Yasuo Kawasaki

Cdc7-dependent phosphorylation of Mer2 facilitates initiation of yeast meiotic recombination

Meiosis ensures genetic diversification of gametes and sexual reproduction. For successful meiosis, multiple events such as DNA replication, recombination, and chromosome segregation must occur coordinately in a strict regulated order. We investigated the meiotic roles of Cdc7 kinase in the initiation of meiotic recombination, namely, DNA double-strand breaks (DSBs) mediated by Spo11 and other coactivating proteins. Genetic analysis using bob1-1 cdc7Delta reveals that Cdc7 is essential for meiotic DSBs and meiosis I progression. We also demonstrate that the N-terminal region of Mer2, a Spo11 ancillary protein required for DSB formation and phosphorylated by cyclin-dependent kinase (CDK), contains two types of Cdc7-dependent phosphorylation sites near the CDK site (Ser30): One (Ser29) is essential for meiotic DSB formation, and the others exhibit a cumulative effect to facilitate DSB formation. Importantly, mutations on these sites confer severe defects in DSB formation even when the CDK phosphorylation is present at Ser30. Diploids of cdc7Delta display defects in the chromatin binding of not only Spo11 but also Rec114 and Mei4, other meiotic coactivators that may assist Spo11 binding to DSB hot spots. We thus propose that Cdc7, in concert with CDK, regulates Spo11 loading to DSB sites via Mer2 phosphorylation.

The DNA Polymerase Domain of polε Is Required for Rapid, Efficient, and Highly Accurate Chromosomal DNA Replication, Telomere Length Maintenance, and Normal Cell Senescence inSaccharomyces cerevisiae

Saccharomyces cerevisiae POL2 encodes the catalytic subunit of DNA polymerase ε. This study investigates the cellular functions performed by the polymerase domain of Pol2p and its role in DNA metabolism. The pol2-16 mutation has a deletion in the catalytic domain of DNA polymerase ε that eliminates its polymerase and exonuclease activities. It is a viable mutant, which displays temperature sensitivity for growth and a defect in elongation step of chromosomal DNA replication even at permissive temperatures. This mutation is synthetic lethal in combination with temperature-sensitive mutants or the 3′- to 5′-exonuclease-deficient mutant of DNA polymerase δ in a haploid cell. These results suggest that the catalytic activity of DNA polymerase ε participates in the same pathway as DNA polymerase δ, and this is consistent with the observation that DNA polymerases δ and ε colocalize in some punctate foci on yeast chromatids during S phase. Thepol2-16 mutant senesces more rapidly than wild type strain and also has shorter telomeres. These results indicate that the DNA polymerase domain of Pol2p is required for rapid, efficient, and highly accurate chromosomal DNA replication in yeast.

Interactions between Mcm10p and other replication factors are required for proper initiation and elongation of chromosomal DNA replication in Saccharomyces cerevisiae

MCM10 is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae. Previous work showed that Mcm10p interacts with the Mcm2–7 protein complex that may be functioning as the replication‐licensing factor. In addition, Mcm10p is required during origin activation and disassembly of the prereplicative complex, which allows smooth passage of replication forks.

Characterization of the Yeast Cdc7p/Dbf4p Complex Purified from Insect Cells ITS PROTEIN KINASE ACTIVITY IS REGULATED BY Rad53p

The yeast Saccharomyces cerevisiaeCdc7p/Dbf4p protein kinase complex was purified to near homogeneity from insect cells. The complex efficiently phosphorylated yeast Mcm2p and less efficiently the remaining Mcm proteins or other replication proteins. Significantly, when pretreated with alkaline phosphatase, Mcm2p became completely inactive as a substrate, suggesting that it must be phosphorylated by other protein kinase(s) to be a substrate for the Cdc7p/Dbf4p complex. Mutant Cdc7p/Dbf4p complexes containing either Cdc7-1p or Dbf4-1∼5p were also partially purified from insect cells and characterized in vitro. Furthermore, the autonomously replicating sequence binding activity of variousdbf4 mutants was also analyzed. These studies suggest that the autonomously replicating sequence-binding and Cdc7p protein kinase activation domains of Dbf4p collaborate to form an active Cdc7p/Dbf4p complex and function during S phase in S. cerevisiae. It is shown that Rad53p phosphorylates the Cdc7p/Dbf4p complex in vitro and that this phosphorylation greatly inhibits the kinase activity of Cdc7p/Dbf4p. This result suggests that Rad53p controls the initiation of chromosomal DNA replication by regulating the protein kinase activity associated with the Cdc7p/Dbf4p complex.

