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John F. X. Diffley - One of the best experts on this subject based on the ideXlab platform.
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separate scfcdc4 recognition elements target Cdc6 for proteolysis in s phase and mitosis
The EMBO Journal, 2001Co-Authors: Gordon R Perkins, Lucy S Drury, John F. X. DiffleyAbstract:The Cdc6 DNA replication initiation factor is targeted for ubiquitin-mediated proteolysis by the E3 ubiquitin ligase SCFCDC4 from the end of G1 phase until mitosis in the budding yeast Saccharomyces cerevisiae. Here we describe a dominant-negative Cdc6 mutant that, when overexpressed, arrests the cell cycle by inhibiting cyclin-dependent kinases (CDKs) and, thus, prevents passage through mitosis. This mutant protein inhibits CDKs more efficiently than wild-type Cdc6, in part because it is completely refractory to SCFCDC4-mediated proteolysis late in the cell cycle and consequently accumulates to high levels. The mutation responsible for this phenotype destroys a putative CDK phosphorylation site near the middle of the Cdc6 primary amino acid sequence. We show that this site lies within a novel Cdc4-interacting domain distinct from a Cdc4-interacting site identified previously near the N-terminus of the protein. We show that both sites can target Cdc6 for proteolysis in late G1/early S phase whilst only the newly identified site can target Cdc6 for proteolysis during mitosis.
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Mutational analysis of conserved sequence motifs in the budding yeast Cdc6 protein.
Journal of Molecular Biology, 2001Co-Authors: Aloys Schepers, John F. X. DiffleyAbstract:Abstract The Cdc6 protein is required to load a complex of Mcm2-7 family members (the MCM complex) into prereplicative complexes at budding yeast origins of DNA replication. Cdc6p is a member of the AAA + superfamily of proteins, which includes the prokaryotic and eukaryotic clamp loading proteins. These proteins share a number of conserved regions of homology and a common three-dimensional architecture. Two of the conserved sequence motifs are the Walker A and B motifs that are involved in nucleotide metabolism and are essential for Cdc6p function in vivo . Here, we analyse mutants in the other conserved sequence motifs. Several of these mutants are temperature-sensitive for growth and are unable to recruit the MCM complex to chromatin at the restrictive temperature. In one such temperature-sensitive mutant, a highly conserved asparagine residue in the sensor I motif was changed to alanine. Overexpression of this mutant protein is lethal. This phenotype is very similar to the phenotype previously described for a mutation in the Walker B motif, suggesting a common role for sensor I and the Walker B motif in Cdc6 function.
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activation of s phase promoting cdks in late g1 defines a point of no return after which Cdc6 synthesis cannot promote dna replication in yeast
Genes & Development, 1996Co-Authors: Simonetta Piatti, John F. X. Diffley, T Bohm, Julie H Cocker, Kim NasmythAbstract:In eukaryotic cells, DNA replication is confined to a discrete period of the cell cycle and does not usually recur until after anaphase. In the budding yeast Saccharomyces cerevisiae, assembly of pre-replication complexes (pre-RCs) at future origins as cells exit mitosis (or later during G,) is necessary for subsequent initiation of DNA replication triggered by activation in late GI of Cdc28lCdkl kinases associated with B-type cyclins Clbl-Clb6. The absence of pre-RCs during G, and M phases could explain why origins of DNA replication fire only once during the cell cycle, even though S-phase-promoting Cdks remain active from the beginning of S phase through the end of M phase. Formation of pre-RCs and their maintenance during G, depend on the synthesis and activity of an unstable protein encoded by Cdc6. We find that Cdc6 synthesis can only promote DNA replication in a restricted window of the cell cycle: between destruction of Clbs after anaphase and activation of Clb5/ and ClbbICdkl in late GI. The latter corresponds to a "point of no return," after which Cdc6 synthesis can no longer promote DNA replication. Cdc6 protein can be made throughout the cell cycle and, in certain circumstances, can accumulate within the nuclei of G, and M phase cells without inducing re-replication. Thus, control over Cdc6 degradation and/or nuclear localization is not crucial for preventing origin re-firing. Our data are consistent with the notion that cells can no longer incorporate de novo synthesized Cdc6 into pre-RCs once ClbICdkl kinases have been activated. We show that Cdc6p associates with ClbICdkl kinases from late G, until late anaphase, which might be important for inhibiting pre-RC assembly during Sf G, , and M phases. Inhibition of pre-RC assembly by the same kinases that trigger initiation explains how origins are prevented from re-firing until Clb kinases are destroyed after anaphase.
