Identification and cloning of two putative subunits of DNA polymerase
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《核酸研究医学期刊》
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, PO Box 016129, Miami, FL 33101-1019, USA
* To whom correspondence should be addressed. Tel: +1 305 243 3105; Fax: +1 305 243 3064; Email: gdurso@miami.edu
ABSTRACT
DNA polymerase epsilon (Pol ) is a multi-subunit enzyme required for the initiation of chromosomal DNA replication. Here, we report the cloning of two fission yeast genes, called dpb3+ and dpb4+ that encode proteins homologous to the two smallest subunits of Pol . Although Dpb4 is not required for cell viability, dpb4 mutants are synthetically lethal with mutations in four genes required for DNA replication initiation, cdc20+ (encoding DNA Pol ), cut5+ (homologous to DPB11/TopBP1), sna41+ (homologous to CDC45) and cdc21+ (encoding Mcm4, a component of the pre-replicative complex). In contrast to Dpb4, Dpb3 is essential for cell cycle progression. A glutathione S-transferase pull-down assay indicates that Dpb3 physically interacts with both Dpb2 and Dpb4, suggesting that Dpb3 associates with other members of the Pol complex. Depletion of Dpb3 leads to an accumulation of cells in S phase consistent with Dpb3 having a role in DNA replication. In addition, many of the cells have a bi-nucleate or multinucleate phenotype, indicating that cell separation is also inhibited. Finally, we have examined in vivo localization of green fluorescent protein (GFP)-tagged Dpb3 and Dpb4 and found that both proteins are localized to the nucleus consistent with their proposed role in DNA replication. However, in the absence of Dpb3, GFP-Dpb4 appears to be more dispersed throughout the cell, suggesting that Dpb3 may be important in establishing or maintaining normal localization of Dpb4.
INTRODUCTION
Eukaryotic chromosomal DNA replication requires the activity of at least three DNA polymerases, alpha (Pol ), delta (Pol ) and epsilon (Pol ). It is generally accepted that Pol is responsible for initiation through the synthesis of RNA/DNA primers, while both Pol and Pol participate in chain elongation (1). We have shown previously that Cdc20, the catalytic subunit of Pol , is composed of an N-terminal region that contains all the domains necessary for polymerase activity, while the C-terminal half of the enzyme contains essential elements with no known biochemical function (2). Interestingly, in yeast, the N-terminal catalytic domains of Pol are dispensable for cell viability (2–4). Based on our earlier observations that temperature-sensitive mutants in the catalytic subunit of Pol arrest in early S phase (5), we proposed that the primary role of Pol is to facilitate assembly of the DNA replication initiation complex. Consistent with this model, we have recently shown that Dpb2, the second largest subunit of Pol , is required for chromosomal DNA replication and binds to replication origins early in S phase (6).
Pol is composed of at least four subunits in yeast and human cells (7–10). In budding yeast, these subunits are encoded by POL2, DPB2, DPB3 and DPB4 (11–13). POL2 and DPB2 are essential for cell viability and mutants in either of these two genes lead to cell cycle arrest and inhibition of DNA replication (11,14). In contrast, neither DPB3 nor DPB4 is essential in S.cerevisiae. However, cells deleted for either of these genes do show subtle defects in nucleotide biosynthesis (12,13). Homologs of all of these proteins have been identified in human cells (15), yet their precise function in chromosomal DNA replication remains obscure.
Both Dpb3 and Dpb4 contain a histone-fold motif (9,12,13). The histone-fold motif, an extended helix–strand–helix, is believed to play a role in both protein–protein and protein–DNA interactions (16,17). Both Drosophila and human Dpb4 have been shown to be a component of the chromatin-accessibility complex (CHRAC), suggesting that Dpb4 may be involved in nucleosome remodeling (18–20). Also, in budding yeast, DPB4 has been identified as a component of ISW2, a CHRAC-like complex that is involved in epigenetic silencing at telomeres (21).
Interestingly, human CHRAC has been reported to stimulate T-antigen-mediated replication of chromatin-bound SV40 DNA in vitro (22), suggesting that CHRAC might provide a remodeling activity that facilitates the initiation of DNA replication on chromatin. It has also been reported that the C-terminal region of Pol interacts with Pol , suggesting that Pol might have a role in sister-chromatid cohesion (14,23). In conclusion, these observations suggest that Pol may have several functions independent of its polymerase activity.
In this paper, we report the cloning and characterization of two fission yeast genes that encode proteins homologous to human and budding yeast Dpb3 and Dpb4. In contrast to S.cerevisiae, we find that Dpb3 is essential for cell viability in S.pombe. Depletion of Dpb3 results in a strong cell cycle delay and accumulation of cells with slightly less than 2C DNA content, suggesting that cells have difficulty completing S phase. Also, a significant percentage of cells depleted for Dpb3 contain two or more nuclei, suggesting loss of Dpb3 results in a partial block to cell separation. GST-pull down assays show that Dpb3 can interact with both Dpb2 and Dpb4, confirming that Dpb3 interacts with the other putative subunits of the Pol complex.
Although Dpb4 was found to be non-essential for cell viability, deletion of dpb4+ is synthetically lethal with mutants defective in the catalytic subunit of Pol , encoded by cdc20+, and with cut5-T401, sna41-912 and cdc21-M68, three mutants defective in DNA replication initiation. These observations are consistent with Dpb4 being important for some aspect of DNA replication.
Finally, we show that both Dpb3 and Dpb4 localize to the nucleus consistent with their proposed role in DNA replication and that normal localization of Dpb4 is dependent on expression of Dpb3.
MATERIALS AND METHODS
Schizosaccharomyces pombe strains and methods
All strains used were derived from 972 (h–) and 975 (h+). Media, growth conditions and standard genetic methods were as described previously (24). Fission yeast cosmids c17G8 (containing dpb3+) and c3D6 (containing dpb4+) were kindly provided by The Sanger Center (Hinxton, UK). DNA content was determined by fluorescence-activated cell sorting as previously described (Sazer and Sherwood, 1990), except that cells were stained with Sytox Green (Molecular Probes) instead of propidium iodide.
Construction of dpb3 and dpb4 mutant strains
The plasmids pKS(+)dpb3::ura4+ and pKS(+)dpb4::ura4+ used for deletion of dpb3+ and dpb4+ were constructed as follows: a 1.8 kb DNA fragment containing the ura4+ gene was isolated from pARC614 digested with HindIII and cloned into pKS(+)Bluescript. Both the upstream (1.6–2.0 kb) and the downstream (1.8–2.0 kb) flanking genomic sequences of each gene were amplified by PCR from cosmid DNA and cloned into pKS(+)Bluescript at the ApaI/XhoI and the XmaI/BamHI restriction sites, respectively, located upstream and downstream of the ura4+ gene. The resulting plasmids were digested with KpnI/NotI to obtain a DNA fragment containing ura4+ and either the dpb3+ or dpb4+ flanking sequences. Each DNA fragment was then transformed into the diploid strain (h+/h– leu1-32/leu1-32 ura4-D18/ura4-D18 ade6-M210/ade6-M216) and stable ura4+ integrants were selected. Replacement of either dpb3+ or dpb4+ with the ura4+ gene was confirmed by the Southern blot analysis. Diploids were induced to sporulate and meiotic segregants were analyzed by tetrad analysis.