Reconstitution of Saccharomyces cerevisiae prereplicative complex assembly in vitro

The assembly of the prereplicative complex (pre‐RC) at the origin of replication in eukaryotes is a highly regulated and highly conserved process that plays a critical role in preventing multiple rounds of DNA replication per cell division cycle. This study analyzes the molecular dynamics of the assembly of Saccharomyces cerevisiae pre‐RC in vitro using ARS1 plasmid DNA and yeast whole cell extracts. In addition, pre‐RC assembly was reconstituted in vitro using ARS1 DNA and purified origin‐recognition complex (ORC), Cdc6p and Cdt1p‐Mcm2‐7p. The results reveal sequential recruitment of ORC, Cdc6p, Cdt1p and Mcm2‐7p on to ARS1 DNA. When Mcm2‐7p is maximally loaded, Cdc6p and Cdt1p are released, suggesting that these two proteins are co‐ordinately regulated during pre‐RC assembly. In extracts from sid2‐21 mutant cells that are deficient in CDT1, ORC and Cdc6p bind to ARS1 but Cdt1p and Mcm2‐7p do not. However, Mcm2‐7p does bind in the presence of exogenous Cdt1p or Cdt1p‐Mcm2‐7p complex. Cdt1p‐Mcm2‐7p complex, which was purified from G1‐, early S or G2/M‐arrested cells, exhibits structure‐specific DNA binding, interacting only with bubble‐ or Y‐shape‐DNA, but the biological significance of this observation is not yet known.

Budding yeast mcm10/dna43 mutant requires a novel repair pathway for viability.

Background: MCM10 is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae. Mcm10p functionally interacts with components of the pre‐replicative complex (Mcm2‐Mcm7 complex and origin recognition complex) as well as the pre‐initiation complex component (Cdc45p) suggesting that it may be a component of the pre‐RC as well as the pre‐IC. Two‐dimensional gel electrophoresis analysis showed that Mcm10p is required not only for the initiation of DNA synthesis at replication origins but also for the smooth passage of replication forks at origins. Genetic analysis showed that MCM10 interacts with components of the elongation machinery such as Polδ and Polɛ, suggesting that it may play a role in elongation replication.

The fifth essential DNA polymerase φ in Saccharomyces cerevisiae is localized to the nucleolus and plays an important role in synthesis of rRNA

We report that POL5 encodes the fifth essential DNA polymerase in Saccharomyces cerevisiae. Pol5p was identified and purified from yeast cell extracts and is an aphidicolin-sensitive DNA polymerase that is stimulated by yeast proliferating cell nuclear antigen (PCNA). Thus, we named Pol5p DNA polymerase φ. Temperature-sensitive pol5-1∼–3 mutants did not arrest at G2/M at the restrictive temperature. Furthermore, the polymerase active-site mutant POL5dn gene complements the lethality of Δpol5. These results suggest that the polymerase activity of Pol5p is not required for the in vivo function of Pol5p. rRNA synthesis was severely inhibited at the restrictive temperature in the temperature-sensitive pol5-3 mutant cells, suggesting that an essential function of Pol5p is rRNA synthesis. Pol5p is localized exclusively to the nucleolus and binds near or at the enhancer region of rRNA-encoding DNA repeating units.

The role of the Saccharomyces cerevisiae Cdc7-Dbf4 complex in the replication checkpoint.

The Cdc7-Dbf4 complex is a conserved serine/threonine protein kinase essential for the initiation of eukaryotic DNA replication. Although an mcm5-bob1 mutation bypasses lethality conferred by mutations in CDC7 or DBF4, the Deltacdc7 mcm5-bob1 mutant is sensitive to hydroxyurea (HU), which induces replication stress. To elucidate the reasons for HU sensitivity conferred by deletion of CDC7, we examined the role of Cdc7-Dbf4 in the replication checkpoint. We found that in Cdc7-Dbf4-deficient cells exposed to replication stress, Rad53 remains in a hypophosphorylated form, anaphase spindle is elongated, and checkpoint-specific transcription is not induced. The hypophosphorylated Rad53 exhibits a low autophosphorylation activity, and recombinant Cdc7-Dbf4 phosphorylates Rad53 in vitro. These results suggest that Cdc7-Dbf4 is required for full activation of Rad53 in response to replication stress.