Fission yeast Cdc23/Mcm10 functions after pre-replicative complex formation to promote Cdc45 chromatin binding

Using a cytological assay to monitor the successive chromatin association of replication proteins leading to replication initiation, we have investigated the function of fission yeast Cdc23/Mcm10 in DNA replication. Inactivation of Cdc23 before replication initiation using tight degron mutations has no effect on Mcm2 chromatin association, and thus pre-replicative complex (pre-RC) formation, although Cdc45 chromatin binding is blocked. Inactivating Cdc23 during an S phase block after Cdc45 has bound causes a small reduction in Cdc45 chromatin binding, and replication does not terminate in the absence of Mcm10 function. These observations show that Cdc23/Mcm10 function is conserved between fission yeast and Xenopus, where in vitro analysis has indicated a similar requirement for Cdc45 binding, but apparently not compared with Saccharomyces cerevisiae, where Mcm10 is needed for Mcm2 chromatin binding. However, unlike the situation in Xenopus, where Mcm10 chromatin binding is dependent on Mcm2-7, we show that the fission yeast protein is bound to chromatin throughout the cell cycle in growing cells, and only displaced from chromatin during quiescence. On return to growth, Cdc23 chromatin binding is rapidly reestablished independently from pre-RC formation, suggesting that chromatin association of Cdc23 provides a link between proliferation and competence to execute DNA replication.     

Essential role of Sna41/Cdc45 in loading of DNA polymerase alpha onto minichromosome maintenance proteins in fission yeast

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 Pol alpha 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 Pol alpha p throughout the cell cycle, whereas it interacts with Mis5p/Mcm6p in the chromatin fractions at the G(1)-S boundary through S phase. In an initiation-defective sna41 mutant, sna41(goa1), interaction of Pol alpha p with Mis5p is not observed, although Pol alpha p loading onto the chromatin that occurs before G(1) START is not affected. These results show that fission yeast Sna41p facilitates the loading of Pol alpha p onto minichromosome maintenance proteins. Our results are consistent with a model in which loading of Pol alpha p onto replication origins occurs through two steps, namely, loading onto chromatin at preSTART and association with prereplicative complexes at G(1)-S through Sna41p, which interacts with minichromosome maintenance proteins in a cell cycle-dependent manner.     

Hsk1 kinase and Cdc45 regulate replication stress-induced checkpoint responses in fission yeast

In fission yeast, replication fork arrest activates the replication checkpoint effector kinase Cds1^Chk2/Rad53 through the Rad3^ATR/Mec1-Mrc1^Claspin pathway. Hsk1, the Cdc7 homologue of fission yeast required for efficient initiation of DNA replication, is also required for Cds1 activation. Hsk1 kinase activity is required for induction and maintenance of Mrc1 hyperphosphorylation, which is induced by replication fork block and mediated by Rad3. Rad3 kinase activity does not change in an hsk1 temperature-sensitive mutant, and Hsk1 kinase activity is not affected by rad3 mutation. Hsk1 kinase vigorously phosphorylates Mrc1 in vitro, predominantly at non-SQ/TQ sites, but this phosphorylation does not seem to affect the Rad3 action on Mrc1. Interestingly, the replication stress-induced activation of Cds1 and hyperphosphorylation of Mrc1 is almost completely abrogated in an initiation-defective mutant of cdc45, but not in an mcm2 or polε mutant. The results suggest that Hsk1-mediated loading of Cdc45 onto replication origins may play important roles in replication stress-induced checkpoint.     

A Yeast GSK-3 Kinase Mck1 Promotes Cdc6 Degradation to Inhibit DNA Re-Replication

Cdc6p is an essential component of the pre-replicative complex (pre-RC), which binds to DNA replication origins to promote initiation of DNA replication. Only once per cell cycle does DNA replication take place. After initiation, the pre-RC components are disassembled in order to prevent re-replication. It has been shown that the N-terminal region of Cdc6p is targeted for degradation after phosphorylation by Cyclin Dependent Kinase (CDK). Here we show that Mck1p, a yeast homologue of GSK-3 kinase, is also required for Cdc6 degradation through a distinct mechanism. Cdc6 is an unstable protein and is accumulated in the nucleus only during G1 and early S-phase in wild-type cells. In mck1 deletion cells, CDC6p is stabilized and accumulates in the nucleus even in late S phase and mitosis. Overexpression of Mck1p induces rapid Cdc6p degradation in a manner dependent on Threonine-368, a GSK-3 phosphorylation consensus site, and SCF^CDC4. We show evidence that Mck1p-dependent degradation of Cdc6 is required for prevention of DNA re-replication. Loss of Mck1 activity results in synthetic lethality with other pre-RC mutants previously implicated in re-replication control, and these double mutant strains over-replicate DNA within a single cell cycle. These results suggest that a GSK3 family protein plays an unexpected role in preventing DNA over-replication through Cdc6 degradation in Saccharomyces cerevisiae. We propose that both CDK and Mck1 kinases are required for Cdc6 degradation to ensure a tight control of DNA replication.     

