Essential Role of Sna41/Cdc45 in Loading of DNA Polymerase alpha  onto Minichromosome Maintenance Proteins in Fission Yeast*

Masashi UchiyamaDagger §, Dominic Griffiths, Ken-ichi AraiDagger ||, and Hisao MasaiDagger **

From the Dagger  Department of Molecular and Developmental Biology, Institute for Medical Science, The University of Tokyo, 4-6-1 Shirokanedai Minato-ku, Tokyo 108-8639, Japan,  Imperial Cancer Research Fund, 44 Lincolns Inn Fields, London WC2A 3PX, United Kingdom, and || Core Research for Evolutional Science and Technology (CREST), Tokyo 108-8639, Japan

Received for publication, January 2, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Assembly of replication complexes at the replication origins is strictly regulated. Cdc45p is known to be a part of the active replication complexes. In Xenopus egg extracts, Cdc45p was shown to be required for loading of DNA polymerase alpha  onto chromatin. The fission yeast cdc45 homologue was identified as a suppressor for nda4 and named sna41. Nevertheless, it is not known how Cdc45p facilitates loading of DNA polymerase alpha  onto chromatin, particularly to prereplicative complexes. To gain novel insight into the function of this protein in fission yeast, we characterized the fission yeast Cdc45 homologue, Sna41p. We have constructed C-terminally epitope-tagged Sna41p and Polalpha p and replaced the endogenous genes with the corresponding tagged genes. Analyses of protein-protein interactions in vivo by the use of these tagged strains revealed the following: Sna41p interacts with Polalpha p throughout the cell cycle, whereas it interacts with Mis5p/Mcm6p in the chromatin fractions at the G1-S boundary through S phase. In an initiation-defective sna41 mutant, sna41goa1, interaction of Polalpha p with Mis5p is not observed, although Polalpha p loading onto the chromatin that occurs before G1 START is not affected. These results show that fission yeast Sna41p facilitates the loading of Polalpha p onto minichromosome maintenance proteins. Our results are consistent with a model in which loading of Polalpha p onto replication origins occurs through two steps, namely, loading onto chromatin at preSTART and association with prereplicative complexes at G1-S through Sna41p, which interacts with minichromosome maintenance proteins in a cell cycle-dependent manner.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chromosomal DNA replication requires a series of complex events including recognition of origins, firing of replication origins, loading of DNA polymerases onto origins, and elongation of newly synthesized DNA. Initiation of DNA replication takes place only at specific loci on the chromosomal DNA, replication origins (1-3). Origin recognition complexes, which have been shown to be associated specifically with replication origins throughout the cell cycle in budding yeast, may serve as hallmarks of the origins. Newly synthesized CDC6/Cdc18p temporally associates with origin recognition complexes at the G1-S boundary (4, 5). This association is followed by the loading of minichromosome maintenance (MCM)1 protein complexes onto the replication origins, leading to the formation of prereplicative complexes (preRCs) (6). After the MCM loading, Cdc6/Cdc18p is phosphorylated by S-phase cyclin-dependent kinase (Cdk), and this phosphorylation leads to the rapid degradation of CDC6/Cdc18p to prevent reinitiation in yeasts (5, 7). Preceding the firing of origins, MCM proteins in preRCs are phosphorylated by CDC7-DBF4 kinase complexes, and CDC45 is loaded onto preRCs at the replication origins (9-11). Finally, replication proteins, such as replication protein A (single-stranded DNA-binding protein) and DNA polymerases, are loaded onto replication complexes (12). After DNA synthesis is initiated, some of the MCM components may leave the complexes, resulting in postreplicative complexes.

The model above is based mostly on findings in budding yeast and in Xenopus egg extracts. Although only limited analyses have been performed in other organisms, much of the data from these analyses are consistent with it. However, considering that the origin structure of budding yeast diverges from that of other eukaryotes, it would be important to study the mechanisms of the initiation in other model systems to clarify the precise mode of initiation in higher eukaryotes. Fission yeast, in which the origin structures may share some similarity with those of higher eukaryotes (13), may serve as another useful model. In fission yeast, the genes encoding six MCM protein homologues (14-18) and origin recognition complexes components have been identified (19-21). In addition, homologues for cdc7, dbf4, and cdc45 have also been identified (22-25).

Although the formation of preRCs is relatively well understood in budding yeast, how replication proteins are assembled into active replication complexes remains mostly unclear in other organisms. DNA polymerase alpha  (polalpha ), the only replicative polymerase equipped with primase activity, plays essential roles in initiation at the origins (26). The second largest subunit of DNA polymerase alpha , b subunit, which undergoes cell cycle-regulated phosphorylation (27, 28), has been suggested to have a regulatory role (29). In budding yeast, unphosphorylated forms of b subunit accumulate only in G1-arrested cells, and this phosphorylation appears to be correlated with CDC6-independent Polalpha loading onto the chromatin fraction (30), but the loading of Polalpha onto preRCs depends on Cdc45p. In Xenopus egg extracts, XCdc45, the Xenopus CDC45 homologue, was shown to be required for Polalpha loading onto the chromatin under the control of S-phase Cdk kinases (31). These findings in two different organisms are apparently inconsistent with each other with regard to the timing and dependence of the Polalpha loading onto the chromatin.