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an essential role for the Cdc6 protein in forming the pre replicative complexes of budding yeast
Nature, 1996Co-Authors: Julie H Cocker, Simonetta Piatti, Kim Nasmyth, Corrado Santocanale, John F. X. DiffleyAbstract:ORIGINS of DNA replication in Saccharomyces cerevisiae are bound by two protein complexes during the cell cycle1,2. Post-replicative complexes closely resemble those generated in vitro by purified origin recognition complex (ORC)1,3–5, which is essential for DNA replication in vivo6–11. Pre-replicative complexes (pre-RCs) are characterized by an extended region of nuelease protection overlapping the ORC footprint1. We show here that the Cdc6 protein (Cdc6p), which is necessary for origin firing in vivo12–16, is essential for the establishment and maintenance of pre-RCs, suggesting that it is a component of these complexes. Without Cdc6p, G1 origins closely resemble post-replicative origins, providing evidence that ORC is also a component of pre-RCs. These results suggest that pre-RCs play an essential role in initiating DNA replication and support a two-step mechanism for the assembly of functional initiation complexes.
K. Helin - One of the best experts on this subject based on the ideXlab platform.
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cell cycle regulated expression of mammalian Cdc6 is dependent on e2f
Molecular and Cellular Biology, 1998Co-Authors: G. Hateboer, A. Wobst, B. O. Petersen, E. Vigo, C. Sardet, Laurent Le Cam, K. HelinAbstract:The E2F transcription factors are essential regulators of cell growth in multicellular organisms, controlling the expression of a number of genes whose products are involved in DNA replication and cell proliferation. In Saccharomyces cerevisiae, the MBF and SBF transcription complexes have functions similar to those of E2F proteins in higher eukaryotes, by regulating the timed expression of genes implicated in cell cycle progression and DNA synthesis. The Cdc6 gene is a target for MBF and SBF-regulated transcription. S. cerevisiae Cdc6p induces the formation of the prereplication complex and is essential for initiation of DNA replication. Interestingly, the Cdc6p homolog in Schizosaccharomyces pombe, Cdc18p, is regulated by DSC1, the S. pombe homolog of MBF. By cloning the promoter for the human homolog of Cdc6p and Cdc18p, we demonstrate here that the cell cycle-regulated transcription of this gene is dependent on E2F. In vivo footprinting data demonstrate that the identified E2F sites are occupied in resting cells and in exponentially growing cells, suggesting that E2F is responsible for downregulating the promoter in early phases of the cell cycle and the subsequent upregulation when cells enter S phase. Our data also demonstrate that the human Cdc6 protein (hCdc6) is essential and limiting for DNA synthesis, since microinjection of an anti-Cdc6 rabbit antiserum blocks DNA synthesis and Cdc6 cooperates with cyclin E to induce entry into S phase in cotransfection experiments. Furthermore, E2F is sufficient to induce expression of the endogenous Cdc6 gene even in the absence of de novo protein synthesis. In conclusion, our results provide a direct link between regulated progression through G1 controlled by the pRB pathway and the expression of proteins essential for the initiation of DNA replication.