Cloning of dpb3+ and dpb4+
The dpb3+ open reading frame (599 bp) was PCR-amplified from cosmid c17G8 using the forward primer (5'-CGCGGATCCGATGGGTGATCCAACAAATAATAATG) and the reverse primer (3'-TCCCCCGGGCTACTCATCACCAGACGCGGAAGAGG) tagged with BamHI and XmaI, respectively (indicated by underline). The amplified product was digested and then cloned in the pRep3X vector at the BamHI and XmaI sites.
To clone the dpb4+ gene, the 803 bp open reading frame was amplified from the cosmid c3D6 using the following primers, 5'-CGCGGATCCGATGAATCAAGATAAATCG and 3'-TCCCCCGGGTTACGAAGAATCATTAAG, containing a BamHI and XmaI restriction sites, respectively. The PCR product generated was then digested and cloned into the pRep3X vector at the same restriction sites.
Construction of a strain expressing dpb3+ under the control of the thiamine-repressible promoter
In order to generate a thiamine-repressible dpb3+ strain, the dpb3+ gene was amplified using the same primers described above, but tagged with BamHI/SacI. The PCR product was digested and inserted at the BamHI/SacI sites in a pJK148 vector that contained the nmt81 promoter cloned at the PstI/BamHI sites. The resulting plasmid was then linearized within the leu1+ gene by digestion with AfeI and transformed into the diploid strain, h+/h– leu1-32/leu1-32 ura4-D18/ura4-D18 ade6-M210/ade6-M216. Stable ura4+ integrants were selected, sporulated and the resulting spores were germinated on minimal plates in the absence of thiamine. Viable leu1+ ura4+ ade– colonies were recovered indicating that the expression of dpb3+ from the nmt81 promoter was sufficient to rescue the dpb3 strain. To shut off dpb3+ expression, cells were then grown in the presence of 10 mM thiamine.
GST pull-down assay
For ectopic expression of proteins, we used the thiamine-repressible nmt1 promoter (25,26). To generate a GST-fusion protein, the dpb3+ coding region was amplified with PCR primers tagged with BamHI and XmaI restriction sites located 5' and 3' to the gene. The amplified fragment was then cloned into the pESP expression vector, downstream of the nmt1 promoter. Similarly, Myc-Dpb4 and Myc-Dpb2 were created by cloning the PCR-amplified genes into pARC616 at the BamHI/XmaI sites, in frame with the Myc-epitope tag. The expression of the Myc-tagged fusion protein in pARC616 is under the control of the weaker nmt41 promoter. Fission yeast cells were transformed with pESP-dpb3+ and either Myc-dpb2+ or Myc-dpb4+ constructs. As a negative control, pESP-dpb3+ was replaced with the pESP vector alone. Colonies containing both plasmids (selected on media lacking leucine and uracil) were isolated and grown in minimal medium supplemented with thiamine (5 μg/ml). The cells (OD 0.5) were then washed extensively with water to remove all traces of thiamine, and the expression of tagged proteins were induced by incubating each strain in media in the absence of thiamine for 18 h at 32°C. The cells were then broken with glass beads and 1 μg of soluble protein was coupled to 50 μl glutathione–Sepharose resin ON at 4°C in 1 ml HB buffer (25 mM MOPS, 1% Triton X-100, 0.1 mM sodium vanadate, 60 mM ?-glycerophosphate, 15 mM p-nitrophenylphosphate, 15 mM MgCl2 and 15 mM EGTA) freshly supplemented with 1 mM phenylmethylsulfonyl fluoride, 0.5 M DTT, and protease inhibitors (Complete Protease Inhibitors Cocktail Tablet, Roche). After washing the resin four times with HB buffer, bound proteins were eluted in 50 μl 2x SDS buffer, boiled and analyzed by 8% SDS–PAGE and western blotting using anti-Myc and anti-GST antibodies.
GFP localization studies
To generate strains that contain Dpb3 and Dpb4 endogenously tagged with green fluorescent protein (GFP), the corresponding genes were PCR-amplified with forward and reverse primers containing BamHI and HindIII sites, respectively, but lacking any stop codon. The amplified DNA fragments were digested and inserted in a pKS(+) Bluescript vector at the BamHI/HindIII sites in frame with GFP at the C-terminal end. The pKS vector used also contained a copy of the sup3-5 allele, a suppressor of the ade6-704 mutant allele. Each of the resulting plasmids were transformed in an ade6-704 strain and stable integrants were selected. Genomic DNA was extracted and the site of integration was confirmed by PCR using a forward primer upstream of each coding region and a reverse primer within the GFP gene. To observe Dpb3–GFP and Dpb4–GFP in live cells, yeast cells were grown to an optical density of <0.5 and analyzed using a Zeiss Axiophot microscope. To visualize nuclei, cells were treated with 1 μg/ml DAPI in Slowfade Component A (Molecular probes). Images were captured and processed using Openlab software.
RESULTS
Dpb3 is required for cell cycle progression
We identified a gene in the S.pombe genome database (accession no. Q10315 ) that encodes a protein that is 34 and 26% identical to human p12 (human Dpb3) and to the N-terminal portion of S.cerevisiae DPB3, respectively. Consequently, the gene was termed dpb3+. Only the N-terminal half of the protein that contains the histone-fold motif (shown in Figure 1A and B) displays any significant homology to the other known orthologs. Human, mouse and Drosophila Dpb3 are considerably smaller (12–15 kD) than Dpb3 from budding or fission yeast. Although both S.pombe and S.cerevisiae Dpb3 contain an extended C-terminal region, a comparison between these two sequences reveals no significant homology within this region of the protein. This suggests that the histone-fold motif is critical for Dpb3 function. Also, although S.pombe Dpb3 shows limited identity to budding yeast DPB3 (<20% overall), the two proteins are nearly identical in size (both are estimated to be 22 kD). There is only one additional protein encoded by the S.pombe genome, called Php5 that is similar to human p12 or S.cerevisiae DPB3. Considering that the estimated mass of this protein (46 kD) is significantly larger than the other homologs, and that it has previously been reported to be a component of a CCAAT-binding factor essential for the expression of the cyc1+ gene, it is unlikely that Php5 represents the third subunit of DNA Pol (27). Therefore, we conclude that the gene we have identified as dpb3+ encodes an ortholog of S.cerevisiae DPB3 and human p12. To determine whether dpb3+ is essential for cell viability, we constructed a diploid strain where a single copy of dpb3+ was replaced by ura4+ (Figure 2A). Construction of the deletion strain was confirmed by Southern blot analysis (Figure 2B). Following sporulation, germinating spores were subjected to tetrad analysis. Most of the meiotic segregants examined gave rise to a 2:2 segregation pattern of viable/non-viable cells indicating that dpb3+ is an essential gene (Figure 2C). As expected, the viable colonies failed to grow in the absence of uracil. In the three tetrads that gave a 1:3 ratio of viable/non-viable cells, the viable colony was ura– suggesting that the remaining ura– spore failed to germinate. Failure of ura– spores to germinate during tetrad analysis is frequently observed. Microscopic analysis of dpb3 germinating spores showed that cells underwent a few rounds of cell division before arresting with an elongated cell (cdc) phenotype, (Figure 2D) indicative of a cell cycle block. Therefore, we concluded that Dpb3 is essential for cell cycle progression.