In this study, we addressed these issues through the characterizations of Sna41p in fission yeast. Fission yeast Polalpha p appears to undergo Cdc18p-independent loading onto chromatin, and this step does not require the function of Sna41p. Nevertheless, Sna41p is still required for interaction of Polalpha p with Mis5p/Mcm6p at the G1-S boundary. These results provide evidence for a model of replication complex assembly in which Polalpha p is loaded onto preRCs in two steps.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Strains

The strains used were all derived from strain SP812 h- ade6-M210 ura4-D18 leu1-32.

Media

Edinburgh minimal medium and appropriate supplements were used for all biochemical analyses.

Molecular Biology Techniques

Unless stated otherwise, all molecular biology techniques were performed as described previously (45). Transformation of yeast strains was performed essentially by the lithium acetate procedure (33).

Constructions of Strains Expressing Tagged Proteins

sna41HA3 tagged strain was constructed using a sna41Delta strain, which will be described elsewhere (49). Three HA epitopes, linked by a single glycine, were inserted just before the termination codon of the sna41 open reading frame by consecutive PCR method. The PCR fragment was directly introduced into sna41Delta strain rescued by pREP3X-Sna41. The transformed cells were plated on yeast extract with supplements containing 5'-fluoroacetic acid. The 5'-fluoroacetic acid-resistant colonies, which could grow without pREP3X-Sna41 plasmid, were tested by PCR, genetic cross, and Western blot. Tandem FLAG tags, linked by a single glycine, were introduced before the termination codon of the polalpha open reading frame as described previously (18), except that pUC18 carrying a ura4 nutritional marker instead of a leu1 marker was used.

Preparations of Cell Extracts

Whole Cell Extracts-- Harvested cells (1 volume) were washed with 3 volumes of cold water three times and then washed by 3 volumes of EB buffer (25 mM Hepes, pH 7.6, 50 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 2 mM dithiothreitol, 0.25% Triton X-100, and proteinase inhibitors) three times. The cells were then resuspended in 1 volume of EB buffer containing NaCl at a concentration indicated. After the addition of the same volume of acid-washed glass beads, cells were broken by a bead beater. After removal of glass beads by filtration, the suspensions were further lysed by sonication and then centrifuged in a microfuge at 15,000 rpm for 25 min. The supernatants were harvested as whole cell extracts. Denatured whole cell extracts were prepared by using DEB buffer (25 mM Hepes, pH 7.6, 6 M urea, and 1% SDS) in place of EB buffer and without the sonication step.

Chromatin-enriched Fractions-- Harvested cells were washed with 3 volumes of cold water three times and with 3 volumes of SB buffer (25 mM Hepes, pH 7.6, 1.2 M sorbitol, 50 or 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, and 2 mM dithiothreitol and proteinase inhibitors) three times. The cells were then resuspended in the same volume of SB buffer containing Zymoryase T100 (Seikagaku-Kogyo) and spheroplasted by a 20-min incubation at 30 °C. After cells were washed with 3 volumes of SB buffer three times, Triton-soluble fractions were extracted by a 30-min incubation at 4 °C in 2 volumes of EBT buffer (25 mM Hepes, pH7.6, 50 or 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, and 2 mM dithiothreitol and proteinase inhibitors) followed by centrifugation at 3,000 rpm for 5 min. The pellets were resuspended in 2 volumes of EBT, and Triton-insoluble fractions (chromatin-enriched fractions) were extracted by sonication, followed by centrifugation at 15,000 rpm for 25 min to ensure the removal of any insoluble aggregates. Immunoprecipitation experiments were performed basically as described previously (32), with some modification. For all processes, EB buffer (with 50 or 150 mM NaCl) was used as washing buffer, and the 1:1 mixture of protein A- and protein G-Sepharose beads was used for precipitation. Extracts were precleared by incubation with Sepharose beads without antibody to suppress nonspecific absorption of proteins to beads. To prepare DNase I-treated extracts, cell suspensions were treated with 0.5 mg/ml DNase I for 15 min after glass bead lysis and filtration. The breakage of DNA was confirmed by agarose gel electrophoresis. The samples were centrifuged at 15,000 rpm for 25 min to obtain supernatants.