The Hect Domain E3 Ligase Tom1 and the F-box Protein Dia2 Control Cdc6 Degradation in G1 Phase

The accurate replication of genetic information is critical to maintaining chromosomal integrity. Cdc6 functions in the assembly of pre-replicative complexes and is specifically required to load the Mcm2-7 replicative helicase complex at replication origins. Cdc6 is targeted for protein degradation by multiple mechanisms in Saccharomyces cerevisiae, although only a single pathway and E3 ubiquitin ligase for Cdc6 has been identified, the SCF^Cdc4 (Skp1/Cdc53/F-box protein) complex. Notably, Cdc6 is unstable during the G₁ phase of the cell cycle, but the ubiquitination pathway has not been previously identified. Using a genetic approach, we identified two additional E3 ubiquitin ligase components required for Cdc6 degradation, the F-box protein Dia2 and the Hect domain E3 Tom1. Both Dia2 and Tom1 control Cdc6 turnover during G₁ phase of the cell cycle and act separately from SCF^Cdc4. Ubiquitination of Cdc6 is significantly reduced in dia2Δ and tom1Δ cells. Tom1 and Dia2 each independently immunoprecipitate Cdc6, binding to a C-terminal region of the protein. Tom1 and Dia2 cannot compensate for each other in Cdc6 degradation. Cdc6 and Mcm4 chromatin association is aberrant in tom1Δ and dia2Δ cells in G₁ phase. Together, these results present evidence for a novel degradation pathway that controls Cdc6 turnover in G₁ that may regulate pre-replicative complex assembly.     

ORC- and Cdc6-dependent complexes at active and inactive chromosomal replication origins in Saccharomyces cerevisiae.

We have developed a genomic footprinting protocol which allows us to examine protein-DNA interactions at single copy chromosomal origins of DNA replication in the budding yeast Saccharomyces cerevisiae. We show that active replication origins oscillate between two chromatin states during the cell cycle: an origin recognition complex (ORC)-dependent post-replicative state and a Cdc6p-dependent pre-replicative state. Furthermore, we show that both post- and pre-replicative complexes can form efficiently on closely apposed replicators. Surprisingly, ARS301 which is active as an origin on plasmids but not in its normal chromosomal location, forms ORC- and Cdc6p-dependent complexes in both its active and inactive contexts. Thus, although ORC and Cdc6p are essential for initiation, their binding is not sufficient to dictate origin use.     

Budding yeast Cdc6p induces re-replication in fission yeast by inhibition of SCF(Pop)-mediated proteolysis

In fission yeast, overexpression of the replication initiator protein Cdc18p induces re-replication, a phenotype characterized by continuous DNA synthesis in the absence of cell division. In contrast, overexpression of Cdc6p, the budding yeast homolog of Cdc18p, does not cause re-replication in S. cerevisiae. However, we have found that Cdc6p has the ability to induce re-replication in fission yeast. Cdc6p cannot functionally replace Cdc18p, but instead interferes with the proteolysis of both Cdc18p and Rum1p, the inhibitor of the protein kinase Cdc2p. This activity of Cdc6p is entirely contained within a short N-terminal peptide, which forms a tight complex with Cdc2p and the F-box/WD-repeat protein Sud1p/Pop2p, a component of the SCF^Pop ubiquitin ligase in fission yeast. These interactions are mediated by two distinct regions within the N-terminal region of Cdc6p and depend on the integrity of its Cdc2p phosphorylation sites. The data suggest that disruption of re-replication control by overexpression of Cdc6p in fission yeast is a consequence of sequestration of Cdc2p and Pop2p, two factors involved in the negative regulation of Rum1p, Cdc18p and potentially other replication proteins. Received: 29 April 1999 / Accepted: 27 June 1999     

Evidence for a Cdc6p-independent mitotic resetting event involving DNA polymerase alpha.