Figure 1. (A) Schematic representation of the conserved Dpb3 proteins from S.pombe, S.cerevisiae, Homo sapiens, Mus musculus and Drosophila melanogaster. Percentage identity to fission yeast Dpb3, within the histone-fold motif, is indicated. (B) Amino acid sequence comparison between the histone-fold motif from S.pombe Dpb3 and its orthologs. Human, mouse and Drosophila are shown as full-length proteins. For S.pombe and S.cerevisiae, only the N-terminal half of the protein is shown because this is the only region that shares extensive homology to Dpb3 from other organisms. Alignment of the proteins was performed using ClustalW. Residues in black indicate identity and residues in gray indicate similarity. The conserved histone-fold motif is indicated by the double overline.
Figure 2. (A) Schematic representation of dpb3 construct. The 0.6 kb dpb3+ gene was replaced with a 1.8 kb ura4+ gene. (B) Southern blot analysis of dpb3 strain, lanes 1 and 2, dpb3/dpb3+; lane 3, wild-type 972. NdeI digestion of genomic DNA generates a fragment of 5.4 kb, indicating that gene replacement was successful. (C) Tetrad analysis confirms dpb3+ is an essential gene. (D) Morphology of cells deleted for dpb3+.
dpb4 is synthetically lethal with DNA replication mutants
The sequence of a putative S.pombe homolog of Dpb4 has been reported previously (9,13). The fission yeast Dpb4 is 210 amino acids long, has an estimated mass of 23 kD and shares 36 and 30% identity to human p17 (human Dpb4) and budding yeast DPB4, respectively. To test whether dpb4+ is essential for viability, we constructed a diploid strain where one copy of dpb4+ was replaced by the ura4+ gene. Following sporulation, germinating spores gave rise to viable colonies in the absence of uracil, demonstrating that dpb4+ is non-essential. Also, the cells deleted for Dpb4 are not temperature sensitive.
To determine whether Dpb4 is involved in DNA synthesis, we have examined whether dpb4 is synthetically lethal with temperature-sensitive DNA replication mutants, including mutants defective for DNA Pol (see Table 1). Cells deleted for dpb4+ were crossed to a variety of mutants and incubated at temperatures ranging from the permissive temperature of 25°C to the restrictive temperature of 36°C. As shown in Table 1, we were unable to recover dpb4 cdc20-IA5 double mutants, suggesting that the mutations are synthetically lethal (confirmed by tetrad analysis). Similarly, when dpb4 was crossed to two additional temperature-sensitive mutants of Pol , cdc20-M10 and cdc20-P7 (28), the restrictive temperature for the double mutants was lower than for the cdc20 mutants alone (30°C for dpb4cdc20-P7 and 32°C for dpb4cdc20-M10, respectively), indicating a synthetic-lethal interaction. As noted earlier, dpb4 cells are not temperature sensitive. We also found that dpb4 mutants were synthetically lethal with three additional replication mutants, sna41-912, cut5-T401 and cdc21-M68 (Table 1). Sna41 is homologous to budding yeast CDC45 and is thought to play a critical role in the loading of DNA polymerases to chromatin during early S phase (29–31). Cut5 was originally identified in fission yeast as a gene required for both DNA replication initiation and coupling of S phase to mitosis (32–34). When incubated at the restrictive temperature of 36°C, cut5 mutants arrest with a 1C DNA content, but fail to establish a checkpoint that blocks entry into mitosis and therefore arrest with a ‘cut’ (cell untimely torn) phenotype. The double mutant dpb4cut5-T401 displayed a similar phenotype at an intermediate temperature of 30°C (data not shown). Cut5 is homologous to S.cerevisiae DPB11 and mammalian TopBP1. Both proteins have been implicated in DNA replication initiation and are thought to be required for the loading of CDC45 and DNA polymerases to chromatin in late G1 (35–37). Similar to Cut5, they have also been shown to be required for the checkpoint control in response to DNA damage (36–40). Cdc21 encodes a protein homologous to MCM4 that is required for the assembly of pre-replicative complexes at the conclusion of anaphase and is likely to be a component of the replicative helicase (41,42). Taken together, these results suggest that Pol might interact directly with proteins required during the early stages of DNA replication initiation.
Table 1. Genetic interactions between dpb4 and DNA replication mutants
Dpb3 interacts physically with both Dpb2 and Dpb4
We used a GST pull-down assay to determine whether Dpb3 can physically interact with Dpb4 or other Pol subunits. For these experiments, we generated strains co-expressing either GST or GST–Dpb3 with Myc-Dpb4. The cells were lysed, and soluble extracts were incubated with glutathione–Sepharose beads. Following incubation, the beads were washed extensively, boiled in sample buffer, run on polyacyrlamide gels and subjected to western blot analysis. We observed that extracts containing GST–Dpb3, but not GST alone, were able to pull down Myc-Dpb4 (Figure 3A), suggesting that Dpb3 can interact with Dpb4 in cell extracts. An interaction between Dpb3 and Dpb4 has also been detected using purified proteins (J. Hurwitz, personal communication).
Figure 3. Dpb3 interacts with both Dpb2 and Dpb4. (A) Lane 1, GST–Dpb3 purification from cells expressing GST–Dpb3 and Myc-Dpb4. Lane 2, GST purification from cells expressing GST and Myc-Dpb4. (B) Lane 1, GST–Dpb3 purification from cells expressing GST–Dpb3 and Myc-Dpb2. Lane 2, GST purification from cells expressing GST and Myc-Dpb2. Top membranes were incubated with anti-GST antibody, and bottom membranes were incubated with anti-Myc antibody.
We also tested whether GST–Dpb3 or GST–Dpb4 could interact with Myc-tagged Dpb2, which encodes the second largest subunit of Pol . Our results indicate that GST–Dpb3, but not GST–Dpb4 (data not shown), can physically interact with Myc-Dpb2 (Figure 3B). In summary, these results suggest that Dpb3 is an essential component of the Pol complex, perhaps acting as a bridge between Dpb2 and Dpb4 subunits. However, it is also possible that GST fused to Dpb4 may interfere with its ability to bind Dpb2.
We also tested whether any of the small subunits can interact with the large catalytic subunit of Pol . Unfortunately, due to the insolubility of Pol , we were unable to detect any interactions by co-immunoprecipitation experiments.
Down-regulation of Dpb3 leads to a cell cycle delay
To further investigate the function of Dpb3, we attempted to isolate temperature-sensitive mutant alleles of dpb3+ by PCR mutagenesis, but unfortunately no conditional mutants were recovered. As an alternative approach, we constructed a dpb3 deletion strain containing an integrated copy of dpb3+ under the control of the thiamine-repressible nmt81 promoter (26). Twenty-four hours after the addition of thiamine, cells appear elongated, suggesting that the cell cycle is delayed (Figure 4A). Over the next 48 h, there is a marginal increase in cell number consistent with a substantial cell cycle block (Figure 4B). These observations suggest that Dpb3 is essential for normal cell cycle progression, and that Dpb3 protein is relatively stable and can only be depleted after several generations. Flow cytometry analysis revealed that from 42 to 72 h, there is an increase in cells in S phase (cells with less than 2C DNA content; Figure 4C), suggesting that cells depleted for Dpb3 have difficulty progressing through S phase. There is also an increase in multinucleate cells (Figure 4A and D), which contributes to the peak at 4C. The increase in multinucleate cells suggests that loss of Dpb3 inhibits cytokinesis in addition to inhibiting DNA replication.
