Fission Yeast sld3+ Is Essential for Cell Growth
We identified a hypothetical protein (GenBank CAA90850.2) in a fission yeast genome database. This protein shares 14% identity and 24% similarity with budding yeast Sld3 (Figure B). The gene encoding the protein was named as sld3+, as a potential counterpart of budding yeast SLD3. The sld3+ encodes a 699-amino acid polypeptide with a calculated molecular mass of ~79 kDa. This protein does not contain characteristic protein motifs, except for putative coiled-coil motifs in regions 142–165 and 253–341 of amino acid positions (Figure A).
Figure 1 Structure of S. pombe SpSld3. (A) Schematic representation of SpSld3 is shown. The shaded boxes indicate the coiled-coil regions predicted by COILS program. Asterisks indicate the positions of amino acids changed by the sld3-10, -41, and -52 mutations. (more ...)
We replaced one of the sld3+ copies in a ura− diploid with the sld3::ura4+. Sporulation and tetrad dissection of the sld3+/sld3::ura4 heterodisruptant yielded two viable and two lethal spores and all viable spores were Ura−. To confirm that the lethality was due to disruption of the sld3+, the sld3+/sld3::ura4+ diploids transformed with pSLD3 plasmid carrying the sld3+ gene were sporulated. Tetrad dissection yielded three and four viable spores, including Ura+ spores. Thus, the sld3+ is indeed essential for cell growth.
Temperature-sensitive sld3 Mutants Are Defective in DNA Replication
To investigate the essential role of Sld3 in cell growth, we isolated temperature-sensitive mutants of sld3. Mutations were introduced by PCR into a fragment carrying the sld3+ and the ura4+ genes and the PCR products were used for transformation of a ura− haploid strain. Among 3000 Ura+ transformants grown at 23°C, three mutants that did not grow at 36°C were isolated. Growth of the mutants at the restrictive temperature was restored by transformation with pSLD3, which suggested that the sld3 gene has the mutations.
Determination of the nucleotide sequence of the mutant sld3 genes revealed that the glutamic acid residue at position 338 is replaced with glycine in the sld3-10, tryptophan at position 310 with arginine in the sld3-52, and the serine at position 153 and the leucine at position 309 with leucine and proline, respectively, in the sld3-41. It should be noted that all of these changes reside in predicted coiled-coil regions, although these amino acids are not conserved between the budding yeast Sld3 and SpSld3, except for one of two sites in the sld3-41 (Figure A).
We first examined growth of the sld3-10 at the restrictive temperature. The increase of the cell number was retarded during 2–4 h at 36°C and arrested after 6 h (Figure A). Cell viability decreased to ~40% at 6 h (Figure B). To determine effects of sld3-10 mutation on DNA replication, we analyzed DNA contents at the restrictive temperature, by using flow cytometry. sld3-10 cells, initially containing 2C DNA as the wild type, produced cells with 1C DNA content after 2 h at 36°C and the population of these cells increased at 4 h (Figure C). In addition, cells with <1C DNA content appeared at 6 h. The other two mutants, sld3-41 and sld3-52, also accumulated cells with 1C DNA content at the restrictive temperature (our unpublished data). Therefore, chromosome DNA is not duplicated in these mutants. SpSld3 is apparently required for DNA replication or for G1–S progression of cell cycle.
Figure 2 Temperature-sensitive growth of the sld3-10. Exponentially growing wild-type (972) and the sld3-10 cells at 23°C were shifted to 36°C and cells collected at indicated time points were analyzed. The cell number (A) and the cell viability (more ...)
With 4,6-diamidino-2-phenylindole staining, we observed that ~10% of sld3-10 cells contained unequally divided nucleus or undivided nucleus cleaved by the septum after 6 h at 36°C, yet such cells were not observed in the wild type (our unpublished data). These cells would correspond to cells with <1C DNA content in flow cytometry analysis and these are presumably formed as a result of nuclear division without completion of DNA replication. These results suggest that the sld3-10 cells have a defect in generation of S-M checkpoint signal.
