chiffon

EVOLUTIONARY HOMOLOGS part 1/2 | part 2/2

Chiffon homologs interact with Cdc7

Yeast Cdc7 protein kinase and Dbf4 protein are both required for the initiation of DNA replication at the G1/S phase boundary of the mitotic cell cycle. Cdc7 kinase function is stage-specific in the cell cycle, but total Cdc7 protein levels remained unchanged. Therefore, regulation of Cdc7 function appears to be the result of posttranslational modification. The mechanism responsible for achieving this specific Cdc7 execution point has been investigated. Cdc7 kinase activity appears to be maximal at the G1/S boundary when measured using either cultures synchronized with alpha factor or Cdc- mutants or with inhibitors of DNA synthesis or mitosis. Therefore, Cdc7 kinase is regulated by a posttranslational mechanism that ensures maximal Cdc7 activity at the G1/S boundary, which is consistent with Cdc7 function in the cell cycle. This cell cycle-dependent regulation could be the result of association with the Dbf4 protein. In this study, the Dbf4 protein is shown to be required for Cdc7 kinase activity because Cdc7 kinase activity is thermolabile in vitro when extracts prepared from a temperature-sensitive dbf4 mutant grown under permissive conditions are used. In addition to employment of the two-hybrid system for protein-protein interactions, in vitro reconstitution assays have demonstrated that the Cdc7 and Dbf4 proteins interact both in vitro and in vivo. A suppressor mutation, bob1-1, that can bypass deletion mutations in both cdc7 and dbf4 has been isolated. However, the bob1-1 mutation cannot bypass all events in G1 phase because it fails to suppress temperature-sensitive cdc4 or cdc28 mutations. This indicates that the Cdc7 and Dbf4 proteins act at a common point in the cell cycle. Therefore, because of the common point of function for the two proteins and the fact that the Dbf4 protein is essential for Cdc7 function, it is proposed that Dbf4 may represent a cyclin-like molecule specific for the activation of Cdc7 kinase (Jackson, 1993).

DNA replication in the budding yeast Saccharomyces cerevisiae initiates from origins of specific DNA sequences during S phase. A screen based on two- and one-hybrid approaches has demonstrated that the product of the DBF4 gene interacts with yeast replication origins in vivo. The Dbf4 protein interacts with and positively regulates the activity of the Cdc7 protein kinase, which is required for entry into S phase in the yeast mitotic cell cycle. The analysis described here suggests a model in which one function of Dbf4 may be to recruit the Cdc7 protein kinase to initiation complexes (Dowell, 1994).

Cdc7/Dbf4 protein kinase is required for the initiation of DNA replication in Saccharomyces cerevisiae. Cdc7/Dbf4 protein kinase is not a cyclin-dependent kinase (CDK), but is regulated in a similar fashion as other CDKs because the Cdc7 kinase subunit is inactive in the absence of the regulatory subunit Dbf4. In contrast to what is known about CDKs, Cdc7/Dbf4 protein kinase has been shown to be an oligomer in the cell in this report. Genetic data that support this claim include interallelic complementation between several cdc7ts alleles and the cdc7T281A allele and also the results of experiments using the two-hybrid system with Cdc7 in both DNA-binding and transactivation domain plasmids. A molecular interaction between two different Cdc7 molecules has been shown by using a HA-tagged Cdc7 protein that differs in size from the wild-type Cdc7 protein: an anti-HA antibody immunoprecipitates both proteins in approximately equal stoichiometry. Analysis of the native molecular weight of Cdc7/Dbf4 protein kinase is consistent with oligomerization of the Cdc7 protein because complexes of about 180 and 300 kDa have been found. Oligomers of Cdc7 protein may exist for the purpose of allosteric regulation or to allow phosphorylation of multiple substrate protein molecules (Shellman, 1998).

Chiffon homologs and the G1/S transition

The initiation of DNA replication in Saccharomyces cerevisiae requires the protein product of the CDC45 gene. Although Cdc45p is present at essentially constant levels throughout the cell cycle, it completes its initiation function in late G1, after START and prior to DNA synthesis. Shortly after mitosis, cells prepare for initiation by assembling prereplicative complexes at their replication origins. These complexes are then triggered at the onset of S phase to commence DNA replication. Cells defective for CDC45 are incapable of activating the complexes to initiate DNA replication. In addition, Cdc45p and Cdc7p/Dbf4p, a kinase implicated in the G1/S phase transition, are dependent on one another for function. These data indicate that CDC45 functions in late G1 phase in concert with CDC7/DBF4 to trigger initiation at replication origins after the assembly of the prereplicative complexes (Owens, 1997).

