Localisation of the DmCdc45 DNA replication factor in the mitotic cycle and during chorion gene amplification

The cdc45 protein was originally identified in Saccharomyces cerevisiae and shown to be essential for initiation of eukaryotic DNA replication. Subsequent isolation and characterisation of the corresponding genes from fission yeast, Xenopus and mammals also support a replication role for the protein in these species. They further suggest that during the course of its function cdc45 interacts with a number of other replication proteins, including minichromosome maintenance proteins, the origin recognition complex and DNA polymerase α. We have cloned the gene coding for cdc45 protein from Drosophila melanogaster. We have analysed the expression pattern of the cdc45 protein throughout the cell cycle and the life cycle using a combination of indirect immunofluorescence and subcellular fractionation. Our data show that cellular localisation and developmental regulation of the protein is consistent with a role in DNA replication. DmCdc45 is predominantly expressed in proliferating cells. In addition, its subcellular location is nuclear during interphase and the protein shows association with chromatin. The chromatin-associated form of the protein shows a post-translational modification, which may be involved in control of the action of the protein. DmCdc45 shows interactions with mcm proteins, however, the interactions detected show some specificity, perhaps suggesting a preferential association with particular mcm proteins. In addition we show that a stoichiometric mcm interaction may not be obligatory for the function of cdc45 in follicle cell replication, because, unlike the mcm proteins, DmCdc45 localises to the chorion amplification foci in the follicle cells of the ovary.     

Characterization of Schizosaccharomyces pombe mcm7(+) and cdc23(+) (MCM10) and interactions with replication checkpoints

MCM proteins are required for the proper regulation of DNA replication. We cloned fission yeast mcm7(+) and showed it is essential for viability; spores lacking mcm7(+) begin S phase later than wild-type cells and arrest with an apparent 2C DNA content. We isolated a novel temperature-sensitive allele, mcm7-98, and also characterized two temperature-sensitive alleles of the fission yeast homolog of MCM10, cdc23(+). mcm7-98 and both cdc23ts alleles arrest with damaged chromosomes and an S phase delay. We find that mcm7-98 is synthetically lethal with the other mcmts mutants but does not interact genetically with either cdc23ts allele. However, cdc23-M36 interacts with mcm4ts. Unlike other mcm mutants or cdc23, mcm7-98 is synthetically lethal with checkpoint mutants Deltacds1, Deltachk1, or Deltarad3, suggesting chromosomal defects even at permissive temperature. Mcm7p is a nuclear protein throughout the cell cycle, and its localization is dependent on the other MCM proteins. Our data suggest that the Mcm3p-Mcm5p dimer interacts with the Mcm4p-Mcm6p-Mcm7p core complex through Mcm7p.     

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.     

Differential requirement of DNA replication factors for subtelomeric ARS consensus sequence protosilencers in Saccharomyces cerevisiae

The establishment of silent chromatin requires passage through S-phase, but not DNA replication per se. Nevertheless, many proteins that affect silencing are bona fide DNA replication factors. It is not clear if mutations in these replication factors affect silencing directly or indirectly via deregulation of S-phase or DNA replication. Consequently, the relationship between DNA replication and silencing remains an issue of debate. Here we analyze the effect of mutations in DNA replication factors (mcm5-461, mcm5-1, orc2-1, orc5-1, cdc45-1, cdc6-1, and cdc7-1) on the silencing of a group of reporter constructs, which contain different combinations of "natural" subtelomeric elements. We show that the mcm5-461, mcm5-1, and orc2-1 mutations affect silencing through subtelomeric ARS consensus sequences (ACS), while cdc6-1 affects silencing independently of ACS. orc5-1, cdc45-1, and cdc7-1 affect silencing through ACS, but also show ACS-independent effects. We also demonstrate that isolated nontelomeric ACS do not recapitulate the same effects when inserted in the telomere. We propose a model that defines the modes of action of MCM5 and CDC6 in silencing.     