Figure 4. Depletion of Dpb3 protein causes a cell cycle delay and an increase in multinucleate cells. dpb3 containing an integrated copy of the dpb3+ under the control of the thiamine-repressible nmt81 promoter was grown in the absence or in the presence of thiamine. (A) Microscopic examination of DAPI-stained cells following the addition of thiamine. Note the increase in the number of multinucleate cells at 24–48 h. (B) There is no significant increase in cell number from 24–72 h following the addition of thiamine. (C) Flow cytometry analysis of DNA content following shut off of dpb3+. (D) The percentage of cells that display two or more nuclei following shut off of dpb3+. As a control (C), cells containing two or more nuclei were counted following shut off of an essential putative splicing factor, ini1+. Cells depleted for Ini1 result in a cell cycle arrest in G2 (55).
Dpb3 is required for proper nuclear localization of Dpb4
To determine the sub-cellular localization of Dpb3 and Dpb4, we constructed yeast strains where GFP was fused to the C-terminal end of Dpb3 or Dpb4 in situ allowing their expression at endogenous levels. Fission yeast cells expressing either Dpb3–GFP or Dpb4–GFP were visualized using direct fluorescence microscopy. We observed that both proteins localized to the nucleus as expected for DNA replication proteins (Figure 5A and B). We also observed that in the absence of Dpb4, the localization of Dpb3–GFP did not change (Figure 5C).
Figure 5. Intracellular localization of Dpb3–GFP and Dpb4–GFP. Fission yeast strains were constructed by fusing either Dpb3 or Dpb4 to GFP to allow expression of each protein at endogenous levels. (A) Localization of Dpb3–GFP. (B) Localization of Dpb4–GFP. (C) Localization of Dpb3–GFP in a dpb4 background. (D) Localization of Dpb4 in the presence of Dpb3 (dpb3nmt81-dpb3+, –thiamine) or (E) in the absence of Dpb3 (dpb3nmt81-dpb3+, +thiamine).
To visualize the localization of Dpb4 in the absence of Dpb3, we isolated a dpb3-nmt81-dpb3+ strain containing Dpb4–GFP. Addition of thiamine to this strain results in cell cycle arrest within 24–48 h. In the absence of thiamine, Dpb4–GFP localizes to the nucleus (Figure 5D). However, when Dpb3 expression is repressed by the addition of thiamine, Dpb4–GFP is not only found in the nucleus but also appears in the cytoplasm, suggesting that Dpb3 may be important to establish or maintain normal localization of Dpb4 (Figure 5E).
DISCUSSION
Pol has been implicated in chromosomal DNA replication, DNA repair, transcriptional silencing and sister-chromatid cohesion (14,43–48). Pol is composed of at least four subunits, two of which, Dpb3 and Dpb4, contain a histone-fold motif suggesting that they may be involved in modifying chromatin (9,12,13). Our own studies of Pol in fission yeast cells suggest that it is required for the assembly of the DNA replication initiation complex. This conclusion was based on observations that temperature-sensitive mutants of Pol arrest in late G1 or early S phase. Also, the fact that the catalytic domains of the large catalytic subunit of Pol are dispensable for cell viability (2,5,49) supports the notion that the C-terminal region of Pol has a unique function in DNA replication that is not dependent on its ability to synthesize DNA. We have also shown that Dpb2, the second largest subunit of Pol , binds to the origin-associated chromatin at the beginning of S phase and is required for DNA replication initiation (6).
In the yeast S.cerevisiae, the two smallest subunits of Pol , encoded by DPB3 and DPB4, are not essential for viability but loss of these genes does appear to have a negative effect on the DNA replication (12,13). Deletion of DPB4 is synthetically lethal with POL2, DPB11, DPB2 and RAD53 mutants, suggesting that DPB4 has some role in chromosomal replication. It has been hypothesized that DPB4 may be important for maintaining stability of the Pol complex (13). Deletion of DPB3 results in an increase in the levels of spontaneous mutations, suggesting that DPB3 is required to maintain high replication fidelity (12).
In contrast to budding yeast, Dpb3 is essential for cell viability in fission yeast. Although we have been unable to detect a specific DNA replication defect in cells depleted of Dpb3, our GST pull-down assays indicate that Dpb3 can interact with both Dpb2 and Dpb4, consistent with it being a subunit of Pol . On the other hand, cells deleted for dpb4+ are viable, but are synthetically lethal with conditional mutants defective in DNA replication initiation. In addition to mutations in cdc20+ (encoding Pol ) and cut5+ (homologous to DPB11/TopBP1), we also found that dpb4 is synthetically lethal with mutations in sna41+ (encoding a protein homologous to CDC45) and in cdc21+ (encoding Mcm4) consistent with Pol having a function early during DNA replication initiation.
In humans, it is not known whether Dpb3 or Dpb4 is essential for cell viability or DNA replication. Pol is not required for SV40 replication in vitro and is not found associated with the replicating viral template during infection (50). However, Pol is found associated with the replicating host chromosomal DNA, suggesting that Pol has a unique role in replication of a eukaryotic chromosome that is dispensable for viral DNA synthesis (50). One possibility is that Pol facilitates the loading of other replication proteins to the site of DNA replication initiation, similar to the role of T antigen in recruiting replication protein A, topoisomerase I and Pol to the SV40 origin (51–53).
Following depletion of Dpb3 protein, we observed a cell cycle delay that correlated with an increase in the percentage of cells in S phase. We also noticed that a substantial number of cells have two or more nuclei suggesting that cytokinesis might be inhibited when Dpb3 levels decrease. This observation is similar to what is observed following depletion of Orc6 from human cells (54). In the case of the Orc6 experiments, the authors suggested that cytokinesis might be more sensitive to changes in Orc6 protein levels than in DNA replication initiation. Therefore, it is possible that in our experiments, depletion of Dpb3 using the thiamine-repressible promoter may not be sufficient to arrest cells in early S phase but is capable of blocking the later stages of cell separation, resulting in an increase in multinucleate cells.
The precise role of Pol in chromosomal replication still needs to be elucidated. With the cloning of all four subunits of the Pol complex from S.pombe, it should now be possible to begin a detailed analysis of protein function, and begin to address how this complex interacts with other replication proteins to promote initiation of DNA replication.
ACKNOWLEDGEMENTS
We thank A. So, F. Verde, J. Sierra-Montes and L. Rodriguez-Menocal for critically reading the manuscript and providing helpful suggestions. We would also like to thank K. Rudd for his invaluable assistance in bioinformatic analysis of Dpb3. M.G.S. was supported by a pre-doctoral research fellowship from the American Heart Association. This work was supported by American Cancer Society RPG-00-262-01-GMC and by grant 1R01-CA-099034 from the National Institutes of Health.
