Abstract
The inhibition of DNA synthesis prevents mitotic entry through the action of the S phase checkpoint. In the yeast Saccharomyces cerevisiae, an essential protein kinase, Spk1/Mec2/Rad53/Sad1, controls the coupling of S phase to mitosis. In an attempt to identify genes that genetically interact with Spk1, we have isolated a temperature-sensitive mutation, rfc5-1, that can be suppressed by overexpression of SPK1. The RFC5 gene encodes a small subunit of replication factor C complex. At the restrictive temperature, rfc5-1 mutant cells entered mitosis with unevenly separated or fragmented chromosomes, resulting in loss of viability. Thus, the rfc5 mutation defective for DNA replication is also impaired in the S phase checkpoint. Overexpression of POL30, which encodes the proliferating cell nuclear antigen, suppressed the replication defect of the rfc5 mutant but not its checkpoint defect. Taken together, these results suggested that replication factor C has a direct role in sensing the state of DNA replication and transmitting the signal to the checkpoint machinery.
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Selected References
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- Baker T. A., Kremenstova E., Luo L. Complete transposition requires four active monomers in the mu transposase tetramer. Genes Dev. 1994 Oct 15;8(20):2416–2428. doi: 10.1101/gad.8.20.2416. [DOI] [PubMed] [Google Scholar]
- Bauer G. A., Burgers P. M. Molecular cloning, structure and expression of the yeast proliferating cell nuclear antigen gene. Nucleic Acids Res. 1990 Jan 25;18(2):261–265. doi: 10.1093/nar/18.2.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Burgers P. M. Saccharomyces cerevisiae replication factor C. II. Formation and activity of complexes with the proliferating cell nuclear antigen and with DNA polymerases delta and epsilon. J Biol Chem. 1991 Nov 25;266(33):22698–22706. [PubMed] [Google Scholar]
- Cullmann G., Fien K., Kobayashi R., Stillman B. Characterization of the five replication factor C genes of Saccharomyces cerevisiae. Mol Cell Biol. 1995 Sep;15(9):4661–4671. doi: 10.1128/mcb.15.9.4661. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hartwell L. H., Weinert T. A. Checkpoints: controls that ensure the order of cell cycle events. Science. 1989 Nov 3;246(4930):629–634. doi: 10.1126/science.2683079. [DOI] [PubMed] [Google Scholar]
- Hennessy K. M., Lee A., Chen E., Botstein D. A group of interacting yeast DNA replication genes. Genes Dev. 1991 Jun;5(6):958–969. doi: 10.1101/gad.5.6.958. [DOI] [PubMed] [Google Scholar]
- Howell E. A., McAlear M. A., Rose D., Holm C. CDC44: a putative nucleotide-binding protein required for cell cycle progression that has homology to subunits of replication factor C. Mol Cell Biol. 1994 Jan;14(1):255–267. doi: 10.1128/mcb.14.1.255. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee S. H., Kwong A. D., Pan Z. Q., Hurwitz J. Studies on the activator 1 protein complex, an accessory factor for proliferating cell nuclear antigen-dependent DNA polymerase delta. J Biol Chem. 1991 Jan 5;266(1):594–602. [PubMed] [Google Scholar]
- Li J. J., Deshaies R. J. Exercising self-restraint: discouraging illicit acts of S and M in eukaryotes. Cell. 1993 Jul 30;74(2):223–226. doi: 10.1016/0092-8674(93)90413-k. [DOI] [PubMed] [Google Scholar]
- Li X., Burgers P. M. Cloning and characterization of the essential Saccharomyces cerevisiae RFC4 gene encoding the 37-kDa subunit of replication factor C. J Biol Chem. 1994 Aug 26;269(34):21880–21884. [PubMed] [Google Scholar]