Budding yeast SLD3
has a genetic interaction with SLD4
(Kamimura et al., 2001
). To investigate such interaction between the sld3+
and the sna41+
gene encoding SpCdc45, sld3
mutants were transformed with a high-copy plasmid carrying sna41+
. The growth of sld3-10
, and -52
at the restrictive temperature was restored by the sna41+
plasmid (Figure D; our unpublished data). In a reciprocal experiment, growth of the sna41-1
, a temperature-sensitive mutant of the sna41
, was restored by transformation with pSLD3 (Figure E). Therefore, strong genetic interactions exist between sld3+
. Taken together with the sequence similarity, sld3+
seems to be a counterpart of budding yeast SLD3
Cell Cycle-dependent Interactions of SpSld3 with SpCdc45 and SpMcm6
Based on genetic interactions between the sld3+ and sna41+, we examined physical interactions between SpSld3 and SpCdc45. Cells expressing SpCdc45-Myc or both SpCdc45-Myc and SpSld3-FLAG from chromosome loci were arrested in the early S phase by adding hydroxyurea (HU), which inhibits deoxyribonucleotide synthesis. SpSld3 was immunoprecipitated in the presence of DNase I and this would reduce nonspecific coprecipitation mediated by DNA. SpCdc45 was coprecipitated from the cells expressing SpSld3-FLAG (Figure A, lane 6), but not from the untagged strain (lane 3) or without anti-FLAG antibody (lane 5). In a reciprocal experiment using an anti-SpCdc45 antibody, SpSld3-FLAG coprecipitated with SpCdc45 (Figure A, lane 8). These observations mean that SpSld3 associates with SpCdc45 in the early S phase.
Figure 3 Coimmunoprecipitation of SpCdc45 and SpMcm6 with SpSld3 in G1–S phases. (A) Cells expressing both SpSld3-FLAG5 and SpCdc45-Myc9 (lanes 4–6 and 7 and 8) or only SpCdc45-Myc9 (lanes 1–3) were cultured in the presence of 10 mM HU (more ...)
Next, we examined the association of SpSld3 with SpCdc45 during the cell cycle. Temperature-sensitive cdc25-22 cells expressing SpSld3-FLAG and SpCdc45-Myc were arrested at the G2/M boundary and released into the cell cycle at the permissive temperature. Analyses of the SpSld3-immunoprecipitates (IPs) by Western blotting showed that SpCdc45 coprecipitated 80–120 min after release (Figure B). The timing of the coprecipitation was shortly after increase in the septation index, which suggests that SpSld3 associates with SpCdc45 mainly in the S phase. On the other hand, SpMcm6 was detected in SpSld3-IPs at 60–100 min, such being earlier than SpCdc45. SpMcm7 also coprecipitated with SpSld3 during the same period as SpMcm6 (our unpublished data). SpSld3 probably associates with MCM proteins before the association with SpCdc45.
SpSld3 migrated as diffused bands during 0–60 min after release from the G2/M block, whereas it formed a sharp band with a higher mobility at 80 min and later time points. To examine the possibility that the slower mobility of SpSld3 was due to the phosphorylation, SpSld3 immunoprecipitated from the nda3-KM311 sld3-flag5
cells arrested in M phase (Hiraoka et al., 1984
) was incubated with CIP. This phosphatase treatment yielded a sharp band with a higher mobility (Figure C, lane 3), whereas the slowly migrating bands remained in the presence of phosphatase inhibitors (Figure C, lane 4). Thus, SpSld3 is phosphorylated in M-phase–arrested cells. The higher mobility band observed during the S phase (Figure B) seems to be a hypophosphorylated form.