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* To whom correspondence should be addressed. Tel: +1 305 243 3105; Fax: +1 305 243 3064; Email: gdurso@miami.edu
ABSTRACT
DNA polymerase epsilon (Pol ) is a multi-subunit enzyme required for the initiation of chromosomal DNA replication. Here, we report the cloning of two fission yeast genes, called dpb3+ and dpb4+ that encode proteins homologous to the two smallest subunits of Pol . Although Dpb4 is not required for cell viability, dpb4 mutants are synthetically lethal with mutations in four genes required for DNA replication initiation, cdc20+ (encoding DNA Pol ), cut5+ (homologous to DPB11/TopBP1), sna41+ (homologous to CDC45) and cdc21+ (encoding Mcm4, a component of the pre-replicative complex). In contrast to Dpb4, Dpb3 is essential for cell cycle progression. A glutathione S-transferase pull-down assay indicates that Dpb3 physically interacts with both Dpb2 and Dpb4, suggesting that Dpb3 associates with other members of the Pol complex. Depletion of Dpb3 leads to an accumulation of cells in S phase consistent with Dpb3 having a role in DNA replication. In addition, many of the cells have a bi-nucleate or multinucleate phenotype, indicating that cell separation is also inhibited. Finally, we have examined in vivo localization of green fluorescent protein (GFP)-tagged Dpb3 and Dpb4 and found that both proteins are localized to the nucleus consistent with their proposed role in DNA replication. However, in the absence of Dpb3, GFP-Dpb4 appears to be more dispersed throughout the cell, suggesting that Dpb3 may be important in establishing or maintaining normal localization of Dpb4.
INTRODUCTION
Eukaryotic chromosomal DNA replication requires the activity of at least three DNA polymerases, alpha (Pol ), delta (Pol ) and epsilon (Pol ). It is generally accepted that Pol is responsible for initiation through the synthesis of RNA/DNA primers, while both Pol and Pol participate in chain elongation (1). We have shown previously that Cdc20, the catalytic subunit of Pol , is composed of an N-terminal region that contains all the domains necessary for polymerase activity, while the C-terminal half of the enzyme contains essential elements with no known biochemical function (2). Interestingly, in yeast, the N-terminal catalytic domains of Pol are dispensable for cell viability (2–4). Based on our earlier observations that temperature-sensitive mutants in the catalytic subunit of Pol arrest in early S phase (5), we proposed that the primary role of Pol is to facilitate assembly of the DNA replication initiation complex. Consistent with this model, we have recently shown that Dpb2, the second largest subunit of Pol , is required for chromosomal DNA replication and binds to replication origins early in S phase (6).
Pol is composed of at least four subunits in yeast and human cells (7–10). In budding yeast, these subunits are encoded by POL2, DPB2, DPB3 and DPB4 (11–13). POL2 and DPB2 are essential for cell viability and mutants in either of these two genes lead to cell cycle arrest and inhibition of DNA replication (11,14). In contrast, neither DPB3 nor DPB4 is essential in S.cerevisiae. However, cells deleted for either of these genes do show subtle defects in nucleotide biosynthesis (12,13). Homologs of all of these proteins have been identified in human cells (15), yet their precise function in chromosomal DNA replication remains obscure.
Both Dpb3 and Dpb4 contain a histone-fold motif (9,12,13). The histone-fold motif, an extended helix–strand–helix, is believed to play a role in both protein–protein and protein–DNA interactions (16,17). Both Drosophila and human Dpb4 have been shown to be a component of the chromatin-accessibility complex (CHRAC), suggesting that Dpb4 may be involved in nucleosome remodeling (18–20). Also, in budding yeast, DPB4 has been identified as a component of ISW2, a CHRAC-like complex that is involved in epigenetic silencing at telomeres (21).
Interestingly, human CHRAC has been reported to stimulate T-antigen-mediated replication of chromatin-bound SV40 DNA in vitro (22), suggesting that CHRAC might provide a remodeling activity that facilitates the initiation of DNA replication on chromatin. It has also been reported that the C-terminal region of Pol interacts with Pol , suggesting that Pol might have a role in sister-chromatid cohesion (14,23). In conclusion, these observations suggest that Pol may have several functions independent of its polymerase activity.
In this paper, we report the cloning and characterization of two fission yeast genes that encode proteins homologous to human and budding yeast Dpb3 and Dpb4. In contrast to S.cerevisiae, we find that Dpb3 is essential for cell viability in S.pombe. Depletion of Dpb3 results in a strong cell cycle delay and accumulation of cells with slightly less than 2C DNA content, suggesting that cells have difficulty completing S phase. Also, a significant percentage of cells depleted for Dpb3 contain two or more nuclei, suggesting loss of Dpb3 results in a partial block to cell separation. GST-pull down assays show that Dpb3 can interact with both Dpb2 and Dpb4, confirming that Dpb3 interacts with the other putative subunits of the Pol complex.
Although Dpb4 was found to be non-essential for cell viability, deletion of dpb4+ is synthetically lethal with mutants defective in the catalytic subunit of Pol , encoded by cdc20+, and with cut5-T401, sna41-912 and cdc21-M68, three mutants defective in DNA replication initiation. These observations are consistent with Dpb4 being important for some aspect of DNA replication.
Finally, we show that both Dpb3 and Dpb4 localize to the nucleus consistent with their proposed role in DNA replication and that normal localization of Dpb4 is dependent on expression of Dpb3.
MATERIALS AND METHODS
Schizosaccharomyces pombe strains and methods
All strains used were derived from 972 (h–) and 975 (h+). Media, growth conditions and standard genetic methods were as described previously (24). Fission yeast cosmids c17G8 (containing dpb3+) and c3D6 (containing dpb4+) were kindly provided by The Sanger Center (Hinxton, UK). DNA content was determined by fluorescence-activated cell sorting as previously described (Sazer and Sherwood, 1990), except that cells were stained with Sytox Green (Molecular Probes) instead of propidium iodide.
Construction of dpb3 and dpb4 mutant strains
The plasmids pKS(+)dpb3::ura4+ and pKS(+)dpb4::ura4+ used for deletion of dpb3+ and dpb4+ were constructed as follows: a 1.8 kb DNA fragment containing the ura4+ gene was isolated from pARC614 digested with HindIII and cloned into pKS(+)Bluescript. Both the upstream (1.6–2.0 kb) and the downstream (1.8–2.0 kb) flanking genomic sequences of each gene were amplified by PCR from cosmid DNA and cloned into pKS(+)Bluescript at the ApaI/XhoI and the XmaI/BamHI restriction sites, respectively, located upstream and downstream of the ura4+ gene. The resulting plasmids were digested with KpnI/NotI to obtain a DNA fragment containing ura4+ and either the dpb3+ or dpb4+ flanking sequences. Each DNA fragment was then transformed into the diploid strain (h+/h– leu1-32/leu1-32 ura4-D18/ura4-D18 ade6-M210/ade6-M216) and stable ura4+ integrants were selected. Replacement of either dpb3+ or dpb4+ with the ura4+ gene was confirmed by the Southern blot analysis. Diploids were induced to sporulate and meiotic segregants were analyzed by tetrad analysis.