Immunofluorescence Studies

An spp1-GFPKan strain expressing Spp1p tagged at the C terminus with the GFP was constructed by the PCR-based method recently described in Ref. 47. The spp1-GFPKan PCR product was transformed into h+/h- ade6-M216/ade6-M210 wild-type diploid strain, and transformants were selected for kanamycin resistance. The transformant was confirmed by Western blotting. For immunostaining with anti-GFP antibody (a gift from K. Sawin), cells were grown in rich media, and ~2.5 × 108 cells were harvested, either by centrifugation or filtration. Cells were fixed by the addition of 10 ml of -80 °C methanol and processed for immunofluorescence essentially as described previously (48). Secondary antibodies (Alexa; Molecular Probes) were used at a 1:1,000 dilution, and cells were visualized using a cooled charge-coupled device camera (Hamamatsu).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sna41p Physically Interacts with Polalpha p in Vivo-- It was reported that, in Xenopus egg extracts, DNA Polalpha p is loaded onto chromatin through interactions with Cdc45p (31). To examine this possibility, we first constructed strains in which triple HA-tagged sna41 and tandem FLAG-tagged polalpha replaced the endogenous genes, as described under "Experimental Procedures" (the tags were at the C terminus in both cases). The presence of the tags did not cause any growth defect in either the sna41HA3 or polalpha FLAG2 strains. The tagged proteins could be detected by anti-HA and anti-FLAG antibodies in the whole cell extracts (Fig. 1A). Sna41HA3p migrated at around 70 kDa on SDS-PAGE, whereas Polalpha FLAG2p appeared as a doublet of 180- and 165-kDa proteins, as described previously (36). To confirm that these bands actually represent the tagged proteins, immunoprecipitates by antibodies against the HA or FLAG epitope were immunoblotted by the antisera against Sna41p or Polalpha p as well as by the epitope antibodies. The same bands reacted with both antibodies (data not shown). Utilizing these strains, we performed immunoprecipitation experiments. When Sna41HA3p was precipitated by anti-HA antibody, Polalpha FLAG2p was co-immunoprecipitated (Fig. 1B). Only the upper band representing the intact p180 Polalpha FLAG2p interacted with Sna41HA3p. Conversely, in the anti-FLAG antibody immunoprecipitate, Sna41HA3p was present (Fig. 1C). Because we sonicated the cells to solubilize chromatin-enriched fractions, it is possible that this interaction is mediated by DNA. To eliminate this possibility, we also examined the interaction after DNase I treatment. As shown in Fig. 1D, even more Polalpha FLAG2p was recovered in the anti-HA immunoprecipitate in DNase I-treated extracts. Therefore, we have concluded that the interaction between Sna41p and Polalpha p is not mediated by DNA.


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Fig. 1.   Sna41p interacts with Polalpha p in vivo. A, expression of the tagged proteins. The integrated tagged strains, polalpha FLAG2 and sna41HA3, were constructed as described under "Experimental Procedures." Exponentially growing polalpha FLAG2 or sna41HA3 cells were harvested, and whole cell extracts were prepared with EB buffer containing 500 mM NaCl or DEB buffer, respectively. Extracts were run on SDS-PAGE and blotted with anti-FLAG2 antibody (top panel) or with anti-HA antibody (bottom panel). Left lane, tagged strain; right lane, untagged wild-type strain. B, Polalpha FLAG2p co-immunoprecipitated with Sna41HA3p. Whole cell extract of sna41HA3 polaFLAG2 strain was used for immunoprecipitation with anti-FLAG antibody (lane 1) or with anti-HA antibody (lane 2). Gel was blotted with anti-FLAG antibody. C, Sna41HA3p co-immunoprecipitated with Polalpha FLAG2p. Cells were arrested for 3.5 h at 36 °C, whole cell extracts were prepared, and immunoprecipitation was conducted with anti-HA antibody (lanes 1 and 2) or with anti-FLAG antibody (lanes 3 and 4). Western blotting was conducted by anti-HA antibody. Tagged, sna41HA3 polalpha FLAG2 cdc20; Untagged, sna41goa1 polalpha FLAG2. D, Sna41HA3p immunoprecipitation after DNase I treatment. Whole cell extracts were treated by sonication (lanes 1 and 3) or with DNase I (lanes 2 and 4). Lanes 1 and 2, 10% of the starting cell extract; lanes 3 and 4, immunoprecipitates with anti-HA antibody. The gels were blotted with anti-FLAG (top panel), anti-Mis5 (middle panel), and anti-HA (bottom panel) antibodies.