In fission yeast, the Hsk1 protein kinase is essential for the initiation of DNA replication. Hsk1 forms a heterodimeric complex with the regulatory subunit, Dfp1. The further characterization of Dfp1 is reported here. Reconstitution experiments with purified proteins indicate that Dfp1 is necessary and sufficient to activate Hsk1 phosphorylation of exogenous substrates, such as the Schizosaccharomyces pombe minichromosome maintenance protein Cdc19. The dfp1(+) gene is essential for viability of S. pombe, and depletion of the Dfp1 protein significantly delays the onset of S phase. Dfp1 is a phosphoprotein in vivo and becomes hyperphosphorylated when cells are blocked in S phase by treatment with the DNA synthesis inhibitor hydroxyurea. Hyperphosphorylation in S phase depends on the checkpoint kinase Cds1. The abundance of Dfp1 varies during progression through the cell cycle. The protein is absent when cells are arrested in G(1) phase. When cells are released into the cell cycle, Dfp1 appears suddenly at the G(1)/S transition, coincident with the initiation of DNA replication. The absence of Dfp1 before S phase is due largely, but not exclusively, to posttranscriptional regulation. It is proposed that cell cycle-regulated activation of Dfp1 expression at the G(1)/S transition results in activation of the Hsk1 protein kinase, which, in turn, leads to the initiation of DNA replication (Brown, 1999).

The nimO predicted protein of Aspergillus nidulans is related structurally and functionally to Dbf4p, the regulatory subunit of Cdc7p kinase in budding yeast. nimOp and Dbf4p are most similar in their C-termini, which contain a PEST motif and a novel, short-looped Cys2-His2 zinc finger-like motif. DNA labeling and reciprocal shift assays using ts-lethal nimO18 mutants have shown that nimO is required for initiation of DNA synthesis and for efficient progression through S phase. nimO18 mutants abrogate a cell cycle checkpoint linking S and M phases by segregating their unreplicated chromatin. This checkpoint defect does not interfere with other checkpoints monitoring spindle assembly and DNA damage (dimer lesions), but does prevent activation of a DNA replication checkpoint. The division of unreplicated chromatin is accelerated in cells lacking a component of the anaphase-promoting complex (bimEAPC1), consistent with the involvement of nimO and APC/C in separate checkpoint pathways. A nimO deletion confers DNA synthesis and checkpoint defects similar to nimO18. Inducible nimO alleles lacking as many as 244 C-terminal amino acids supported hyphal growth, but not asexual development, when overexpressed in a ts-lethal nimO18 strain. However, the truncated alleles cannot rescue a nimO deletion, indicating that the C terminus is essential and suggesting some type of interaction among nimO polypeptides (James, 1999).

Using a reconstituted DNA replication assay from yeast, it has been demonstrated that two kinase complexes are essential for the promotion of replication in vitro. An active Clb/Cdc28 kinase complex, or its vertebrate equivalent, is required in trans to stimulate initiation in G(1)-phase nuclei, whereas the Dbf4/Cdc7 kinase complex must be provided by the template nuclei themselves. Dbf4p, the regulatory subunit of Cdc7p, accumulates during late G(1) phase; it becomes chromatin associated prior to Clb/Cdc28 activation, and assumes a punctate pattern of localization that is similar to, and dependent on, the origin recognition complex (ORC). The association of Dbf4p with a detergent-insoluble chromatin fraction in G(1)-phase nuclei requires ORC but not Cdc6p or Clb/Cdc28 kinase activity, and correlates with competence for initiation. A model is proposed in which Dbf4p targets Cdc7p to the prereplication complex prior to the G(1)/S transition, by a pathway parallel to, but independent of, the Cdc6p-dependent recruitment of MCMs (Pasero, 1999).

