers resistance to G418 in C. fasciculata. A clone expressing drug resistance pGEM7AAAAAAAAAAA CfaTOP2(1-1186) from extrachromosomal pdN-1 plasmid was examined for AAAAAAAAAAA AAA possible expression of the amino-terminal portion of topoIImt. AAAAAAAAAAA AAASacII BamHI In addition to full-length topoIImt, western blots of C. fascic- AAAA AAA ulata (pdN-1) show the presence of a 45 kDa protein recog- pdN-1 AAAA AAA nized by rabbit polyclonal antibodies against topoIImt consis- BamHI HindIII 11.0 kb EcoRI AAAAAAAAA tent with the molecular mass predicted based on the location AAAAAAAAA of an in-frame stop codon within the NEO cassette (Fig. 3). Some less intense bands present in both lanes of Fig. 3 appear TOP2 3'DS AAAAAAAAANEO cassette AAAAAA to be non-specific proteins recognized by this polyclonal AAAAAA antiserum. CfaTOP2(2625-3717) BamHI In addition to the 4.8 kb TOP2 mRNA expressed from chro- SacII mosomal copies of TOP2, cells carrying the pdN-1 plasmid Fig. 2. Plasmid pdN-1. Sequences containing the amino-terminal or also express a truncated TOP2 mRNA of approximately 2.0 carboxy-terminal third of the CfaTOP2 gene in the pdN-1 map are kb, which corresponds to the size predicted based on the uti- designated as CfaTOP2 (1-1186) or CfaTOP2 (2625-3717), lization of the most frequently used TOP2 splice acceptor site respectively. TOP2 5′ US and TOP2 3′ DS refer to the upstream and located at −670 relative to the ATG initiation codon (unpub- downstream sequences flanking the CfaTOP2 gene. The NEO lished results) and the polyadenylation site contained in the cassette, which replaces the middle third of the CfaTOP2 gene Leishmania major DHFR -TS 5′ flanking sequence of the NEO confers G418 resistance to C. fasciculata cells carrying the plasmid cassette (Kapler et al., 1990). pdN-1. We have investigated the possibility that sequences required in cis for the periodic expression of TOP2 might be present in DNA replication gene that is periodically expressed during the pdN-1 plasmid. C. fasciculata (pdN-1) cells were syn- progression through the cell cycle. chronized in the same manner as the wild-type C. fasciculata and analyzed by northern blotting at 30 minute intervals. As Expression of a disrupted TOP2 gene in plasmid shown in Fig. 4, both the plasmid-expressed 2.0 kb TOP2 pdN-1 mRNA and the endogenous 4.8 kb TOP2 message are A 6.1 kb genomic fragment containing the TOP2 gene and 5′ expressed with the same periodicity as observed for the wild- and 3′ flanking sequences cloned in plasmid pt2-1 (Pasion et type TOP2 mRNA in Fig. 1. Both the 4.8 and 2.0 kb transcripts al., 1992) was disrupted by insertion of a neomycin phospho- are expressed at a high level at the time of release from the transferase drug-resistance cassette (NEO) between two SacII hydroxyurea block and then again at 180 minutes after release. sites within the TOP2 coding sequence to yield the plasmid Both transcript levels go through minima at 90-120 minutes and then again at 300 minutes. A minor RNA species migrating at approximately 1.35 kb possibly represents an mRNA resulting from the utilization of alternate splice acceptor sites

Fig. 4. Northern analysis of hydroxyurea-synchronized C. Fig. 3. C. fasciculata transformant Cf (pdN-1) expresses p45, a fasciculata transformant Cf (pdN-1). Total cell RNA was isolated truncated topoIImt. Lysates of wild-type C. fasciculata and the from synchronized Cf (pdN-1) cells at 30 minute intervals after transformant Cf (pdN-1) cells were analyzed by western blot and release from hydroxyurea arrest and analyzed (25 µg each lane) by stained with rabbit polyclonal anti-topoIImt antibodies. Each lane northern blotting. The apparent sizes of the transcripts detected are contains 2.5×106 cell equivalents. The positions of full length indicated to the right of the blot. Times of sampling (in minutes) are topoIImt and the 45 kDa truncated topoIImt are indicated. shown above alternate lanes. Trypanosomatid DNA replication genes 3519 in the TOP2 5′ flanking sequence located at −77 and −67 cycle. Such a factor might be similar to the Drosophila tra-2 relative to the TOP2 initiation codon (unpublished results). The protein whose binding to dsx