Sna41p and Polalpha p Are Present at a Constant Level and Interact with Each Other throughout the Cell Cycle-- In the synchronized cultures of the sna41HA3 polalpha FLAG2 strain released from HU arrest, the levels of Polalpha FLAG2p (32) and Mis5p (33) remained unchanged. The amount of Sna41HA3p also stayed constant regardless of cell cycle stage (Fig. 2A). We also examined the amounts of Polalpha FLAG2p, Mis5p, and Sna41HA3p in various cdc mutants arrested at different cell cycle stages. We chose cdc10, cdc20, cdc22, cdc23, poldelta (cdc6), cdc17, and cdc25 to obtain cells arrested at specific cell cycle stages. cdc10 encodes a component of the transcription factor essential for cdc18 expression and arrests the cell cycle at G1 START. cdc20 encodes the DNA polymerase epsilon  catalytic subunit and arrests the cell cycle at the G1-S boundary just before the HU arrest point. cdc22 encodes the large subunit of ribonucleotide reductase and arrests the cell cycle at the point same as the HU arrest. cdc23 is the homologue of budding yeast MCM10 and arrests the cell cycle at S phase after the HU arresting point. poldelta encodes the large subunit of DNA polymerase delta  and similarly arrests the cell cycle at S phase. cdc17 encodes DNA ligase and arrests the cell cycle at the S-G2 boundary. cdc25, encoding a protein phosphatase that activates Cdc2p, arrests the cell cycle at G2-M. As shown in Fig. 2B, the amounts of Polalpha FLAG2p, Mis5p, and Sna41HA3p were similar in these extracts arrested at various cell cycle stages. We also examined the interaction between Polalpha p and Sna41p in cultures synchronously released from HU block. We found that this interaction could be observed regardless of cell cycle stage (Fig. 3).


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Fig. 2.   Expressions of Polalpha p, Mis5p, and Sna41p during the cell cycle. A, the sna41HA3 polalpha FLAG2 cells were arrested in HU for 3.5 h and released into the cell cycle. After a 30-min recovery, the samples were collected every 15 min. The septation and mitotic indices were measured under a microscope. Whole cell extracts were prepared from all the samples. Top graph, the vertical axis indicates the septation index (black-diamond ) or mitotic index (open circle ). Bottom panels show Polalpha FLAG2p, Mis5p, and Sna41HA3p in the whole cell extracts at each time after release from HU block. The gels (from top to bottom) were blotted by anti-FLAG, anti-Mis5, and anti-HA antibodies, respectively. B, the exponentially growing sna41HA3 strain under cdc10, cdc20, cdc22, cdc23, poldelta , cdc17, or cdc25 background was arrested for 4 h at 36 °C, and the whole cell extract was prepared from each strain. The gels were blotted with anti-FLAG, anti-Mis5, and anti-HA antibodies (top three panels). The bottom panel shows the CBB staining of the same gel to show the loading of approximately equal amount of proteins in each lane.


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Fig. 3.   Sna41p interacts with Mis5p in S phase. sna41HA3 polalpha FLAG2 strain was synchronized by HU arrest for 3.5 h and subsequent release from the block. After a 30-min recovery, the samples were collected every 15 min. Whole cell extracts were prepared from all of the samples, and Sna41HA3p was immunoprecipitated by anti-HA antibody. Left and right vertical axes of the top graph indicate, respectively, the percentage of septation index () or mitotic index (open circle ) and the relative extent of Mis5p interaction with Sna41HA3p (black-diamond ). To generate the latter values, the band intensities of the second and third panels were quantified, and the ratio of Mis5p to Sna41HA3p was calculated with the value at 1.5 h taken as 1. After electrophoresis on SDS-PAGE, the gel was blotted with anti-FLAG, anti-Mis5p, and anti-HA antibodies (top three panels). The panel indicated as CBB stain shows the CBB staining of the same gel to show the presence of roughly equal amount of proteins in each lane. In the bottom panel, immunoprecipitates containing an equal amount of Sna41HA3p were blotted with anti-Mis5p antibody.

