Cdc7p-Dbf4p Regulates Mitotic Exit by Inhibiting Polo Kinase

Graduate Program in Cell and Molecular Biology, Michigan State University, East Lansing, MI, USA.
PLoS Genetics (Impact Factor: 8.17). 06/2009; 5(5):e1000498. DOI: 10.1371/journal.pgen.1000498
Source: PubMed

ABSTRACT Author Summary
Cdc7p-Dbf4p is a two-subunit enzyme required to copy the genetic material present on every chromosome in a process termed DNA replication. Dbf4p is an essential regulatory subunit of this enzyme that likely directs the Cdc7p subunit to its targets within the cell. We found that Dbf4p physically interacts with another protein called Polo that acts during mitosis, a later step in the cell cycle when the newly copied chromosomes are equally divided to mother and daughter cells. Polo is a master regulator of mitosis and impacts many other proteins required for cell division. We determined that Cdc7p-Dbf4p is a Polo inhibitor and, further, that Cdc7p-Dbf4p delayed or prevented chromosome segregation when errors occurred during the cell division process. Interestingly, Dbf4p may bind the Polo substrate-binding domain using a type of interaction not previously described. Thus, we have uncovered a new activity for Cdc7p-Dbf4p in the cell cycle to inhibit chromosome segregation, and these findings impact multiple fields that investigate how cells accurately copy and segregate their chromosomes.

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    • "Rsc2 could also bind the polo-box domain (PBD) of Cdc5, which normally binds substrates previously primed by phosphorylation by another kinase (Elia et al., 2003a). Indeed, Rsc2- HA3 bound to a recombinant GST-PBD fusion protein (Miller et al., 2009) but not to GST alone (Fig. 9 "
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    • "In addition to its highly conserved function in origin firing, other, less well understood, functions have been reported for Cdc7 kinase. These include activation of the ATR-Chk1 pathway in response to DNA damage and DNA replication stress (Takeda et al, 1999; Costanzo et al, 2003; Dierov et al, 2004; Tenca et al, 2007; Kim et al, 2008), cohesin loading onto chromatin required for chromosomal segregation in mitosis (Takahashi et al, 2008), regulation of exit from mitosis (Miller et al, 2009) and double-strand break formation during meiotic recombination (Matos et al, 2008). As the two-step-replication model excludes the formation of replication-competent origins once S phase has started, it has been argued on the basis of experimental evidence that a putative cell cycle checkpoint could delay progression from G1 into S phase if replication initiation is perturbed (Blow and Gillespie, 2008). "
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    ABSTRACT: Cdc7-Dbf4 is a two-subunit kinase required for initiating DNA replication. The Dbf4 regulatory subunit is required for Cdc7 kinase activity. Previous studies have shown that the C termini of Dbf4 orthologs encode a single (putative) C(2)H(2) zinc (Zn) finger, referred to as "motif C." By mutational analysis we show that the Zn finger is not required for the essential function of Dbf4. However, deletion and point mutants altering conserved Zn-finger residues exhibit a substantially slowed S-phase, DNA damage sensitivity, and a hypo-mutagenic phenotype following UV irradiation. Using two-hybrid and biochemical assays, we show that the Dbf4 Zn finger interacts with Cdc7 and stimulates its kinase activity. However, a separable Dbf4 region also mediates an interaction with Cdc7 such that only the loss of both Cdc7-interacting regions results in lethality. In contrast, an N-terminal BRCT-like domain is not required for induced mutagenesis nor does it interact with Cdc7. By making chimeric Dbf4 proteins that contain known BRCT domains in Saccharomyces cerevisiae, we show that the BRCT domain from Rev1, a translesion DNA polymerase, can uniquely substitute for the Dbf4 BRCT domain. Thus, we have mapped regions on budding yeast Dbf4 required for binding and activating Cdc7 kinase. Our data also suggest that the Dbf4 and Rev1 BRCT domains interact with a common protein or structure, although the precise function of both domains and their binding partners remains elusive.
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