Activation of budding yeast replication origins and suppression of lethal DNA damage effects on origin function by ectopic expression of the co-chaperone protein Mge1

Initiation of DNA replication in eukaryotes requires the origin recognition complex (ORC) and other proteins that interact with DNA at origins of replication. In budding yeast, the temperature-sensitive orc2-1 mutation alters these interactions in parallel with defects in initiation of DNA replication and in checkpoints that depend on DNA replication forks. Here we show that DNA-damaging drugs modify protein-DNA interactions at budding yeast replication origins in association with lethal effects that are enhanced by the orc2-1 mutation or suppressed by a different mutation in ORC. A dosage suppressor screen identified the budding yeast co-chaperone protein Mge1p as a high copy suppressor of the orc2-1-specific lethal effects of adozelesin, a DNA-alkylating drug. Ectopic expression of Mge1p also suppressed the temperature sensitivity and initiation defect conferred by the orc2-1 mutation. In wild type cells, ectopic expression of Mge1p also suppressed the lethal effects of adozelesin in parallel with the suppression of adozelesin-induced alterations in protein-DNA interactions at origins, stimulation of initiation of DNA replication, and binding of the precursor form of Mge1p to nuclear chromatin. Mge1p is the budding yeast homologue of the Escherichia coli co-chaperone protein GrpE, which stimulates initiation at bacterial origins of replication by promoting interactions of initiator proteins with origin sequences. Our results reveal a novel, proliferation-dependent cytotoxic mechanism for DNA-damaging drugs that involves alterations in the function of initiation proteins and their interactions with DNA.     

Schizosaccharomyces pombe Noc3 is essential for ribosome biogenesis and cell division but not DNA replication

The initiation of eukaryotic DNA replication is preceded by the assembly of prereplication complexes (pre-RCs) at chromosomal origins of DNA replication. Pre-RC assembly requires the essential DNA replication proteins ORC, Cdc6, and Cdt1 to load the MCM DNA helicase onto chromatin. Saccharomyces cerevisiae Noc3 (ScNoc3), an evolutionarily conserved protein originally implicated in 60S ribosomal subunit trafficking, has been proposed to be an essential regulator of DNA replication that plays a direct role during pre-RC formation in budding yeast. We have cloned Schizosaccharomyces pombe noc3(+) (Spnoc3(+)), the S. pombe homolog of the budding yeast ScNOC3 gene, and functionally characterized the requirement for the SpNoc3 protein during ribosome biogenesis, cell cycle progression, and DNA replication in fission yeast. We showed that fission yeast SpNoc3 is a functional homolog of budding yeast ScNoc3 that is essential for cell viability and ribosome biogenesis. We also showed that SpNoc3 is required for the normal completion of cell division in fission yeast. However, in contrast to the proposal that ScNoc3 plays an essential role during DNA replication in budding yeast, we demonstrated that fission yeast cells do enter and complete S phase in the absence of SpNoc3, suggesting that SpNoc3 is not essential for DNA replication in fission yeast.     

Fission yeast cdc21, a member of the MCM protein family, is required for onset of S phase and is located in the nucleus throughout the cell cycle.

The fission yeast cdc21 protein belongs to the MCM family, implicated in the once per cell cycle regulation of chromosome replication. In budding yeast, proteins in this family are eliminated from the nucleus during S phase, which has led to the suggestion that they may serve to distinguish unreplicated from replicated DNA, as in the licensing factor model. We show here that, in contrast to the situation in budding yeast, cdc21 remains in the nucleus after S phase, as is found for related proteins in mammalian cells. We suggest that regulation of nuclear import of these proteins may not be an essential aspect of their function in chromosome replication. To determine the function of cdc21+, we have analysed the phenotype of a gene deletion. cdc21+ is required for entry into S phase and, unexpectedly, a proportion of cells depleted of the gene product are able to enter mitosis in the absence of DNA replication. These results are consistent with the view that individual proteins in the MCM family are required for all initiation events, and defective initiation may impair the coordination between mitosis and S phase.     

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.     

Evidence for sequential action of cdc7 and cdk2 protein kinases during initiation of DNA replication in Xenopus egg extracts

To investigate how the protein kinase cdc7 stimulates DNA replication in metazoans, a soluble cell-free replication system derived from Xenopus eggs was used. DNA was incubated in egg cytosol to form prereplication complexes and then in nucleoplasmic extract to initiate DNA synthesis. We find that cdc7 is greatly enriched in nucleoplasmic extract and that this high concentration is essential for efficient DNA replication, supporting previous models that the nucleus activates replication indirectly by sequestering essential components. cdc7 binds to chromatin at the G(1)/S transition before initiation occurs, and it dissociates from chromatin as S phase progresses. The chromatin association of cdc7 requires chromatin-bound MCM. In turn, cdc7 is required to load the initiation factor cdc45 onto the DNA. Finally, efficient replication is observed when chromatin is exposed first to cdc7 and then to cdk2 but not when it is exposed to cdk2 before cdc7. Therefore, the cdc7- and cdk2-dependent initiation steps can be separated, indicating the existence of a novel, stable initiation intermediate. Moreover, the data suggest that cdk2 can only act after cdc7 has executed its function.     