Localization of SpSld3 at Chromosomal Replication Origin during G1–S Phases
SpMcm6 localizes to the replication origin during G1–S phases (Ogawa et al., 1999
). Because SpSld3 associates with SpMcm6, we asked whether SpSld3 is associated with the replication origin during a specific phase of the cell cycle. We did chromatin ChIP assays (Ogawa et al., 1999
) by using cdc25-22
cells expressing SpSld3-FLAG, synchronized as described above. The cells were cross-linked with formaldehyde at indicated time points and DNA fragments associated with SpSld3-FLAG were recovered by immunoprecipitation. SpSld3 association was analyzed by PCR for the chromosomal replication origin ars2004
(Okuno et al., 1997
), together with two non-ARS regions. The three fragments were amplified to a similar extent from total cellular DNA used as a template (Figure A). On the other hand, when immunoprecipitated DNA was used as a template, the ars2004
fragment was preferentially amplified during 80–120 min after release (Figure A) and this period corresponded to G1–S phases monitored by an increase in the septation index. Preferential amplification of the ars2004
fragment depended on the FLAG-tagged SpSld3 and on cross-linking treatment (our unpublished data). These observations mean that SpSld3 associates with the ars2004
origin during G1–S phases.
Figure 4 SpSld3 associates with origin DNA in G1–S phases.(A) The cdc25-22 cells expressing SpSld3-FLAG5 and SpCdc45-Myc9 were synchronized, as described for Figure B. At every 20 min, cells were fixed with formaldehyde and DNA fragments (more ...)
To examine timing of the origin-association of SpSld3 more precisely, we did ChIP assays by using the cdc10
strain in which Cdc18 or Cdt1 is not expressed and MCM proteins are not loaded onto the chromatin at the restrictive temperature (Ogawa et al., 1999
; Nishitani et al., 2000
cells expressing SpSld3-FLAG were incubated at 36°C for 3 h to arrest the cell cycle in the early G1 phase. The ars2004
fragment was not preferentially amplified from immunoprecipitates of SpSld3 (Figure B). To verify that the ChIP assay functioned for the cells incubated at the high temperature, cdc25-22
cells released from G2/M boundary were arrested in the early S phase in the presence of HU, and then incubated at 36°C for 1 h. The ars2004
fragment was selectively amplified from DNA immunoprecipitated with SpSld3 to an extent similar to the sample not incubated at 36°C (our unpublished data). These results suggest that the SpSld3 is not loaded onto the replication origin at the cdc10
-arrest point in the G1 phase.
We then examined the origin association of SpSld3 in hsk1-89
mutant, a temperature-sensitive mutant of Hsk1 kinase (Takeda et al., 2001
). It was reported that DDK activity is not required for chromatin loading of MCM but is required for Cdc45 loading in Xenopus
egg extracts (Jares and Blow, 2000
; Walter, 2000
). Thus, in hsk1-89
cells, the cell cycle is expected to arrest before initiation, in late G1 phase. hsk1-89
mutant cells expressing SpSld3-FLAG were incubated at the restrictive temperature (30°C) for 2 h then subjected to ChIP assay. The ars2004
fragment was preferentially amplified from the SpMcm6-IPs (Figure C), thus confirming that DDK is not required for MCM loading in fission yeast. The specific amplification of ars2004
fragment was not observed using the SpSld3-IPs as a template (Figure C). Because DDK activity is reduced by the hsk1-89
mutation (Takeda et al., 2001
), the association of SpSld3 with the replication origin depends on DDK activity. The SpSld3 associates with origins in the late G1 phase, after or on the DDK-execution point.
Chromatin Loading of SpCdc45 Is Impaired by the sld3-10 Mutation
To determine the role of SpSld3 in initiation of DNA replication, we investigated whether the sld3-10 mutation would affect the loading of MCM and SpCdc45 onto chromatin. We first examined the association of MCM and SpCdc45 proteins with chromatin in cell cycle progression. We used cdc25-22 cells expressing SpCdc45-Myc and SpSld3-FLAG synchronized as described above. At the indicated time points, the chromatin-enriched fraction was separated from the soluble-protein fraction and analyzed by Western blotting. As shown in Figure A, SpOrc4, a subunit of SpORC, was present in the chromatin-enriched fraction throughout the cell cycle. SpMcm6 was associated with chromatin from 40 to 120 min after release, which corresponds to the G1–S phases. SpMcm7 was detected in the chromatin-enriched fraction during the same period as SpMcm6 (our unpublished data). On the other hand, SpCdc45 appeared in the chromatin-enriched fraction at 60 min, peaked at 80–100 min after release, and was then reduced (Figure A). Therefore, SpCdc45 associates with chromatin later than do MCM proteins. We did not detect SpSld3 in the chromatin-enriched fraction or in the soluble fraction, probably because the amounts of the protein in these fractions were below detection limits (our unpublished data).