Eukaryotic DNA replication is limited to once per cell cycle because cyclin-dependent kinases (cdks), which are required to fire origins, also prevent re-replication. Components of the replication apparatus, therefore, are 'reset' by cdk inactivation at the end of mitosis. In budding yeast, assembly of Cdc6p-dependent pre-replicative complexes (pre-RCs) at origins can only occur during G1 because it is blocked by cdk1 (Cdc28) together with B cyclins (Clbs). Here we describe a second, separate process which is also blocked by Cdc28/Clb kinase and, therefore, can only occur during G1; the recruitment of DNA polymerase alpha-primase (pol alpha) to chromatin. The recruitment of pol alpha to chromatin during G1 is independent of pre-RC formation since it can occur in the absence of Cdc6 protein. Paradoxically, overproduction of Cdc6p can drive both dephosphorylation and chromatin association of pol alpha. Overproduction of a mutant in which the N-terminus of Cdc6 has been deleted is unable to drive pol alpha chromatin binding. Since this mutant is still competent for pre-RC formation and DNA replication, we suggest that Cdc6p overproduction resets pol alpha chromatin binding by a mechanism which is independent of that used in pre-RC assembly.     

Hsk1 kinase and Cdc45 regulate replication stress-induced checkpoint responses in fission yeast

In fission yeast, replication fork arrest activates the replication checkpoint effector kinase Cds1^Chk2/Rad53 through the Rad3^ATR/Mec1-Mrc1^Claspin pathway. Hsk1, the Cdc7 homolog of fission yeast required for efficient initiation of DNA replication, is also required for Cds1 activation. Hsk1 kinase activity is required for induction and maintenance of Mrc1 hyperphosphorylation, which is induced by replication fork block and mediated by Rad3. Rad3 kinase activity does not change in an hsk1 temperature-sensitive mutant, and Hsk1 kinase activity is not affected by rad3 mutation. Hsk1 kinase vigorously phosphorylates Mrc1 in vitro, predominantly at non-SQ/TQ sites, but this phosphorylation does not seem to affect the Rad3 action on Mrc1. Interestingly, the replication stress-induced activation of Cds1 and hyperphosphorylation of Mrc1 is almost completely abrogated in an initiation-defective mutant of cdc45, but not significantly in an mcm2 or polε mutant. These results suggest that Hsk1-mediated loading of Cdc45 onto replication origins may play important roles in replication stress-induced checkpoint.Key words: Cdc7, Cdc45, checkpoint, DNA replication, Mrc1     

Budding yeast Cdc6p induces re-replication in fission yeast by inhibition of SCFPop-mediated proteolysis

In fission yeast, overexpression of the replication initiator protein Cdc18p induces re-replication, a phenotype characterized by continuous DNA synthesis in the absence of cell division. In contrast, overexpression of Cdc6p, the budding yeast homolog of Cdc18p, does not cause re-replication in S. cerevisiae. However, we have found that Cdc6p has the ability to induce re-replication in fission yeast. Cdc6p cannot functionally replace Cdc18p, but instead interferes with the proteolysis of both Cdc18p and Rum1p, the inhibitor of the protein kinase Cdc2p. This activity of Cdc6p is entirely contained within a short N-terminal peptide, which forms a tight complex with Cdc2p and the F-box/WD-repeat protein Sud1p/Pop2p, a component of the SCF^Pop ubiquitin ligase in fission yeast. These interactions are mediated by two distinct regions within the N-terminal region of Cdc6p and depend on the integrity of its Cdc2p phosphorylation sites. The data suggest that disruption of re-replication control by overexpression of Cdc6p in fission yeast is a consequence of sequestration of Cdc2p and Pop2p, two factors involved in the negative regulation of Rum1p, Cdc18p and potentially other replication proteins.     

Xenopus Cut5 is essential for a CDK-dependent process in the initiation of DNA replication

Fission yeast Cut5/Rad4 and its budding yeast homolog Dpb11 are required for both DNA replication and the S-phase checkpoint. Here, we have investigated the role of the Xenopus homolog of Cut5 in the initiation of DNA replication using Xenopus egg extracts. Xenopus Cut5, which shows sequence similarity to DmMus101 and HsTopBP1, is essential for DNA replication in the egg extracts. It is required for the chromatin binding of Cdc45 and DNA polymerases, but not for the formation of pre-replicative complexes or the elongation stage of DNA replication. The chromatin binding of Cut5 consists of two distinct modes. S-phase cyclin-dependent kinase (S-CDK)-independent binding is sufficient for DNA replication while S-CDK-dependent binding is dispensable. Further, S-CDK acts after the chromatin binding of Cut5 and before the binding of Cdc45. These results demonstrate that the chromatin binding of Cut5 is required for the action of S-CDK, which in turn triggers the formation of pre-initiation complexes of DNA replication.     