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Cell cycle regulated expression of mammalian Cdc6 is dependent on E2F
Molecular and Cellular Biology, 1998Co-Authors: G. Hateboer, A. Wobst, B. O. Petersen, L. Le Cam, E. Vigo, C. Sardet, K. HelinAbstract:The E2F transcription factors are essential regulators of cell growth in multicellular organisms, controlling the expression of a number of genes whose products are involved in DNA replication and cell proliferation. In Saccharomyces cerevisiae, the MBF and SBF transcription complexes have functions similar to those of E2F proteins in higher eukaryotes, by regulating the timed expression of genes implicated in cell cycle progression and DNA synthesis. The Cdc6 gene is a target for MBF and SEP-regulated transcription. S. cerevisiae Cdc6p induces the formation of the prereplication complex and is essential for initiation of DNA replication. Interestingly, the Cdc6p homolog in Schizosaccharomyces pombe, Cdc18p, is regulated by DSC1, the S. pombe homolog of MBF. By cloning the promoter for the human homolog of Cdc6p and Cdc18p, we demonstrate here that the cell cycle-regulated transcription of this gene is dependent on E2F. In vivo footprinting data demonstrate that the identified E2F sites are occupied in resting cells and in exponentially growing cells, suggesting that E2F is responsible for downregulating the promoter in early phases of the cell cycle and the subsequent upregulation when cells enter S phase. Our data also demonstrate that the human Cdc6 protein (hCdc6) is essential and limiting for DNA synthesis, since microinjection of an anti-Cdc6 rabbit antiserum blocks DNA synthesis and Cdc6 cooperates with cyclin E to induce entry into S phase in cotransfection experiments. Furthermore, E2F is sufficient to induce expression of the endogenous Cdc6 gene even in the absence of de novo protein synthesis. In conclusion, our results provide a direct link between regulated progression through G(1) controlled by the PRB pathway and the expression of proteins essential for the initiation of DNA replication.
D Koshland - One of the best experts on this subject based on the ideXlab platform.
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addition of extra origins of replication to a minichromosome suppresses its mitotic loss in Cdc6 and cdc14 mutants of saccharomyces cerevisiae
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: E Hogan, D KoshlandAbstract:Abstract Many cell division cycle (cdc) mutants of Saccharomyces cerevisiae exhibit elevated mitotic loss of pDK243, a 14-kilobase minichromosome with a centromere and one autonomous replicating sequence (ARS). Tandem copies of different ARSs were added to pDK243. The addition of these ARS clusters to pDK243 had no effect on its mitotic loss in cdc7 (protein kinase), cdc9 (DNA ligase), or cdc16 or cdc17 (DNA polymerase) mutants. However, in Cdc6 and cdc14 mutants, the mitotic loss of pDK243 with an ARS cluster was suppressed by a factor of 6-8 compared to pDK243 without the cluster. This suppression was dependent upon the number of ARSs in the cluster and the integrity of the ARS consensus sequence in each ARS of the cluster. ARSs are known to be DNA replication origins. Therefore, the suppression of mini-chromosome loss by ARSs in Cdc6 and cdc14 mutants suggests that these mutants are defective in the initiation of DNA replication. Since the Cdc6 protein appears to act at the G1/S phase transition, the Cdc6 protein may be a factor required at the beginning of S phase to initiate DNA replication at origins. In contrast, the CDC14 protein acts after mitosis. We suggest that the CDC14 protein performs a function late in the cell cycle that may be required for efficient initiation of DNA replication during S phase of the next cell cycle.
Kim Nasmyth - One of the best experts on this subject based on the ideXlab platform.
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loading of an mcm protein onto dna replication origins is regulated by Cdc6p and cdks
Cell, 1997Co-Authors: Tomoyuki U Tanaka, Dunja Knapp, Kim NasmythAbstract:Abstract In eukaryotic cells, firing of DNA replication origins normally does not recur until after M phase. This characteristic is thought to be due to the properties of "initiation" proteins like Orc, Cdc6, and Mcms. Using formaldehyde cross-linking, we show that Cdc6p and Mcm7p associate specifically with replication origins during G1 but not during G2 in S. cerevisiae. Mcm7p's association with origins depends on Cdc6p. Ectopic expression of Cdc6p enables it to associate with origins during G2, but this fails to recruit Mcm7p. Our data suggest that the loading of Mcm proteins onto origins is regulated by two mechanisms: first, by Cdc6p occupancy, and second, by S- and M-CDKs, whose activity during S, G2, and M phases prevents Mcm loading.
