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内容記述 |
In Saccharomyces cerevisiae, chromosomal DNA replication initiates at a restricted region known as the autonomously replicating sequence (ARS). The origin recognition complex (Orc) binds ARS throughout the cell cycle and the minichromosome maintenance (Mcm) proteins are recruited by Cdc6 from late M phase to G<SUB>1</SUB> phase to form the pre-replicative complex (pre-RC). Then, the Sld3-Cdc45 complex joins the pre-RC and ARS region is unwound in cooperation with Cdk and Cdc7/Dbf4 protein kinases. Finally, DNA polymerases are recruited to ARS and this step requires Dpb11, which forms a complex with DNA polymerase ε (Pol ε). However, how the Dpb11-Polε complex is recruited to ARS has not been known. In this article, we describe novel replication proteins that seem to mediate between the Sld3-Cdc45 and the Dpb11 -Pol ε complexes at the initiation step of DNA replication. <br /> To gain an insight into the function of Dpb11, we have isolated sld (synthetic lethality with dpb11-1) mutations. One of the SLD genes, SLD5 encodes a 34-kDa protein, which amino acid sequence is well conserved among eukaryotic organisms and is essential for cell growth. Since original sld5-1 mutation itself did not show any defect in cell growth, we isolated four thermosensitive sld5 mutations. At the restrictive temperature, all the thermosensitive mutant cells arrested with a dumbbell-shape with a single nucleus with a DNA content between 1C and 2C, suggesting that Sld5 is required for DNA replication. Since all of them divided more than once after temperature shift up, we had not characterized them in detail.<br /> To identify factors interacting with Sld5, we isolated PSF1 (Partner of SLD five) as a multicopy suppressor of the sld5- 12thermosensitive mutation. The PSF1 gene encodes a 24-kDa protein, which amino acid sequence is well conserved among eukaryotic organisms and is essential for cell growth. To understand the function of PSF1, a thermosensitive mutation, psf1-1, was isolated and characterized. At the restrictive temperature, psf1-1 cells arrested with a dumbbell shape with a single nucleus as observed in sld5 thermosensitive mutants. To investigate whether DNA is synthesized at the restrictive temperature, psf1-1 cells were arrested in G<SUB>1</SUB> phase with α-factor and released at the restrictive temperature. DNA content of psf1-1 cells had not increased for 120 min, and then gradually reached 2C while wild-type cells reached 2C DNA content by 80 min, suggesting that Psf1 is required for an early step of DNA replication. Since cells defective in initiation of DNA replication begin to lose viability immediately after cells start budding at the restrictive temperature, we determined the point at which the cells start losing viability. At the restrictive temperature, psf1-1 cells as well as sld5-12 cells started losing viability when cells started budding. It suggests that Psf1 participates in the initiation step of DNA replication. We also arrested psf1-1 cells in S phase with HU or in M phase with nocodazole and then released them at the restrictive temperature. In both cases, cells entered subsequent G<SUB>1</SUB> phase and arrested with 1C DNA content while psf1-1 cells after release from HU-arrest reached 2C DNA content 30 min later than wild type cells. Since HU blocks late-origin firing, it is likely that Psf1 is essential for all origin-firing during S phase progression and chromosome DNA replicates only from early-firing origins in psf1-1 cells released at the restrictive temperature from HU. <br /> The transcript-level of PSF1 is reported to fluctuate during the cell cycle and to peak at G<SUB>1</SUB>/Ｓ phase boundary. We thus examined the protein-level of Psf1 by western blotting using tagged Psf1 and found that the Psf1 protein level is roughly constant during the cell cycle. Then, we determined cellular localization of the Psf1 protein during the cell cycle by indirect immuno-fluorescent microscopy and revealed that the Psf1 protein is localized in nucleus throughout the cell cycle. As Psf1 is a nuclear protein required for DNA replication, we examined whether the Psf1 protein associates with ARS region in vivo using chromatin immunoprecipitation (CHIP) assay. The CHIP assay revealed that Psf1 associates with early ARSs, ARS1 and ARS305 from 60 min after release from α-factor and 75 min with late ARS, ARS501. Psf1 also reassociated with non-ARS fragments in later period. We further showed that Sld5 associates with ARS1 at the same timing as Psf1, suggesting that Sld5 and Psf1 associate with ARS together. During the CHIP assay, we also found that Psf1 coimmunoprecipitates with Rfa when Psf1 associates with chromosome DNA. Rfa binds single-stranded DNA, which appears in unwound origins and at replication forks during DNA replication. Therefore, these results suggest that Psf1 associates with replication origins and folks. <br /> Our genetic analysis strongly suggests that Psf1 interacts with Sld5 because thermosensitive growth of psf1-1 and sld5-12 was suppressed by high-copy SLD5 and PSF1, and the psf1-1 mutation was synthetically lethal with sld5-12. Two-hybrid analysis also showed an interaction between Psf1 and Sld5. We therefore performed coimmunoprecipitation assay and demonstrated that Psf1 co-immunoprecipitates with Sld5 in G<SUB>1</SUB>, S and M phases. We further showed that the Sld5-Psf1 complex is hardly detected in psf1-1 cells. Since psf1-1 cells are defective in DNA replication, it seems likely that complex formation between Psf1 and Sld5 is required for chromosomal DNA replication. <br /> Our two-hybrid assay further showed that Psf1 interacts with Dpb11, Dpb2 and Sld3. The Sld3-Cdc45 complex associates with ARS and this association is required for unwinding of ARS region. Dpb2 is a second largest subunit of Pol ε that forms a complex with Dpb11 and associates with ARS after unwinding of ARS. Thus, Psf1 interacts with the proteins that associate with ARS before and after its unwinding. We therefore propose that the Sld5-Psf1 complex mediates between Cdc45-Sld3 and Dpb11-Pol ε complexes for recruitment of DNA polymerases to ARS. |
要旨・審査要旨 / Abstract, Screening Result (306.1 kB)