Ordered assembly of Sld3, GINS and Cdc45 is distinctly regulated by DDK and CDK for activation of replication origins

Initiation of chromosome DNA replication in eukaryotes is tightly regulated through assembly of replication factors at replication origins. Here, we investigated dependence of the assembly of the initiation complex on particular factors using temperature-sensitive fission yeast mutants. The psf3-1 mutant, a GINS component mutant, arrested with unreplicated DNA at the restrictive temperature and the DNA content gradually increased, suggesting a defect in DNA replication. The mutation impaired GINS complex formation, as shown by pull-down experiments. Chromatin immunoprecipitation assays indicated that GINS integrity was required for origin loading of Psf2, Cut5 and Cdc45, but not Sld3. In contrast, loading of Psf2 onto origins depended on Sld3 and Cut5 but not on Cdc45. These results suggest that Sld3 functions furthest upstream in initiation complex assembly, followed by GINS and Cut5, then Cdc45. Consistent with this conclusion, Cdc7-Dbf4 kinase (DDK) but not cyclin-dependent kinase (CDK) was required for Sld3 loading, whereas recruitment of the other factors depended on both kinases. These results suggest that DDK and CDK regulate distinct steps in activation of replication origins in fission yeast.     

Cdc6 and DNA replication: Limited to humble origins

The budding yeast Cdc6 protein is important for regulating DNA replication intiation. Cdc6p acts at replication origins, and cdc6-1 mutants arrest with unreplicated DNA and show elevated minichromosome loss rates. Overexpression of the related Cdc 18 protein in fission yeast results in DNA rereplication; however, Cdc6p overexpression does not cause this result. A recent paper⁽¹⁾ further defines the role of Cdc6p in DNA replication. Cdc6p only promotes DNA replication between the end of mitosis and late G₁, and although the Cdc6 protein is highly unstable, neither degradation nor nuclear localization is critical for limiting DNA replication to this interval.     

A novel intermediate in initiation complex assembly for fission yeast DNA replication

Assembly of initiation factors on individual replication origins at onset of S phase is crucial for regulation of replication timing and repression of initiation by S-phase checkpoint control. We dissected the process of preinitiation complex formation using a point mutation in fission yeast nda4-108/mcm5 that shows tight genetic interactions with sna41(+)/cdc45(+). The mutation does not affect loading of MCM complex onto origins, but impairs Cdc45-loading, presumably because of a defect in interaction of MCM with Cdc45. In the mcm5 mutant, however, Sld3, which is required for Cdc45-loading, proficiently associates with origins. Origin-association of Sld3 without Cdc45 is also observed in the sna41/cdc45 mutant. These results suggest that Sld3-loading is independent of Cdc45-loading, which is different from those observed in budding yeast. Interestingly, returning the arrested mcm5 cells to the permissive temperature results in immediate loading of Cdc45 to the origin and resumption of DNA replication. These results suggest that the complex containing MCM and Sld3 is an intermediate for initiation of DNA replication in fission yeast.     

Assembly of a complex containing Cdc45p, replication protein A, and Mcm2p at replication origins controlled by S-phase cyclin-dependent kinases and Cdc7p-Dbf4p kinase

In Saccharomyces cerevisiae, replication origins are activated with characteristic timing during S phase. S-phase cyclin-dependent kinases (S-CDKs) and Cdc7p-Dbf4p kinase are required for origin activation throughout S phase. The activation of S-CDKs leads to association of Cdc45p with chromatin, raising the possibility that Cdc45p defines the assembly of a new complex at each origin. Here we show that both Cdc45p and replication protein A (RPA) bind to Mcm2p at the G(1)-S transition in an S-CDK-dependent manner. During S phase, Cdc45p associates with different replication origins at specific times. The origin associations of Cdc45p and RPA are mutually dependent, and both S-CDKs and Cdc7p-Dbf4p are required for efficient binding of Cdc45p to origins. These findings suggest that S-CDKs and Cdc7p-Dbf4p promote loading of Cdc45p and RPA onto a preformed prereplication complex at each origin with preprogrammed timing. The ARS1 association of Mcm2p, but not that of the origin recognition complex, is diminished by disruption of the B2 element of ARS1, a potential origin DNA-unwinding element. Cdc45p is required for recruiting DNA polymerase alpha onto chromatin, and it associates with Mcm2p, RPA, and DNA polymerase epsilon only during S phase. These results suggest that the complex containing Cdc45p, RPA, and MCMs is involved in origin unwinding and assembly of replication forks at each origin.     