- Li X., Burgers P. M. Molecular cloning and expression of the Saccharomyces cerevisiae RFC3 gene, an essential component of replication factor C. Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):868–872. doi: 10.1073/pnas.91.3.868. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McAlear M. A., Howell E. A., Espenshade K. K., Holm C. Proliferating cell nuclear antigen (pol30) mutations suppress cdc44 mutations and identify potential regions of interaction between the two encoded proteins. Mol Cell Biol. 1994 Jul;14(7):4390–4397. doi: 10.1128/mcb.14.7.4390. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Murray A. W. Creative blocks: cell-cycle checkpoints and feedback controls. Nature. 1992 Oct 15;359(6396):599–604. doi: 10.1038/359599a0. [DOI] [PubMed] [Google Scholar]
- Navas T. A., Zhou Z., Elledge S. J. DNA polymerase epsilon links the DNA replication machinery to the S phase checkpoint. Cell. 1995 Jan 13;80(1):29–39. doi: 10.1016/0092-8674(95)90448-4. [DOI] [PubMed] [Google Scholar]
- Noskov V., Maki S., Kawasaki Y., Leem S. H., Ono B., Araki H., Pavlov Y., Sugino A. The RFC2 gene encoding a subunit of replication factor C of Saccharomyces cerevisiae. Nucleic Acids Res. 1994 May 11;22(9):1527–1535. doi: 10.1093/nar/22.9.1527. [DOI] [PMC free article] [PubMed] [Google Scholar]
- O'Donnell M., Onrust R., Dean F. B., Chen M., Hurwitz J. Homology in accessory proteins of replicative polymerases--E. coli to humans. Nucleic Acids Res. 1993 Jan 11;21(1):1–3. doi: 10.1093/nar/21.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pai E. F., Kabsch W., Krengel U., Holmes K. C., John J., Wittinghofer A. Structure of the guanine-nucleotide-binding domain of the Ha-ras oncogene product p21 in the triphosphate conformation. Nature. 1989 Sep 21;341(6239):209–214. doi: 10.1038/341209a0. [DOI] [PubMed] [Google Scholar]
- Savitsky K., Bar-Shira A., Gilad S., Rotman G., Ziv Y., Vanagaite L., Tagle D. A., Smith S., Uziel T., Sfez S. A single ataxia telangiectasia gene with a product similar to PI-3 kinase. Science. 1995 Jun 23;268(5218):1749–1753. doi: 10.1126/science.7792600. [DOI] [PubMed] [Google Scholar]
- Stern D. F., Zheng P., Beidler D. R., Zerillo C. Spk1, a new kinase from Saccharomyces cerevisiae, phosphorylates proteins on serine, threonine, and tyrosine. Mol Cell Biol. 1991 Feb;11(2):987–1001. doi: 10.1128/mcb.11.2.987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sugimoto K., Sakamoto Y., Takahashi O., Matsumoto K. HYS2, an essential gene required for DNA replication in Saccharomyces cerevisiae. Nucleic Acids Res. 1995 Sep 11;23(17):3493–3500. doi: 10.1093/nar/23.17.3493. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tsurimoto T., Stillman B. Replication factors required for SV40 DNA replication in vitro. I. DNA structure-specific recognition of a primer-template junction by eukaryotic DNA polymerases and their accessory proteins. J Biol Chem. 1991 Jan 25;266(3):1950–1960. [PubMed] [Google Scholar]
- Weinert T. A., Hartwell L. H. Cell cycle arrest of cdc mutants and specificity of the RAD9 checkpoint. Genetics. 1993 May;134(1):63–80. doi: 10.1093/genetics/134.1.63. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weinert T. A., Hartwell L. H. The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cerevisiae. Science. 1988 Jul 15;241(4863):317–322. doi: 10.1126/science.3291120. [DOI] [PubMed] [Google Scholar]
- Weinert T. A., Kiser G. L., Hartwell L. H. Mitotic checkpoint genes in budding yeast and the dependence of mitosis on DNA replication and repair. Genes Dev. 1994 Mar 15;8(6):652–665. doi: 10.1101/gad.8.6.652. [DOI] [PubMed] [Google Scholar]