Essential role of MCM proteins in premeiotic DNA replication

A critical event in eukaryotic DNA replication involves association of minichromosome maintenance (MCM2-7) proteins with origins, to form prereplicative complexes (pre-RCs) that are competent for initiation. The ability of mutants defective in MCM2-7 function to complete meiosis had suggested that pre-RC components could be irrelevant to premeiotic S phase. We show here that MCM2-7 proteins bind to chromatin in fission yeast cells preparing for meiosis and during premeiotic S phase in a manner suggesting they in fact are required for DNA replication in the meiotic cycle. This is confirmed by analysis of a degron mcm4 mutant, which cannot carry out premeiotic DNA replication. Later in meiosis, Mcm4 chromatin association is blocked between meiotic nuclear divisions, presumably accounting for the absence of a second round of DNA replication. Together, these results emphasize similarity between replication mechanisms in mitotic and meiotic cell cycles.     

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     

Apoptosis-like yeast cell death in response to DNA damage and replication defects

In budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast and other unicellular organisms, DNA damage and other stimuli can induce cell death resembling apoptosis in metazoans, including the activation of a recently discovered caspase-like molecule in budding yeast. Induction of apoptotic-like cell death in yeasts requires homologues of cell cycle checkpoint proteins that are often required for apoptosis in metazoan cells. Here, we summarize these findings and our unpublished results which show that an important component of metazoan apoptosis recently detected in budding yeast-reactive oxygen species (ROS)-can also be detected in fission yeast undergoing an apoptotic-like cell death. ROS were detected in fission and budding yeast cells bearing conditional mutations in genes encoding DNA replication initiation proteins and in fission yeast cells with mutations that deregulate cyclin-dependent kinases (CDKs). These mutations may cause DNA damage by permitting entry of cells into S phase with a reduced number of replication forks and/or passage through mitosis with incompletely replicated chromosomes. This may be relevant to the frequent requirement for elevated CDK activity in mammalian apoptosis, and to the recent discovery that the initiation protein Cdc6 is destroyed during apoptosis in mammals and in budding yeast cells exposed to lethal levels of DNA damage. Our data indicate that connections between apoptosis-like cell death and DNA replication or CDK activity are complex. Some apoptosis-like pathways require checkpoint proteins, others are inhibited by them, and others are independent of them. This complexity resembles that of apoptotic pathways in mammalian cells, which are frequently deregulated in cancer. The greater genetic tractability of yeasts should help to delineate these complex pathways and their relationships to cancer and to the effects of apoptosis-inducing drugs that inhibit DNA replication.     

Origins and complexes: the initiation of DNA replication

Eukaryotic DNA is organized for replication as multiple replicons. DNA synthesis in each replicon is initiated at an origin of replication. In both budding yeast, Saccharomyces cerevisiae and fission yeast, Schizosaccharomyces pombe, origins contain specific sequences that are essential for initiation, although these differ significantly between the two yeasts with those of S. pombe being more complex then those of S. cerevisiae. However, it is not yet clear whether the replication origins of plants contain specific essential sequences or whether origin sites are determined by features of chromatin structure. In all eukaryotes there are several biochemical events that must take place before initiation can occur. These are the marking of the origins by the origin recognition complex (ORC), the loading onto the origins, in a series of steps, of origin activation factors including the MCM proteins, and the initial denaturation of the double helix to form a replication "bubble". Only then can the enzymes that actually initiate replication, primase and DNA polymerase-alpha, gain access to the template. In many cells this complex series of events occurs only once per cell cycle, ensuring that DNA is not re-replicated within one cycle. However, regulated re-replication of DNA within one cell cycle (DNA endoreduplication) is relatively common in plants, indicating that the "once-per-cycle" controls can be overridden.     

Chromotin binding, nuclear localization and phosphorylation of Xenopus cdc21 are cell-cycle dependent and associated with the control of initiation of DNA replication.