Fission Yeast Cdc24 Is a Replication Factor C- and Proliferating Cell Nuclear Antigen-Interacting Factor Essential for S-Phase Completion

At the nonpermissive temperature the fission yeast cdc24-M38 mutant arrests in the cell cycle with incomplete DNA replication as indicated by pulsed-field gel electrophoresis. The cdc24⁺ gene encodes a 501-amino-acid protein with no significant homology to any known proteins. The temperature-sensitive cdc24 mutant is effectively rescued by pcn1⁺, rfc1⁺ (a fission yeast homologue of RFC1), and hhp1⁺, which encode the proliferating cell nuclear antigen (PCNA), the large subunit of replication factor C (RFC), and a casein kinase I involved in DNA damage repair, respectively. The Cdc24 protein binds PCNA and RFC1 in vivo, and the domains essential for Cdc24 function and for RFC1 and PCNA binding colocalize in the N-terminal two-thirds of the molecule. In addition, cdc24⁺ genetically interacts with the gene encoding the catalytic subunit of DNA polymerase , which is stimulated by PCNA and RFC, and with those encoding the fission yeast counterparts of Mcm2, Mcm4, and Mcm10. These results indicate that Cdc24 is an RFC- and PCNA-interacting factor required for DNA replication and might serve as a target for regulation.     

Consequences of abnormal CDK activity in S phase

Cyclin Dependent Kinases (CDKs) are important regulators of DNA replication. In this work we have investigated the consequences of increasing or decreasing the CDK activity in S phase. To this end we identified S-phase regulators of the fission yeast CDK, Cdc2, and used appropriate mutants to modulate Cdc2 activity. In fission yeast Mik1 has been thought to be the main regulator of Cdc2 activity in S phase. However, we find that Wee1 has a major function in S phase and thus we used wee1 mutants to investigate the consequences of increased Cdc2 activity. These wee1 mutants display increased replication stress and, particularly in the absence of the S-phase checkpoint, accumulate DNA damage. Notably, more cells incorporate EdU in a wee1^? strain as compared to wildtype, suggesting altered regulation of DNA replication. In addition, a higher number of cells contain chromatin-bound Cdc45, an indicator of active replication forks. In addition, we found that Cdc25 is required to activate Cdc2 in S phase and used a cdc25 mutant to explore a situation where Cdc2 activity is reduced. Interestingly, a cdc25 mutant has a higher tolerance for replication stress than wild-type cells, suggesting that reduced CDK activity in S phase confers resistance to at least some forms of replication stress.     

An E2 enzyme Ubc11 is required for ubiquitination of Slp1/Cdc20 and spindle checkpoint silencing in fission yeast

For ordered mitotic progression, various proteins have to be regulated by an ubiquitin ligase, the anaphase-promoting complex or cyclosome (APC/C) with appropriate timing. Recent studies have implied that the activity of APC/C also contributes to release of mitotic checkpoint complexes (MCCs) from its target Cdc20 in the process of silencing the spindle assembly checkpoint (SAC). Here we describe a temperature-sensitive mutant (ubc11-P93L) in which cell cycle progression is arrested at mitosis. The mutant grows normally at the restrictive temperature when SAC is inactivated, suggesting that the arrest is not due to abnormal spindle assembly, but rather due to prolonged activation of SAC. Supporting this notion, MCCs remain bound to APC/C even when SAC is satisfied. The ubc11⁺ gene encodes one of the two E2 enzymes required for progression through mitosis in fission yeast. Remarkably, Slp1 (a fission yeast homolog of Cdc20), which is degraded in an APC/C-dependent manner, stays stable throughout the cell cycle in the ubc11-P93L mutant lacking the functional SAC. Other APC/C substrates, in contrast, were degraded on schedule. We have also found that a loss of Ubc4, the other E2 required for progression through mitosis, does not affect the stability of Slp1. We propose that each of the two E2 enzymes is responsible for collaborating with APC/C for a specific set of substrates, and that Ubc11 is responsible for regulating Slp1 with APC/C for silencing the SAC.