Sna41p Interacts with Mis5p Only in the Chromatin-enriched Fractions in Early S Phase-- The physiological interactions between CDC45 and MCM complex have been suggested in Saccharomyces cerevisiae (8, 10). Therefore, we have examined the interaction between Sna41HA3p and MCM complex. We examined one of the MCM proteins, Mis5p/SpMcm6p (15, 38). First, we performed immunoprecipitation experiments in exponentially growing cells. We observed co-immunoprecipitation of Sna41HA3p and Mis5p with either anti-Mis5p antibody (38) (data not shown) or anti-HA antibody (Fig. 1D). Because DNase I treatment did not affect the amount of Mis5p immunoprecipitated by anti-HA antibody, the interaction between Mis5p and Sna41HA3p is not mediated by DNA. Next, we analyzed the interaction in cultures synchronized by HU. The interaction between Sna41HA3p and Mis5p was cell cycle-regulated and correlated with the septation index (Fig. 3). Because Schizosaccharomyces pombe undergoes septation in G1-S phase, we concluded that the interaction took place in S phase. To further examine this regulation, we next tested the interaction in cell cycle-arrested cultures. Sna41HA3 strain was arrested by various cell cycle mutants, cdc10-129, cdc20-M10, cdc22-M45, cdc23-M36 (35), poldelta ts01 (36), cdc17-K42, and cdc25-22 (37). Sna41HA3p interacted with Mis5p in cdc20-, cdc22-, and cdc23-arrested extracts (Fig. 4A). Interestingly, although both cdc23 and poldelta mutants arrest the cell cycle with late S-phase DNA contents, only weak interaction was observed only in poldelta . In addition, we tested whether these interactions were affected by the protein localization. The Triton-soluble and -insoluble (the chromatin-enriched) fractions were prepared from the arrested cdc mutant cells. Interestingly, the interaction was only observed in Triton-insoluble fractions. Despite the presence of significant amount of Mis5p and Sna41p, virtually no interaction was observed in triton soluble fractions (Fig. 4B). Because Sna41p appears to be exclusively localized in nucleus throughout the cell cycle (data not shown), the interaction between Sna41HA3p and Mis5p is likely to take place exclusively on the chromatin. Taken together with the results in the previous section that Sna41HA3p interacts with Polalpha p throughout the cell cycle, the role of CDC45-related proteins in assembly of replication machinery, as proposed previously (31), may be accomplished by the physical interaction between Cdc45p and MCM at the G1-S boundary.


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Fig. 4.   Sna41p interacts with Mis5p exclusively in chromatin-enriched fractions. Exponentially growing sna41HA3 strains under cdc10, cdc20, cdc22, cdc23, poldelta , cdc17, and cdc25 backgrounds were arrested for 3.5 h at 36 °C. A, Sna41HA3p and Mis5p interact in cdc20-, cdc22-, and cdc23-arrested extracts. The whole cell extracts were prepared with EB buffer containing 50 mM NaCl for each strain. The immunoprecipitates were separated by SDS-PAGE and blotted by anti-FLAG (top panel), anti-Mis5p (middle panel), and anti-HA (bottom panel) antibodies. B, interaction between Sna41HA3p and Mis5p is detected in Triton-insoluble fractions but not in Triton-soluble fractions. The cells were harvested and spheroplasted. The Triton-soluble and -insoluble fractions were prepared as described under "Experimental Procedures." The immunoprecipitated samples (top and middle panels) and untreated extracts (bottom panel) were separated by SDS-PAGE, and the filter was blotted with either anti-Mis5p (top and bottom panels) or anti-HA (middle panel) antibody. Left six lanes, Triton-soluble fractions; right six lanes, Triton-insoluble (chromatin-enriched) fractions. In both A and B, immunoprecipitation was performed by anti-HA antibody after the protein concentrations of the extracts were adjusted.

sna41goa1 Mutant Has No Defect in Chromatin Loading of Polalpha p-- In Xenopus egg extracts, XCdc45 was shown to be required for chromatin loading of Polalpha p. Independently, in budding yeast, Polalpha complexes were found to undergo a so-called "mitotic resetting" event and to be loaded onto chromatin fractions before CDC6 loading in budding yeast (30). These two results suggest that Sna41p may play essential roles in a similar mitotic resetting event of Polalpha p in fission yeast. To address this question, we examined the chromatin loading of Sna41HA3p, Mis5p, and Polalpha FLAG2p in HU synchronized extracts. The Sna41HA3 polalpha FLAG2 strain was arrested by HU for 4 h and released into cell cycle from the block. Then, chromatin-enriched fractions were prepared at various times after release. As reported previously, the amount of Mis5p on the chromatin was cell cycle-regulated and correlated with the septation index (33). The amount of Sna41HA3p and Polalpha FLAG2p in the same fractions also fluctuated, as did Mis5p, and generally increased during S phase (Fig. 5A). However, it was difficult from this experiment to conclude on the order of the loading of these three proteins onto the chromatin. To clarify the timing of chromatin loading, we examined the chromatin loading in cell cycle-arrested extracts. Sna41HA3 polalpha FLAG2 strains in combination with the cell cycle mutants cdc10-129, cdc20-M10, cdc22-M45, cdc23-M36, poldelta ts01, and cdc25-22 were used to arrest the cell cycle. The chromatin-enriched fractions were analyzed by Western blotting. Mis5p was recovered in chromatin-enriched fractions from cdc20-, cdc22-, cdc23-, and poldelta -arrested cells (Fig. 5B), in agreement with the recent report (33). Sna41HA3p was recovered mainly in cdc10 and cdc20 as well as in cdc22 and in cdc23 in reduced amount. Polalpha FLAG2p was detected in cdc10-, cdc20-, and cdc22-arrested fractions. Because no Mis5p was detected in cdc10, Sna41HA3p and Polalpha FLAG2p loading onto chromatin might be controlled by a mechanism different from that for MCMs. Because cdc10 mutant fails to progress into S phase due to the lack of Cdc18p expression (38), we could conclude that, unlike Mis5p, Sna41p and Polalpha p loading onto the chromatins is independent of Cdc18p. We also examined the chromatin loading of Polalpha FLAG2p and Mis5p in sna41goa1 mutant, which we recently isolated as an initiation-defective temperature-sensitive mutant and identified as an allele of sna41 (49). The defect in Sna41p did not affect the chromatin loading of Polalpha FLAG2p and Mis5p (Fig. 5B). Interestingly, Polalpha FLAG2p in the chromatin-enriched fractions was most abundant in the sna41goa1 mutant. This is unexpected, but we can clearly conclude that Sna41p is not required for Polalpha p loading onto chromatin.