Cloning of dpb3+ and dpb4+
The dpb3+ open reading frame (599 bp) was PCR-amplified from cosmid c17G8 using the forward primer (5'-CGCGGATCCGATGGGTGATCCAACAAATAATAATG) and the reverse primer (3'-TCCCCCGGGCTACTCATCACCAGACGCGGAAGAGG) tagged with BamHI and XmaI, respectively (indicated by underline). The amplified product was digested and then cloned in the pRep3X vector at the BamHI and XmaI sites.
To clone the dpb4+ gene, the 803 bp open reading frame was amplified from the cosmid c3D6 using the following primers, 5'-CGCGGATCCGATGAATCAAGATAAATCG and 3'-TCCCCCGGGTTACGAAGAATCATTAAG, containing a BamHI and XmaI restriction sites, respectively. The PCR product generated was then digested and cloned into the pRep3X vector at the same restriction sites.
Construction of a strain expressing dpb3+ under the control of the thiamine-repressible promoter
In order to generate a thiamine-repressible dpb3+ strain, the dpb3+ gene was amplified using the same primers described above, but tagged with BamHI/SacI. The PCR product was digested and inserted at the BamHI/SacI sites in a pJK148 vector that contained the nmt81 promoter cloned at the PstI/BamHI sites. The resulting plasmid was then linearized within the leu1+ gene by digestion with AfeI and transformed into the diploid strain, h+/h– leu1-32/leu1-32 ura4-D18/ura4-D18 ade6-M210/ade6-M216. Stable ura4+ integrants were selected, sporulated and the resulting spores were germinated on minimal plates in the absence of thiamine. Viable leu1+ ura4+ ade– colonies were recovered indicating that the expression of dpb3+ from the nmt81 promoter was sufficient to rescue the dpb3 strain. To shut off dpb3+ expression, cells were then grown in the presence of 10 mM thiamine.
GST pull-down assay
For ectopic expression of proteins, we used the thiamine-repressible nmt1 promoter (25,26). To generate a GST-fusion protein, the dpb3+ coding region was amplified with PCR primers tagged with BamHI and XmaI restriction sites located 5' and 3' to the gene. The amplified fragment was then cloned into the pESP expression vector, downstream of the nmt1 promoter. Similarly, Myc-Dpb4 and Myc-Dpb2 were created by cloning the PCR-amplified genes into pARC616 at the BamHI/XmaI sites, in frame with the Myc-epitope tag. The expression of the Myc-tagged fusion protein in pARC616 is under the control of the weaker nmt41 promoter. Fission yeast cells were transformed with pESP-dpb3+ and either Myc-dpb2+ or Myc-dpb4+ constructs. As a negative control, pESP-dpb3+ was replaced with the pESP vector alone. Colonies containing both plasmids (selected on media lacking leucine and uracil) were isolated and grown in minimal medium supplemented with thiamine (5 μg/ml). The cells (OD 0.5) were then washed extensively with water to remove all traces of thiamine, and the expression of tagged proteins were induced by incubating each strain in media in the absence of thiamine for 18 h at 32°C. The cells were then broken with glass beads and 1 μg of soluble protein was coupled to 50 μl glutathione–Sepharose resin ON at 4°C in 1 ml HB buffer (25 mM MOPS, 1% Triton X-100, 0.1 mM sodium vanadate, 60 mM ?-glycerophosphate, 15 mM p-nitrophenylphosphate, 15 mM MgCl2 and 15 mM EGTA) freshly supplemented with 1 mM phenylmethylsulfonyl fluoride, 0.5 M DTT, and protease inhibitors (Complete Protease Inhibitors Cocktail Tablet, Roche). After washing the resin four times with HB buffer, bound proteins were eluted in 50 μl 2x SDS buffer, boiled and analyzed by 8% SDS–PAGE and western blotting using anti-Myc and anti-GST antibodies.
GFP localization studies
To generate strains that contain Dpb3 and Dpb4 endogenously tagged with green fluorescent protein (GFP), the corresponding genes were PCR-amplified with forward and reverse primers containing BamHI and HindIII sites, respectively, but lacking any stop codon. The amplified DNA fragments were digested and inserted in a pKS(+) Bluescript vector at the BamHI/HindIII sites in frame with GFP at the C-terminal end. The pKS vector used also contained a copy of the sup3-5 allele, a suppressor of the ade6-704 mutant allele. Each of the resulting plasmids were transformed in an ade6-704 strain and stable integrants were selected. Genomic DNA was extracted and the site of integration was confirmed by PCR using a forward primer upstream of each coding region and a reverse primer within the GFP gene. To observe Dpb3–GFP and Dpb4–GFP in live cells, yeast cells were grown to an optical density of <0.5 and analyzed using a Zeiss Axiophot microscope. To visualize nuclei, cells were treated with 1 μg/ml DAPI in Slowfade Component A (Molecular probes). Images were captured and processed using Openlab software.
RESULTS
Dpb3 is required for cell cycle progression
We identified a gene in the S.pombe genome database (accession no. Q10315 ) that encodes a protein that is 34 and 26% identical to human p12 (human Dpb3) and to the N-terminal portion of S.cerevisiae DPB3, respectively. Consequently, the gene was termed dpb3+. Only the N-terminal half of the protein that contains the histone-fold motif (shown in Figure 1A and B) displays any significant homology to the other known orthologs. Human, mouse and Drosophila Dpb3 are considerably smaller (12–15 kD) than Dpb3 from budding or fission yeast. Although both S.pombe and S.cerevisiae Dpb3 contain an extended C-terminal region, a comparison between these two sequences reveals no significant homology within this region of the protein. This suggests that the histone-fold motif is critical for Dpb3 function. Also, although S.pombe Dpb3 shows limited identity to budding yeast DPB3 (<20% overall), the two proteins are nearly identical in size (both are estimated to be 22 kD). There is only one additional protein encoded by the S.pombe genome, called Php5 that is similar to human p12 or S.cerevisiae DPB3. Considering that the estimated mass of this protein (46 kD) is significantly larger than the other homologs, and that it has previously been reported to be a component of a CCAAT-binding factor essential for the expression of the cyc1+ gene, it is unlikely that Php5 represents the third subunit of DNA Pol (27). Therefore, we conclude that the gene we have identified as dpb3+ encodes an ortholog of S.cerevisiae DPB3 and human p12. To determine whether dpb3+ is essential for cell viability, we constructed a diploid strain where a single copy of dpb3+ was replaced by ura4+ (Figure 2A). Construction of the deletion strain was confirmed by Southern blot analysis (Figure 2B). Following sporulation, germinating spores were subjected to tetrad analysis. Most of the meiotic segregants examined gave rise to a 2:2 segregation pattern of viable/non-viable cells indicating that dpb3+ is an essential gene (Figure 2C). As expected, the viable colonies failed to grow in the absence of uracil. In the three tetrads that gave a 1:3 ratio of viable/non-viable cells, the viable colony was ura– suggesting that the remaining ura– spore failed to germinate. Failure of ura– spores to germinate during tetrad analysis is frequently observed. Microscopic analysis of dpb3 germinating spores showed that cells underwent a few rounds of cell division before arresting with an elongated cell (cdc) phenotype, (Figure 2D) indicative of a cell cycle block. Therefore, we concluded that Dpb3 is essential for cell cycle progression.