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activation of s phase promoting cdks in late g1 defines a point of no return after which Cdc6 synthesis cannot promote dna replication in yeast
Genes & Development, 1996Co-Authors: Simonetta Piatti, John F. X. Diffley, T Bohm, Julie H Cocker, Kim NasmythAbstract:In eukaryotic cells, DNA replication is confined to a discrete period of the cell cycle and does not usually recur until after anaphase. In the budding yeast Saccharomyces cerevisiae, assembly of pre-replication complexes (pre-RCs) at future origins as cells exit mitosis (or later during G,) is necessary for subsequent initiation of DNA replication triggered by activation in late GI of Cdc28lCdkl kinases associated with B-type cyclins Clbl-Clb6. The absence of pre-RCs during G, and M phases could explain why origins of DNA replication fire only once during the cell cycle, even though S-phase-promoting Cdks remain active from the beginning of S phase through the end of M phase. Formation of pre-RCs and their maintenance during G, depend on the synthesis and activity of an unstable protein encoded by Cdc6. We find that Cdc6 synthesis can only promote DNA replication in a restricted window of the cell cycle: between destruction of Clbs after anaphase and activation of Clb5/ and ClbbICdkl in late GI. The latter corresponds to a "point of no return," after which Cdc6 synthesis can no longer promote DNA replication. Cdc6 protein can be made throughout the cell cycle and, in certain circumstances, can accumulate within the nuclei of G, and M phase cells without inducing re-replication. Thus, control over Cdc6 degradation and/or nuclear localization is not crucial for preventing origin re-firing. Our data are consistent with the notion that cells can no longer incorporate de novo synthesized Cdc6 into pre-RCs once ClbICdkl kinases have been activated. We show that Cdc6p associates with ClbICdkl kinases from late G, until late anaphase, which might be important for inhibiting pre-RC assembly during Sf G, , and M phases. Inhibition of pre-RC assembly by the same kinases that trigger initiation explains how origins are prevented from re-firing until Clb kinases are destroyed after anaphase.
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an essential role for the Cdc6 protein in forming the pre replicative complexes of budding yeast
Nature, 1996Co-Authors: Julie H Cocker, Simonetta Piatti, Kim Nasmyth, Corrado Santocanale, John F. X. DiffleyAbstract:ORIGINS of DNA replication in Saccharomyces cerevisiae are bound by two protein complexes during the cell cycle1,2. Post-replicative complexes closely resemble those generated in vitro by purified origin recognition complex (ORC)1,3–5, which is essential for DNA replication in vivo6–11. Pre-replicative complexes (pre-RCs) are characterized by an extended region of nuelease protection overlapping the ORC footprint1. We show here that the Cdc6 protein (Cdc6p), which is necessary for origin firing in vivo12–16, is essential for the establishment and maintenance of pre-RCs, suggesting that it is a component of these complexes. Without Cdc6p, G1 origins closely resemble post-replicative origins, providing evidence that ORC is also a component of pre-RCs. These results suggest that pre-RCs play an essential role in initiating DNA replication and support a two-step mechanism for the assembly of functional initiation complexes.
Karen A. Heichman - One of the best experts on this subject based on the ideXlab platform.
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CDC16 Controls Initiation at Chromosome Replication Origins
Molecular Cell, 1998Co-Authors: Karen A. Heichman, James M. RobertsAbstract:Abstract The Cdc28p cyclin-dependent kinase is thought to both catalyze the onset of DNA replication and prevent rereplication by blocking the reassembly of initiation complexes at replication origins. Budding yeast with mutations in the CDC16 gene represent an exception to this model, because they rereplicate DNA despite being in a G2-like arrest with continually elevated Cdc28p kinase activity. We show, in contradiction to Pichler et al. 1997, that the extra DNA that accumulates in cdc16 mutants is largely chromosomal, as we originally reported. Two-dimensional DNA electrophoresis shows that cdc16 mutants reinitiate DNA synthesis from normal chromosome replication origins, and density transfer experiments show that multiple chromosomal locations are affected. Rereplication from origins requires both Cdc6p and Cdc46/Mcm5p, initiation proteins that had been thought to be inactivated by the Cdc28p kinase. These results establish that CDC16 is required to prevent inappropriate firing of replication origins.
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Cdc6 and DNA replication: limited to humble origins.
BioEssays, 1996Co-Authors: Karen A. HeichmanAbstract:The budding yeast Cdc6 protein is important for regulating DNA replication intiation. Cdc6p acts at replication origins, and Cdc6-1 mutants arrest with unreplicated DNA and show elevated minichromosome loss rates. Overexpression of the related Cdc 18 protein in fission yeast results in DNA rereplication; however, Cdc6p overexpression does not cause this result. A recent paper(1) further defines the role of Cdc6p in DNA replication. Cdc6p only promotes DNA replication between the end of mitosis and late G1, and although the Cdc6 protein is highly unstable, neither degradation nor nuclear localization is critical for limiting DNA replication to this interval.