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Fig. 5.   Polalpha FLAG2p is loaded onto the chromatin-enriched fractions earlier than Mis5p in a manner independent of sna41 function. A, Sna41HA3 polalpha FLAG2 strain was arrested by HU for 4 h and released. Chromatin-enriched fractions were prepared and run on SDS-PAGE. Left vertical axis of the top graph indicates the percentage of the septation index (black-diamond ) or mitotic index (open circle ). B, Sna41HA3 polalpha FLAG2 strains containing a cell cycle mutation indicated at the top (except for sna41goa1) were analyzed. sna41goa1 contains only polalpha FLAG2p. In both A and B, EB buffer containing 150 mM NaCl was used to prepare the extracts, and the gels were blotted with anti-FLAG (top panels), anti-Mis5p (upper middle panels), or anti-HA (lower middle panels) antibody. The bottom panels show CBB staining of the same gel.

sna41goa1 Is Defective in Association of Polalpha p with MCMs-- Although we showed that chromatin loading of Polalpha was not affected by sna41goa1 mutation, the physical and genetic interactions among Sna41p, Polalpha p, and Mis5p must be the key to reveal sna41 functions in G1-S transition. The arresting point of sna41goa1 is close to that of cdc20 or cdc22 (data not shown), and interaction of Sna41HA3p with MCMs takes place mainly at this cell cycle point. Taking these facts into account, we hypothesized that Sna41p is not required for chromatin loading of Polalpha p but is still required for association of Polalpha p with replicative complexes at the origins of DNA replication.

Recently, Griffiths et al. (39) isolated and characterized S. pombe DNA primase 1, spp1, encoding a subunit of DNA polymerase alpha  enzyme complexes. The amount of this protein remains constant throughout the cell cycle, and the protein is localized in the nucleus at all cell cycle stages. However, when Spp1GFPp is stained with anti-GFP antibody by indirect immunofluorescence, the nuclear staining of Spp1GFPp disappears at the G1-S boundary or at the HU-arrested stage. We found that the staining pattern of Spp1GFPp in sna41goa1 stayed basically the same as the G2 status even after 5 h at the nonpermissive temperature, despite the fact that sna41goa1 arrests the cell cycle at the G1-S boundary very close to cdc22 or the HU arresting point (Fig. 6). This observation suggests that some G1-S-specific alterations of Polalpha complexes may not occur in sna41goa1, consistent with our proposal that sna41p is required for interaction of Polalpha p with preRC.


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Fig. 6.   G1-S specific regulation of Polalpha FLAG2p is absent in sna41goa1. A, exponentially growing spp1-GFPKan or spp1-GFPKan sna41goa1 cells were arrested either by 10 mM HU or by temperature shift to 36 °C, respectively. The cells were fixed by methanol and then processed for immunofluorescence with anti-GFP antibody and staining by 4',6-diamidino-2-phenylindole. First row, exponentially growing spp1-GFPKan sna41goa1at 25 °C; second and third rows, spp1-GFPKan sna41goa1 arrested at 36 °C for 3 h; fourth row, spp1-GFPKan sna41goa1 arrested at 36 °C for 5 h; fifth and sixth rows, spp1-GFPKan (sna41+) arrested by HU for 2.5 h. B, quantitation of GFP staining. In the merged image, the cells containing nuclei with yellow staining were counted as positive, and those containing nuclei with red staining were counted as negative, and fractions of the cells with positive staining are presented. Approximately 200 cells were counted.

