WITHDRAWN: The MCM2p-binding is crucial for the function of CDC7-DBF4 protein kinase during the initiation of chromosomal DNA replication in budding yeast.
This manuscript was withdrawn at the request of the authors.
Journal Article↗
Biomedical subjects
Yasuo Kawasaki
Publications and source records attributed to Yasuo Kawasaki.
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.
Cell Cycle Proteins↗
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 Pol delta and Pol epsilon, suggesting that it may play a role in elongation replication. RESULTS: We show that the mcm10 mutation causes replication fork pausing not only at potentially active origins but also at silent origins. We screened for mutations that are lethal in combination with mcm10-1 and obtained seven mutants named slm1-slm6 for synthetically lethal with mcm10. These mutants comprised six complementation groups that can be divided into three classes. Class 1 includes genes that encode components of the pre-RC and pre-IC and are represented by SLM3, 4 and 5 which are allelic to MCM7, MCM2 and CDC45, respectively. Class 2 includes genes involved in the processing of Okazaki fragments in lagging strand synthesis and is represented by SLM1, which is allelic to DNA2. Class 3 includes novel DNA repair genes represented by SLM2 and SLM6. CONCLUSIONS: The viability of the mcm10-1 mutant is dependent on a novel repair pathway that may participate either in resolving accumulated replication intermediates or the damage caused by blocked replication forks. These results are consistent with the hypothesis that Mcm10p is required for the passage of replication forks through obstacles such as those created by pre-RCs assembled at active or inactive replication origins.
Carrier Proteins↗
The fifth essential DNA polymerase phi 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 phi. Temperature-sensitive pol5-1-- -3 mutants did not arrest at G(2)/M at the restrictive temperature. Furthermore, the polymerase active-site mutant POL5dn gene complements the lethality of Delta pol5. 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.
Amino Acid Sequence↗
The DNA polymerase domain of pol(epsilon) is required for rapid, efficient, and highly accurate chromosomal DNA replication, telomere length maintenance, and normal cell senescence in Saccharomyces cerevisiae.
Saccharomyces cerevisiae POL2 encodes the catalytic subunit of DNA polymerase epsilon. 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 epsilon 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 delta in a haploid cell. These results suggest that the catalytic activity of DNA polymerase epsilon participates in the same pathway as DNA polymerase delta, and this is consistent with the observation that DNA polymerases delta and epsilon colocalize in some punctate foci on yeast chromatids during S phase. The pol2-16 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.
Base Sequence↗