Cell fusion experiments performed nearly 30 years ago showed that S-phase cytosol provides a diffusible substance that can activate DNA replication in G1-phase nuclei, but not in G2-phase nuclei. This led to two proposals: (1) that nuclei can assume at least two states -- one in which they are competent to initiate DNA replication and another in which they are not, and (2) that a trans-acting, S-phase promoting factor triggers initiation. Although these phenomena were subsequently reconstituted in replication assays based on Xenopus egg extracts, the identification of the genes and proteins that participate in these events was primarily achieved through the genetic analysis of the G1/S transition in yeast. The components of the pre-RC (ORC, Cdc6p, MCMs, and Cdc45p) were genetically identified and demonstrated to be essential for the initiation of DNA synthesis in yeast. Moreover, mutations in two universally conserved Ser/Thr kinases, Cdc28p and Cdc7p, and their unstable regulatory subunits, arrest or delay passage from G1 to S phase. Inactivation of CDC7 in mid-S phase also impairs initiation at late-firing origins, suggesting that this kinase does not only act at the G1/S transition. To explain this observation it was proposed that the Dbf4/Cdc7 kinase acts locally at origins to promote initiation. The mode of action of Dbf4/Cdc7 kinase is distinctly different from that of Clb/Cdc28, which behaves as a diffusible SPF. More importantly, Dbf4p, the Cdc7p regulatory subunit, associates with prereplicative chromatin in an ORC-dependent manner. Its binding, which peaks in late G1 phase, is nonetheless independent of Cdc6 and MCM complex loading. The presence of Dbf4p in G1-phase nuclei correlates with the potential of these templates for the initiation of DNA synthesis in vitro (Pasero, 1999).

Using extracts and template nuclei from mutant yeast strains, it has been found that either an S-phase or mitotic B-type cyclin/Cdc28 complex, or its vertebrate equivalent, is necessary to stimulate semiconservative DNA replication in G1-phase nuclei in vitro. The addition of kinase alone is sufficient to stimulate replication slightly, although another component of the nuclear extract is limiting for maximal replication efficiency. The results from in vitro replication are fully consistent with the timing of Clb5p and Clb6p expression and genetic evidence implicating the Clb5,6/Cdc28 kinase in the promotion of replication in vivo. However, it appears that yeast is rather permissive as to the nature of the Cdk that can function to initiate DNA replication. In a G1-phase extract with high Sic1p levels, purified Xenopus CycB/Cdc2 kinase stimulates replication more efficiently than the purified Clb5/Cdc28 kinase. This may either indicate a need for both the Clb5 and Clb6/Cdc28 complexes, or reflect inhibition of the exogenously added Clb5/Cdc28 by the high levels of Sic1p. Although the synthesis of Dbf4p also peaks in late G1 and early S phase, the presence of active Dbf4/Cdc7 kinase is not required in trans to activate replication in wild-type G1-phase nuclei. Dbf4/Cdc7 is therefore not, formally speaking, a component of the diffusible S-phase-promoting factor (SPF) (Pasero, 1999).

Unlike Cdc7p, which maintains a constant level through the cell cycle, Dbf4p is a highly unstable protein, particularly when cells are blocked by an alpha-factor arrest in early G1. Using nuclei from a synchronized culture in which DBF4 expression can be shut off, it has been shown that the competence of a late G1-phase nucleus to support DNA replication in vitro correlates with the presence of this factor. This is true even when a wild-type S-phase extract is added to promote replication. In support of this conclusion, the conditional inactivation of either Cdc7p or Dbf4p compromises the replicative ability of G1-phase templates, whereas cdc28-deficient G1-phase nuclei replicate efficiently in Cdk1-containing extracts (Pasero, 1999).

Dbf4p can interact with a complex that recognizes the ARS consensus, as indicated by a one-hybrid assay; this suggests that at least part of the regulatory function of Dbf4p might be to target Cdc7p to origins. Consistent with this hypothesis, immunolocalization of Dbf4p reveals ORC-like foci in late G1 and early S phase, and this punctate distribution is compromised in orc2-1 mutant strains. A subpopulation of Dbf4p copurifies with Orc2p in an insoluble nuclear fraction and, like ORC, resists solubilization by DNase I digestion. Importantly, the association of Dbf4p with the chromatin fraction in late G1 requires an intact ORC complex, but not Cdc6p or MCM-bound proteins. Although it cannot be ruled out that a subfraction of Dbf4p can bind the assembled MCM complex, the data clearly define a Cdc6/MCM-independent association that requires intact ORC (Pasero, 1999).