We have already shown that Sna41HA3p interacts with Polalpha FLAG2p regardless of cell cycle stages and with Mis5p at the G1-S boundary in chromatin-enriched fractions. Therefore, it is possible that Polalpha complexes are loaded onto replicative complexes with the aid of Sna41p, which may function as a bridge. To further test this possibility, we first examined the interaction of Polalpha FLAG2p with Mis5p in the HU-synchronized culture. Polalpha FLAG2p was precipitated by anti-FLAG antibody, and the interactions were analyzed by Western blotting. Consistent with the results in Fig. 2, Polalpha FLAG2p interacted with Sna41p regardless of the cell cycle stage. In contrast, Mis5p interaction fluctuated during the cell cycle (Fig. 7A). The extent of the interaction correlated with septation index. This result clearly shows that Polalpha FLAG2p interacted with Mis5p only during S phase. These results strongly suggest the possibility that Sna41p mediates the loading of Polalpha complexes onto the replication complexes. This possibility could be examined by analyzing the interaction of Polalpha p with MCM in the sna41goa1 mutant because the mutant contains a very low level of Sna41p at the nonpermissive temperature due to its instability (data not shown). Because sna41goa1 and cdc20-M10 arrest the cell cycle at a very similar point, we examined the interaction between Polalpha FLAG2p and Mis5p in sna41goa1- and cdc20-arrested extracts. In the cdc20-arrested extract, Mis5p was coprecipitated with polalpha FLAG2p. In contrast, Mis5p was not co-precipitated in the sna41goa1-arrested extract (Fig. 7B). In conclusion, we show here that Sna41p function is essential for interaction of Polalpha p with Mis5p at the initiation of DNA replication.


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Fig. 7.   Interaction of Polalpha FLAG2p with Mis5p occurs during S phase and is dependent on Sna41p. A, sna41HA3 polalpha FLAG2 strain was arrested by 10 mM HU for 4 h and released. EB buffer containing 150 mM NaCl was used to prepare the whole cell extracts. Left vertical axes of the top graph indicate the percentage of septation index () or mitotic index (open circle ). Immunoprecipitates with anti-FLAG antibody were blotted with anti-FLAG (top panel), anti-Mis5 (middle panel), and anti-HA (bottom panel) antibodies. B, polalpha FLAG2 cdc20-M10 (left lanes) and polalpha FLAG2 sna41goa1 (right lanes) strains were shifted to 36 °C for 3.5 h, and then the whole cell extracts were prepared using EB buffer containing 150 mM NaCl. The whole cell extracts (left panels) and immunoprecipitates by anti-FLAG antibody (right panels) were blotted with anti-FLAG (top panel) or anti-Mis5p (second panel) antibody. The bottom left panel is CBB staining.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It was previously reported that DNA polymerase alpha  is loaded onto chromatin through Cdc45 protein in Xenopus egg extracts. Furthermore, through characterization of a new G1 arrest mutant, sna41goa1, we found genetic interactions between sna41 and polalpha (49). These findings lead us to examine physical interactions between Sna41p and Polalpha p. We found that Sna41p and Polalpha p interacted throughout the cell cycle. Sna41p also interacted with Mis5p, the fission yeast homologue of MCM6, during S phase. Sna41p and Polalpha p were loaded onto the chromatin-enriched fractions in cdc10-arrested extract. Sna41p is not required for chromatin loading of Polalpha p but is still required for its interaction with Mis5p in S phase.

The Interaction between Sna41p and Mis5p is Cell Cycle- and Localization-dependent-- In budding yeast, physical and genetic interactions of CDC45 with MCMs have been reported (8, 10). Although the interaction is known to take place at the G1-S boundary, what exactly triggers this interaction has remained unclear. We have found that Sna41p and Mis5p interact exclusively in the chromatin-enriched fractions in S. pombe. At present, we cannot completely rule out the possibility that this interaction is mediated indirectly by DNA linking the two proteins. However, we think this possibility is rather unlikely because the average sizes of DNA are less than 300 base pairs after sonication of the Triton-insoluble fractions and also because we detected the interaction in DNase I-treated extracts (Fig. 1D).

It is probable that the interaction between these two proteins is regulated by a third component such as Hsk1p-Dfp1/Him1 (22-24, 40), S-phase Cdk, or unknown mediator(s). Two different bands of Mis5p were detected on SDS-PAGE when extracts were prepared by the spheroplast method. In addition, the upper band was maximized in the sna41goa1-arrested fraction (Fig. 5B) and minimized in cdc25 or cdc10 (data not shown). Although we cannot conclude at this moment that this mobility shift is due to phosphorylation, this possibility needs to be examined in the future experiments.