Figure 5 Association of SpCdc45 with chromatin is reduced in sld3-10 mutant. (A) Cell cycle-dependent association of SpCdc45 with chromatin is shown. The cdc25-22 cells expressing SpSld3-FLAG5 and SpCdc45-Myc9 were synchronized, as described for Figure (more ...)
To examine the effect of the sld3-10 mutation on association of SpCdc45 with the chromatin, mutant cells expressing SpOrc1-FLAG and SpCdc45-Myc were incubated at 36°C for 3 h. As a control, the wild-type cells were arrested at the early S phase by incubating at 36°C for 3 h in the presence of HU. Flow cytometry revealed that the mutant and the HU-treated wild-type strains accumulated cells with a 1C DNA content (Figure B). The results of Western blotting showed that approximately one-third of SpOrc1 and one-tenth each of SpMcm6, SpMcm7, and SpCdc45 were in the chromatin-enriched fraction of the HU-arrested wild-type cells (Figure C). In contrast, SpCdc45 was not detected in the chromatin-enriched fraction of sld3-10 extracts, whereas SpOrc1, SpMcm6, and SpMcm7 were detected as the HU-arrested wild type (Figure C). Therefore, the sld3-10 mutant has a defect in loading or maintenance of SpCdc45 onto chromatin. Consistent with these results, SpCdc45 did not coprecipitate with SpMcm6 at the restrictive temperature of sld3-10 (Figure D). These results suggest that SpSld3 is required for loading of SpCdc45 onto the pre-RCs at replication origins.
Requirement of SpSld3 during S Phase
Because MCM and Cdc45 proteins are required for elongation of DNA replication (Labib et al., 2000
; Tercero et al., 2000
), we determined whether SpSld3 is required after entry into S phase. Wild-type and sld3-10
cells were arrested by HU at 23°C and then incubated at 36°C to inactivate the mutant protein. On removal of HU at 36°C, the DNA content of the wild-type cells increased from 1C to 2C within 30 min (Figure B). In contrast, sld3-10
cells increased the DNA content very slowly after release. Although the DNA content of sld3-10
cells increased to nearly 2C DNA after 3 h, the cell number did not increase (Figure C), probably because DNA synthesis was not completed, as suggested by the broad peak seen on flow-cytometry results. The severe retardation of DNA replication may result from a defect in elongation in addition to a defect in initiation of late-firing origins, which had not been activated in the HU-arrested cells. The gradual loss of viability after release (Figure D) might be due to irreversible defects in elongation of DNA replication such as aberrant DNA synthesis.
Figure 6 SpCdc45 is dissociated from the S-phase–arrested chromatin in sld3-10. (A) Wild-type and sld3-10 cells expressing SpOrc1-FLAG5 and SpCdc45-Myc9 were cultured for 4 h in the presence of 10 mM HU at the permissive temperature (23°C) and (more ...)
To investigate the role of SpSld3 during the S phase, we examined effects of sld3-10 mutation on stability of chromatin association of MCM and SpCdc45 proteins. In HU-arrested wild-type cells, the amounts of chromatin-bound SpOrc1, SpMcm6, and SpCdc45 remained unchanged after subsequent incubation at 36°C for 1 h (Figure E, lane 6). In contrast, in the sld3-10 cells, the amount of SpCdc45 associated with the chromatin was decreased by incubation at 36°C (Figure E, lane 12). The amount of SpMcm6 in the chromatin fraction was not significantly affected, as shown by quantification of immunostained signals (Figure F). These results show that the SpSld3 is required for maintenance of SpCdc45 on chromatin during the S phase.