Figure 1. (A) Schematic representation of the conserved Dpb3 proteins from S.pombe, S.cerevisiae, Homo sapiens, Mus musculus and Drosophila melanogaster. Percentage identity to fission yeast Dpb3, within the histone-fold motif, is indicated. (B) Amino acid sequence comparison between the histone-fold motif from S.pombe Dpb3 and its orthologs. Human, mouse and Drosophila are shown as full-length proteins. For S.pombe and S.cerevisiae, only the N-terminal half of the protein is shown because this is the only region that shares extensive homology to Dpb3 from other organisms. Alignment of the proteins was performed using ClustalW. Residues in black indicate identity and residues in gray indicate similarity. The conserved histone-fold motif is indicated by the double overline.
Figure 2. (A) Schematic representation of dpb3 construct. The 0.6 kb dpb3+ gene was replaced with a 1.8 kb ura4+ gene. (B) Southern blot analysis of dpb3 strain, lanes 1 and 2, dpb3/dpb3+; lane 3, wild-type 972. NdeI digestion of genomic DNA generates a fragment of 5.4 kb, indicating that gene replacement was successful. (C) Tetrad analysis confirms dpb3+ is an essential gene. (D) Morphology of cells deleted for dpb3+.
dpb4 is synthetically lethal with DNA replication mutants
The sequence of a putative S.pombe homolog of Dpb4 has been reported previously (9,13). The fission yeast Dpb4 is 210 amino acids long, has an estimated mass of 23 kD and shares 36 and 30% identity to human p17 (human Dpb4) and budding yeast DPB4, respectively. To test whether dpb4+ is essential for viability, we constructed a diploid strain where one copy of dpb4+ was replaced by the ura4+ gene. Following sporulation, germinating spores gave rise to viable colonies in the absence of uracil, demonstrating that dpb4+ is non-essential. Also, the cells deleted for Dpb4 are not temperature sensitive.
To determine whether Dpb4 is involved in DNA synthesis, we have examined whether dpb4 is synthetically lethal with temperature-sensitive DNA replication mutants, including mutants defective for DNA Pol (see Table 1). Cells deleted for dpb4+ were crossed to a variety of mutants and incubated at temperatures ranging from the permissive temperature of 25°C to the restrictive temperature of 36°C. As shown in Table 1, we were unable to recover dpb4 cdc20-IA5 double mutants, suggesting that the mutations are synthetically lethal (confirmed by tetrad analysis). Similarly, when dpb4 was crossed to two additional temperature-sensitive mutants of Pol , cdc20-M10 and cdc20-P7 (28), the restrictive temperature for the double mutants was lower than for the cdc20 mutants alone (30°C for dpb4cdc20-P7 and 32°C for dpb4cdc20-M10, respectively), indicating a synthetic-lethal interaction. As noted earlier, dpb4 cells are not temperature sensitive. We also found that dpb4 mutants were synthetically lethal with three additional replication mutants, sna41-912, cut5-T401 and cdc21-M68 (Table 1). Sna41 is homologous to budding yeast CDC45 and is thought to play a critical role in the loading of DNA polymerases to chromatin during early S phase (29–31). Cut5 was originally identified in fission yeast as a gene required for both DNA replication initiation and coupling of S phase to mitosis (32–34). When incubated at the restrictive temperature of 36°C, cut5 mutants arrest with a 1C DNA content, but fail to establish a checkpoint that blocks entry into mitosis and therefore arrest with a ‘cut’ (cell untimely torn) phenotype. The double mutant dpb4cut5-T401 displayed a similar phenotype at an intermediate temperature of 30°C (data not shown). Cut5 is homologous to S.cerevisiae DPB11 and mammalian TopBP1. Both proteins have been implicated in DNA replication initiation and are thought to be required for the loading of CDC45 and DNA polymerases to chromatin in late G1 (35–37). Similar to Cut5, they have also been shown to be required for the checkpoint control in response to DNA damage (36–40). Cdc21 encodes a protein homologous to MCM4 that is required for the assembly of pre-replicative complexes at the conclusion of anaphase and is likely to be a component of the replicative helicase (41,42). Taken together, these results suggest that Pol might interact directly with proteins required during the early stages of DNA replication initiation.
Table 1. Genetic interactions between dpb4 and DNA replication mutants
Dpb3 interacts physically with both Dpb2 and Dpb4
We used a GST pull-down assay to determine whether Dpb3 can physically interact with Dpb4 or other Pol subunits. For these experiments, we generated strains co-expressing either GST or GST–Dpb3 with Myc-Dpb4. The cells were lysed, and soluble extracts were incubated with glutathione–Sepharose beads. Following incubation, the beads were washed extensively, boiled in sample buffer, run on polyacyrlamide gels and subjected to western blot analysis. We observed that extracts containing GST–Dpb3, but not GST alone, were able to pull down Myc-Dpb4 (Figure 3A), suggesting that Dpb3 can interact with Dpb4 in cell extracts. An interaction between Dpb3 and Dpb4 has also been detected using purified proteins (J. Hurwitz, personal communication).
Figure 3. Dpb3 interacts with both Dpb2 and Dpb4. (A) Lane 1, GST–Dpb3 purification from cells expressing GST–Dpb3 and Myc-Dpb4. Lane 2, GST purification from cells expressing GST and Myc-Dpb4. (B) Lane 1, GST–Dpb3 purification from cells expressing GST–Dpb3 and Myc-Dpb2. Lane 2, GST purification from cells expressing GST and Myc-Dpb2. Top membranes were incubated with anti-GST antibody, and bottom membranes were incubated with anti-Myc antibody.
We also tested whether GST–Dpb3 or GST–Dpb4 could interact with Myc-tagged Dpb2, which encodes the second largest subunit of Pol . Our results indicate that GST–Dpb3, but not GST–Dpb4 (data not shown), can physically interact with Myc-Dpb2 (Figure 3B). In summary, these results suggest that Dpb3 is an essential component of the Pol complex, perhaps acting as a bridge between Dpb2 and Dpb4 subunits. However, it is also possible that GST fused to Dpb4 may interfere with its ability to bind Dpb2.
We also tested whether any of the small subunits can interact with the large catalytic subunit of Pol . Unfortunately, due to the insolubility of Pol , we were unable to detect any interactions by co-immunoprecipitation experiments.
Down-regulation of Dpb3 leads to a cell cycle delay
To further investigate the function of Dpb3, we attempted to isolate temperature-sensitive mutant alleles of dpb3+ by PCR mutagenesis, but unfortunately no conditional mutants were recovered. As an alternative approach, we constructed a dpb3 deletion strain containing an integrated copy of dpb3+ under the control of the thiamine-repressible nmt81 promoter (26). Twenty-four hours after the addition of thiamine, cells appear elongated, suggesting that the cell cycle is delayed (Figure 4A). Over the next 48 h, there is a marginal increase in cell number consistent with a substantial cell cycle block (Figure 4B). These observations suggest that Dpb3 is essential for normal cell cycle progression, and that Dpb3 protein is relatively stable and can only be depleted after several generations. Flow cytometry analysis revealed that from 42 to 72 h, there is an increase in cells in S phase (cells with less than 2C DNA content; Figure 4C), suggesting that cells depleted for Dpb3 have difficulty progressing through S phase. There is also an increase in multinucleate cells (Figure 4A and D), which contributes to the peak at 4C. The increase in multinucleate cells suggests that loss of Dpb3 inhibits cytokinesis in addition to inhibiting DNA replication.