Polalpha p Loading onto DNA Replication Origin May Require Two Independent Steps-- We have found that sna41goa1 suppresses the growth defect of polalpha ts13 (49). Thus, it is most likely that this genetic interaction is attributed to the physical interaction between Sna41p and Polalpha p. In accordance with this prediction, we found that these two proteins interact with each other. This is consistent with the result in Xenopus and the recent in vitro observation in human (41). In Xenopus egg extracts, DNA polymerase alpha  is loaded onto chromatin at the G1-S boundary in a manner dependent on XCdc45 and Cdk activity (31). In contrast, budding yeast DNA polymerase alpha  is loaded at the end of mitosis independent of CDC6 (30), although another report claims that the Polalpha b subunit is loaded onto chromatin at G1-S (42). These somewhat contradictory results may be due to the different cell cycle operations in yeast and Xenopus eggs. DNA replication and mitosis are alternating practically without gap periods in Xenopus egg extracts, whereas yeast cell cycles are under strict G1 regulations. Diversity in the methods for preparation of chromatin fractions may also account in part for these discrepancies. However, our data may provide a more unified view for the loading of DNA polymerase alpha . We found that Polalpha p loading on the chromatin-enriched fractions is observed even in cdc10- or sna41goa1-arrested extract (Fig. 5B). This clearly shows that this step is not dependent on either Cdc18p or Sna41p. This result is consistent with the observation in budding yeast. We also showed that interaction of Polalpha p with MCMs at the G1-S boundary depends on functional Sna41p (Fig. 7). Recently, Mis5p/Mcm6p was shown to be loaded at or near autonomously replicating sequences (33). Although further confirmation is needed, it is most likely that association of Polalpha p with MCMs facilitates relocation of the former protein to replication origins. This result is more consistent with the finding in Xenopus egg extracts. Taken together, our data support the following model (Fig. 8) of loading of DNA polymerase alpha  onto replication complexes in the proliferating cell cycle of somatic cells. The first step may depend on Polalpha b subunit and inactivation of M-phase Cdk. The fact that Polalpha p and Sna41p recovered in the chromatin-enriched fractions are relatively resistant to extraction with DNase I digestion (data not shown) suggests that the first step of loading may deliver these proteins not simply onto the chromatin but onto a particular nuclear structure or nuclear matrix. Because Sna41p and Polalpha p can interact with each other throughout the cell cycle, the two proteins may be loaded onto chromatin as a complex. However, because Polalpha p is loaded onto chromatin even in the sna41goa1 mutant, the association of Sna41p and Polalpha p is unlikely to be essential for Polalpha p loading. This step may be required for accumulation of the proteins involved in DNA synthesis at the specific nuclear structures. Then, interaction of Sna41p with MCMs brings DNA polymerase alpha  onto replication complexes at the origins in the second step. This step may be regulated by S-phase Cdk and/or by Cdc7 kinase. It should be noted that our results do not exclude the possibility that the observed interactions are mediated by other proteins. In fact, both Mis5p and Polalpha p are parts of protein complexes (six-protein MCM complex and four-protein DNA polymerase alpha  complex, respectively) that are essential components for a larger replication machinery, and therefore it may be more likely that Sna41p interacts with these complexes as well as other components in the replication apparatus.


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Fig. 8.   Two-step model for loading of the DNA polymerase alpha /primase complex onto replication origins. DNA polymerase alpha /primase and Sna41p form a complex throughout the cell cycle. In early G1, both proteins are loaded onto chromatin or some kind of nuclear structure (Nuclear Matrix?). This step may depend on DNA polymerase alpha  b subunit, as was shown in budding yeast. At the G1-S boundary, Polalpha complex associates with replication origins through Sna41p-mediated interaction with MCMs.

The model can explain the recent finding in S. cerevisiae that CDC45 and DNA polymerase alpha  are loaded onto late-firing origins later than early ones (43, 44) by postulating that origin-specific phosphorylation events activate association of Cdc45 with MCM. The data presented in this work indicate that physical interactions between MCM-Cdc45p-Polalpha p constitute an important step for assembly of active replication machinery. More biochemical studies are needed to understand the nature of these interactions and the roles of these complexes in initiation of DNA replication.

    ACKNOWLEDGEMENTS

We thank Susan Forsburg for the generous gift of anti-Mis5 antibody. The anti-polalpha antibody and strains were kindly provided by Teresa Wang. We thank all members of our laboratory for useful discussion.

    FOOTNOTES

* This work was supported in part by grants-in-aid for scientific research on priority areas from the Ministry of Education, Science, Sports and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by Japan Society for the Promotion of Science Research Fellowships for Young Scientists.

** To whom correspondence should be addressed: Dept. of Cell Biology, Tokyo Metropolitan Inst. of Medical Science, 3-18-22 Honkomagome, Bunkyo-Ku, Tokyo 113-8613, Japan. Tel.: 81-3-5685-2264; Fax: 81-3-5685-2932; E-mail: hmasai@rinshoken.or.jp.

Published, JBC Papers in Press, May 8, 2001, DOI 10.1074/jbc.M100007200

    ABBREVIATIONS

The abbreviations used are: MCM, minichromosome maintenance proteins; preRC, prereplicative complex; Cdk, cyclin-dependent kinase; polalpha , DNA polymerase alpha ; HA, hemagglutinin; PCR, polymerase chain reaction; GFP, green fluorescent protein; PAGE, polyacrylamide gel electrophoresis; HU, hydroxyurea; CBB, Coomasie Brilliant Blue R-250.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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