pre-mRNA appears to be higher molecular mass species migrating at 5.85, 7.7 and 10 necessary for sex-specific splicing and polyadenylation kb appear to represent RNAs expressed from the extrachro- (Hedley and Maniatis, 1991). mosomal plasmid, which are polyadenylated at sites within So far, a promoter responsible for expression of TOP2 has plasmid sequences. These latter species are not observed in not been identified, however, since many protein-coding genes cells lacking the plasmid or in cells in which the disrupted in trypanosomes are transcribed polycistronically from TOP2 gene is integrated into one of the chromosomal copies putative upstream promoters (Johnson et al., 1987), transcrip- of TOP2 (S. G. Pasion and O. S. Ray, unpublished results). tion may possibly initiate at some distance. Even in the event that the chromosomal TOP2 might be expressed as a polycistronic transcript, the periodic accumulation of the 2.0 kb DISCUSSION TOP2 transcript expressed from the pdN-1 plasmid indicates that cis-acting sequences necessary for regulation are The coordination of nuclear and mitochondrial DNA replica- contained within the deletion clone. With the availability of the tion in trypanosomatids is unique among eukaryotes, where pdN-1 plasmid, it will now be possible to identify and charac- mitochondrial DNA replication is typically not restricted to any terize the cis elements required for the periodic expression of one period of the cell cycle (Guttes et al., 1967). Our results the TOP2 gene. show that the steady state levels of the mRNAs of a mito- In light of the early divergence of trypanosomes from the chondrial replication gene as well as those of nuclear replica- main eukaryotic lineage, relative to the divergence of plants, tion genes are cell cycle regulated in the trypanosomatid C. fas- animals and fungi (Sogin, 1991), the cell cycle regulation of ciculata. These results are similar to those found for nuclear DNA replication gene expression may be a common feature of DNA replication genes in S. cerevisiae where at least 18 genes all eukaryotes. The experiments presented here suggest that encoding DNA replication proteins are expressed periodically coordinate expression of nuclear and mitochondrial DNA (Johnston and Lowndes, 1992). These include genes encoding replication genes may be responsible, at least in part, for the subunits of DNA polymerase α, DNA polymerase δ and DNA nearly-coincident initiation of nuclear and mitochondrial DNA primase, PCNA (proliferating cell nuclear antigen, a pol δ pro- replication in trypanosomatids. Further dissection of the regu- cessivity factor) and DNA ligase. In addition, some genes latory network responsible for the periodic expression of these encoding enzymes involved in the production of dNTPs are genes will be required to establish where the factors involved also expressed periodically. These include thymidylate kinase, in coordinating nuclear and mitochondrial S phases fit into the thymidylate synthetase and ribonucleotide reductase. These hierarchy of regulatory molecules controlling progression genes, as well as the B-type cyclin genes, are all activated at through the cell cycle. or soon after cells undergo START. This set of periodically expressed replication and cell cycle control genes appears to We thank K. Hurst for performing preliminary studies on the be coordinately regulated by a transcription factor (DSC1) that hydroxyurea synchronization method. This work was supported by recognizes an upstream element consisting of one or more NIH grant AI20080 to D.S.R. and by predoctoral National Research copies of the 6 bp sequence ACGCGT, the MluI restriction Service awards in Cellular and Molecular Biology (L.M.B.) from NIH grant GM07185 and in Genetics and Regulatory Mechanisms (S.G.P. sequence. Similar ACGCGT elements and near matches are and G.W.B.) from NIH grant GM07104. also involved in regulating a DNA synthesis gene in Schizosac- charomyces pombe. In this case, binding sites for the regulatory factor are present at sites located both upstream and down- REFERENCES stream of the transcription start site (Lowndes et al., 1992). 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