Figure 4. Depletion of Dpb3 protein causes a cell cycle delay and an increase in multinucleate cells. dpb3 containing an integrated copy of the dpb3+ under the control of the thiamine-repressible nmt81 promoter was grown in the absence or in the presence of thiamine. (A) Microscopic examination of DAPI-stained cells following the addition of thiamine. Note the increase in the number of multinucleate cells at 24–48 h. (B) There is no significant increase in cell number from 24–72 h following the addition of thiamine. (C) Flow cytometry analysis of DNA content following shut off of dpb3+. (D) The percentage of cells that display two or more nuclei following shut off of dpb3+. As a control (C), cells containing two or more nuclei were counted following shut off of an essential putative splicing factor, ini1+. Cells depleted for Ini1 result in a cell cycle arrest in G2 (55).
Dpb3 is required for proper nuclear localization of Dpb4
To determine the sub-cellular localization of Dpb3 and Dpb4, we constructed yeast strains where GFP was fused to the C-terminal end of Dpb3 or Dpb4 in situ allowing their expression at endogenous levels. Fission yeast cells expressing either Dpb3–GFP or Dpb4–GFP were visualized using direct fluorescence microscopy. We observed that both proteins localized to the nucleus as expected for DNA replication proteins (Figure 5A and B). We also observed that in the absence of Dpb4, the localization of Dpb3–GFP did not change (Figure 5C).
Figure 5. Intracellular localization of Dpb3–GFP and Dpb4–GFP. Fission yeast strains were constructed by fusing either Dpb3 or Dpb4 to GFP to allow expression of each protein at endogenous levels. (A) Localization of Dpb3–GFP. (B) Localization of Dpb4–GFP. (C) Localization of Dpb3–GFP in a dpb4 background. (D) Localization of Dpb4 in the presence of Dpb3 (dpb3nmt81-dpb3+, –thiamine) or (E) in the absence of Dpb3 (dpb3nmt81-dpb3+, +thiamine).
To visualize the localization of Dpb4 in the absence of Dpb3, we isolated a dpb3-nmt81-dpb3+ strain containing Dpb4–GFP. Addition of thiamine to this strain results in cell cycle arrest within 24–48 h. In the absence of thiamine, Dpb4–GFP localizes to the nucleus (Figure 5D). However, when Dpb3 expression is repressed by the addition of thiamine, Dpb4–GFP is not only found in the nucleus but also appears in the cytoplasm, suggesting that Dpb3 may be important to establish or maintain normal localization of Dpb4 (Figure 5E).
DISCUSSION
Pol has been implicated in chromosomal DNA replication, DNA repair, transcriptional silencing and sister-chromatid cohesion (14,43–48). Pol is composed of at least four subunits, two of which, Dpb3 and Dpb4, contain a histone-fold motif suggesting that they may be involved in modifying chromatin (9,12,13). Our own studies of Pol in fission yeast cells suggest that it is required for the assembly of the DNA replication initiation complex. This conclusion was based on observations that temperature-sensitive mutants of Pol arrest in late G1 or early S phase. Also, the fact that the catalytic domains of the large catalytic subunit of Pol are dispensable for cell viability (2,5,49) supports the notion that the C-terminal region of Pol has a unique function in DNA replication that is not dependent on its ability to synthesize DNA. We have also shown that Dpb2, the second largest subunit of Pol , binds to the origin-associated chromatin at the beginning of S phase and is required for DNA replication initiation (6).
In the yeast S.cerevisiae, the two smallest subunits of Pol , encoded by DPB3 and DPB4, are not essential for viability but loss of these genes does appear to have a negative effect on the DNA replication (12,13). Deletion of DPB4 is synthetically lethal with POL2, DPB11, DPB2 and RAD53 mutants, suggesting that DPB4 has some role in chromosomal replication. It has been hypothesized that DPB4 may be important for maintaining stability of the Pol complex (13). Deletion of DPB3 results in an increase in the levels of spontaneous mutations, suggesting that DPB3 is required to maintain high replication fidelity (12).
In contrast to budding yeast, Dpb3 is essential for cell viability in fission yeast. Although we have been unable to detect a specific DNA replication defect in cells depleted of Dpb3, our GST pull-down assays indicate that Dpb3 can interact with both Dpb2 and Dpb4, consistent with it being a subunit of Pol . On the other hand, cells deleted for dpb4+ are viable, but are synthetically lethal with conditional mutants defective in DNA replication initiation. In addition to mutations in cdc20+ (encoding Pol ) and cut5+ (homologous to DPB11/TopBP1), we also found that dpb4 is synthetically lethal with mutations in sna41+ (encoding a protein homologous to CDC45) and in cdc21+ (encoding Mcm4) consistent with Pol having a function early during DNA replication initiation.
In humans, it is not known whether Dpb3 or Dpb4 is essential for cell viability or DNA replication. Pol is not required for SV40 replication in vitro and is not found associated with the replicating viral template during infection (50). However, Pol is found associated with the replicating host chromosomal DNA, suggesting that Pol has a unique role in replication of a eukaryotic chromosome that is dispensable for viral DNA synthesis (50). One possibility is that Pol facilitates the loading of other replication proteins to the site of DNA replication initiation, similar to the role of T antigen in recruiting replication protein A, topoisomerase I and Pol to the SV40 origin (51–53).
Following depletion of Dpb3 protein, we observed a cell cycle delay that correlated with an increase in the percentage of cells in S phase. We also noticed that a substantial number of cells have two or more nuclei suggesting that cytokinesis might be inhibited when Dpb3 levels decrease. This observation is similar to what is observed following depletion of Orc6 from human cells (54). In the case of the Orc6 experiments, the authors suggested that cytokinesis might be more sensitive to changes in Orc6 protein levels than in DNA replication initiation. Therefore, it is possible that in our experiments, depletion of Dpb3 using the thiamine-repressible promoter may not be sufficient to arrest cells in early S phase but is capable of blocking the later stages of cell separation, resulting in an increase in multinucleate cells.
The precise role of Pol in chromosomal replication still needs to be elucidated. With the cloning of all four subunits of the Pol complex from S.pombe, it should now be possible to begin a detailed analysis of protein function, and begin to address how this complex interacts with other replication proteins to promote initiation of DNA replication.
ACKNOWLEDGEMENTS
We thank A. So, F. Verde, J. Sierra-Montes and L. Rodriguez-Menocal for critically reading the manuscript and providing helpful suggestions. We would also like to thank K. Rudd for his invaluable assistance in bioinformatic analysis of Dpb3. M.G.S. was supported by a pre-doctoral research fellowship from the American Heart Association. This work was supported by American Cancer Society RPG-00-262-01-GMC and by grant 1R01-CA-099034 from the National Institutes of Health.
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