The question remains unresolved: what are the minimal components required to form a replication-competent origin complex? Clearly, the association of the MCM complex with chromatin and the pre-RC requires the presence of both ORC and Cdc6p. Cdc6p is a highly labile protein, which has to be synthesized in late G1 following an alpha-factor block or other events that deplete Cdc6p from the cell. The ability of Cdc6p to load MCM proteins is inhibited by high Clb/Cdc28 kinase activity. It has been proposed that association of Dbf4p with chromatin and its potential targeting of Cdc7p to the pre-RC defines a second pathway necessary for creation of an initiation competent state at origins in G1. In contrast to MCMs, Dbf4p recruitment is clearly independent of Cdc6p, and can occur either in the absence or presence of Clb/Cdc28 activity. Like Cdc6p, Dbf4p is highly labile, and is depleted in stationary phase and pheromone-blocked cells. Unlike Cdc6p, however, part of Dbf4p persists in the insoluble nuclear fraction throughout much of S phase. It has been suggested that two steps are required to form an initiation-competent nucleus: Cdc7p loading through Dbf4p and MCM loading through Cdc6p. Both pathways converge on the MCM complex: the ORC-Cdc6p-MCM pathway assembles MCMs at origins; the ORC-Dbf4p pathway is likely to target the Cdc7p kinase to its critical target, Mcm2p. This model provides a mechanism to restrict Dbf4/Cdc7 kinase action to those MCMs assembled at origins (Pasero, 1999).

Deletion of the Dbf4p amino terminus renders the protein nonfunctional. This deletion removed not only the ARS-targeting domain, but also the only NLS in the protein, thus its inactivity may have reflected the lack of nuclear localization. In a preliminary study, a series of subdomains of the Dbf4p amino terminus was overexpressed, to see if Dbf4p-binding sites at origins could be saturated. Constructs encoding the full origin-targeting domain cause cell growth arrest when induced, although in liquid culture this is manifest only when cells are recovering from stationary phase or from alpha-factor arrest. This shows that overexpression of the Dbf4N-terminus is not lethal per se, and suggests that competition for origin targeting is most successful when the endogenous Dbf4p levels drop, and the targeting of Cdc7p must be achieved de novo (Pasero, 1999).

The best evidence that the targeting of Dbf4p through ORC may be a regulated event, is demonstrated by the release of Dbf4p from the insoluble nuclear fraction in cells arrested in S phase by hydroxyurea. This displacement is Rad53p dependent, and results in a highly stabilized, soluble form of Dbf4p. Dbf4p also appears to be displaced from chromatin during DNA synthesis in vitro, although in this case the displaced Dbf4p is rapidly degraded. Thus, the Rad53p modification of Dbf4p (Santocanale, 1998 and J.F. Diffley, pers. comm. to Pasero, 1998) may serve to stabilize the released factor, whereas release of Dbf4p during a normal S phase may serve to deplete the nuclear pool of Dbf4p to prevent reformation of an active pre-RC. Because the amount of active Cdc7 kinase appears to be regulated primarily through the level of its labile Dbf4p cofactor, a targeting and stabilization of the complex by its association with origins may be essential for achieving a critical concentration of the kinase at its essential sites of action. Such a mechanism is particularly relevant in view of the low abundance and relatively low affinity shown by Cdc7p for its substrates in vitro. Future studies will address the critical question of how Dbf4p synthesis and degradation are controlled, because it is predicted that these will be closely linked to proper cell cycle progression and recovery from a resting or G0 state (Pasero, 1999).

The Cdc7-Dbf4 kinase is essential for regulating initiation of DNA replication in Saccharomyces cerevisiae. A human Cdc7 homolog, HsCdc7 has been identified as has a human Dbf4 homolog, HsDbf4. HsDbf4 binds to HsCdc7 and activates HsCdc7 kinase activity when HsDbf4 and HsCdc7 are coexpressed in insect and mammalian cells. HsDbf4 protein levels are regulated during the cell cycle with a pattern that matches that of HsCdc7 protein kinase activity. They are low in G1, increase during G1-S, and remain high during S and G2-M. Purified baculovirus-expressed HsCdc7-HsDbf4 selectively phosphorylates the MCM2 subunit of the minichromosome maintenance (MCM) protein complex isolated by immunoprecipitation with MCM7 antibodies in vitro. Two-dimensional tryptic phosphopeptide-mapping analysis of in vivo 32P-labeled MCM2 from HeLa cells reveals that several major tryptic phosphopeptides of MCM2 comigrate with those of MCM2 phosphorylated by HsCdc7-HsDbf4 in vitro, suggesting that MCM2 is a physiological HsCdc7-HsDbf4 substrate. Immunoneutralization of HsCdc7-HsDbf4 activity by microinjection of anti-HsCdc7 antibodies into HeLa cells blocks initiation of DNA replication. These results indicate that the HsCdc7-HsDbf4 kinase is directly involved in regulating the initiation of DNA replication by targeting MCM2 protein in mammalian cells (Jiang, 1999).

Multiple functions of Chiffon homologs

chiffon Evolutionary homologs continues: part 2/2

chiffon: Biological Overview | Developmental Biology | Effects of Mutation | References

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