A Xenopus homologue of Schizosaccharomyces pombe cdc21 has been characterized as a new member of the MCM family of proteins. The cdc21 protein exhibits cell-cycle dependent chromatin binding and phosphorylation in association with S-phase control. Cdc21 binds to decondensing chromatin at the end of mitosis, localizing to numerous foci which form prior to reconstitution of the nuclear membrane. The association of cdc21 with chromatin occurs in membrane-free high speed extracts and is resistant to detergent extraction. The spatial organization of the cdc21 foci resembles that of pre-replication centres though no co-localization with RP-A was observed. Cdc21 remains bound to chromatin during the initiation of DNA replication and is displaced as the DNA replication forks progress. These subnuclear changes in localization correlate with cell-cycle-regulated changes in phosphorylation. Cdc21 binds to chromatin in an underphosphorylated state, but in early S phase the nuclear localized cdc21 is partially phosphorylated before it is displaced from the chromatin. Cytoplasmic cdc21 remains underphosphorylated but at the beginning of mitosis the entire pool of cdc21 is hyperphosphorylated, possibly by the cdc2/cyclin B kinase. These properties identify Xenopus cdc21 as a possible component of the DNA licensing factor.     

A single fission yeast mitotic cyclin B p34cdc2 kinase promotes both S-phase and mitosis in the absence of G1 cyclins.

Deletion of the fission yeast mitotic B-type cyclin gene cdc13 causes cells to undergo successive rounds of DNA replication. We have used a strain which expresses cdc13 conditionally to investigate re-replication. Activity of Start genes cdc2 and cdc10 is necessary and p34cdc2 kinase is active in re-replicating cells. We tested to see whether other cyclins were required for re-replication using cdc13delta. Further deletion of cig1 and puc1 had no effect, but deletion of cig2/cyc17 caused a severe delay in re-replication. Deletion of cig1 and cig2/cyc17 together abolished re-replication completely and cells arrested in G1. This, and analysis of the temperature sensitive cdc13-117 mutant, suggests that cdc13 can effectively substitute for the G1 cyclin activity of cig2/cyc17. We have characterized p56cdc13 activity and find evidence that in the absence of G1 cyclins, S-phase is delayed until the mitotic p34cdc2-p56cdc13 kinase is sufficiently active. These data suggest that a single oscillation of p34cdc2 kinase activity provided by a single B-type cyclin can promote ordered progression into both DNA replication and mitosis, and that the level of cyclin-dependent kinase activity may act as a master regulator dictating whether cells undergo S-phase or mitosis.     

Overexpression of a novel gene , Cms1, can rescue the growth arrest of a Saccharomyces cerevisiae mcm10 suppressor

INTRODUCTIONDNA replication is a fundamenial process thatmust occur only once at each ce1l cycle. This restrictcontrol appears to be achieved through the coordi-nated actiVities of numerous proteins. The buddingyeast Saccharompes cerevhaae provides an excellenteukaryotic model fOr study of proteins invo1ved inthe control of DNA replication.In the budding yeast, minichromosome mainte-nance (MCM) proteins, MCM2-7, are a family of strsequence-related proteins that play crucia1 roles inr…     

SpSld3 is required for loading and maintenance of SpCdc45 on chromatin in DNA replication in fission yeast

Initiation of DNA replication in eukaryotic cells is regulated through the ordered assembly of replication complexes at origins of replication. Association of Cdc45 with the origins is a crucial step in assembly of the replication machinery, hence can be considered a target for the regulation of origin activation. To examine the process required for SpCdc45 loading, we isolated fission yeast SpSld3, a counterpart of budding yeast Sld3 that interacts with Cdc45. SpSld3 associates with the replication origin during G1-S phases and this association depends on Dbf4-dependent (DDK) kinase activity. In the corresponding period, SpSld3 interacts with minichromosome maintenance (MCM) proteins and then with SpCdc45. A temperature-sensitive sld3-10 mutation suppressed by the multicopy of the sna41+ encoding SpCdc45 impairs loading of SpCdc45 onto chromatin. In addition, this mutation leads to dissociation of preloaded Cdc45 from chromatin in the hydroxyurea-arrested S phase, and DNA replication upon removal of hydroxyurea is retarded. Thus, we conclude that SpSld3 is required for stable association of Cdc45 with chromatin both in initiation and elongation of DNA replication. The DDK-dependent origin association suggests that SpSld3 is involved in temporal regulation of origin firing.     

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.