Cyclin-Specific Control of Ribosomal DNA Segregation

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Molecular and Cellular Biology (Impact Factor: 4.78). 07/2008; 28(17):5328-36. DOI: 10.1128/MCB.00235-08
Source: PubMed


Following chromosome duplication in S phase of the cell cycle, the sister chromatids are linked by cohesin. At the onset of anaphase, separase cleaves cohesin and thereby initiates sister chromatid separation. Separase activation results from the destruction of its inhibitor, securin, which is triggered by a ubiquitin ligase called the anaphase-promoting complex (APC). Here, we show in budding yeast that securin destruction and, thus, separase activation are not sufficient for the efficient segregation of the repetitive ribosomal DNA (rDNA). We find that rDNA segregation also requires the APC-mediated destruction of the S-phase cyclin Clb5, an activator of the protein kinase Cdk1. Mutations that prevent Clb5 destruction are lethal and cause defects in rDNA segregation and DNA synthesis. These defects are distinct from the mitotic-exit defects caused by stabilization of the mitotic cyclin Clb2, emphasizing the importance of cyclin specificity in the regulation of late-mitotic events. Efficient rDNA segregation, both in mitosis and meiosis, also requires APC-dependent destruction of Dbf4, an activator of the protein kinase Cdc7. We speculate that the dephosphorylation of Clb5-specific Cdk1 substrates and Dbf4-Cdc7 substrates drives the resolution of rDNA in early anaphase. The coincident destruction of securin, Clb5, and Dbf4 coordinates bulk chromosome segregation with segregation of rDNA.

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    • "This would provide a molecular mechanism to explain how the APC controls lifespan and how the cell overcomes Fob1's potential negative effects on rDNA recombination and longevity. Recent work has demonstrated that the APC is required for rDNA silencing, assembly, and segregation in budding and fission yeast (Sullivan et al. 2008; Dubey et al. 2009; Rodríguez-Sánchez et al. 2011). Thus, the identification of Fob1 as a novel APC target would provide insight into how the APC may influence rDNA biology. "
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    ABSTRACT: Genomic stability, stress response and nutrient signaling all play critical, evolutionarily conserved roles in lifespan determination. However, the molecular mechanisms coordinating these processes with longevity remain unresolved. Here we investigate the involvement of the yeast Anaphase Promoting Complex (APC) in longevity. The APC governs passage through M and G1 via ubiquitin-dependent targeting of substrate proteins, and is associated with cancer and premature aging when defective. Our 2-hybrid screen utilizing Apc5 as bait recovered the lifespan determinant Fob1 as prey. Fob1 is unstable specifically in G1, cycles throughout the cell cycle in a manner similar to Clb2 (an APC target), and is stabilized in APC (apc5(CA)) and proteasome (rpn10) mutants. Deletion of FOB1 increased RLS in WT, apc5(CA) and apc10 cells, and suppressed apc5(CA) cell cycle progression and rDNA recombination defects. Alternatively, increased FOB1 expression decreased RLS in WT cells, but did not reduce the already short apc5(CA) RLS, suggesting an epistatic interaction between apc5(CA) and fob1. Mutation to a putative L-Box (Fob1(E420V)), a Destruction Box-like motif, abolished Fob1 modifications, stabilized the protein, and increased rDNA recombination. Our work provides a mechanistic role played by the APC to promote replicative longevity and genomic stability in yeast.
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    • "The MII arrest is partly (but not completely) rescued by rec8Δ, indicating that diploids homozygous for dfp1-r35 have additional defects beyond difficulties in regulating Rec8. These likely include disruptions in recombination and the transcriptional program, as we observe, or changes in chromosome or rDNA segregation as reported by Bailis and Forsburg, and Sullivan et al. (Bailis and Forsburg, 2004; Sullivan et al., 2008). "
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    ABSTRACT: The DDK complex is a conserved kinase complex, consisting of a catalytic subunit, Hsk1 (Cdc7), and its regulatory subunit Dfp1 (Dbf4). This kinase is essential for DNA replication. In this work, we show that dfp1-r35, which truncates the Dfp1 C-terminus zinc finger, causes severe meiotic defects, including reduced spore viability, reduced formation of programmed double strand breaks, altered expression of meiotic genes, and disrupted chromosome segregation. There is a high frequency of dyad formation. Mutants are also defective in the phosphorylation and degradation of the meiotic cohesion, Rec8, resulting in a failure to proceed through the MII division. These defects are more pronounced in a haploid meiosis model than in a normal diploid meiosis. Thus, several critical meiotic functions are linked specifically to the C-terminus of Dfp1, which may target specific substrates for phosphorylation by Hsk1.
    Full-text · Article · Jul 2013 · Biology Open
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    • "We attempted to test the meiotic function of Clb5 lacking its amino-terminal 96 amino acids (which includes the destruction box sequence ); however, despite repeated attempts, we were unable to recover viable strains that had correctly installed this truncation at the CLB5 locus. Clb5 lacking amino acids 1–95 was previously found to be lethal to mitotically proliferating cells (Sullivan et al. 2008), and we interpret our inability to construct this strain to reflect the toxicity imposed by stabilized Clb5. We were able to construct strains harboring a Clb5–Clb3 fusion lacking amino acid residues 1–96 (C53DT2, see Figure 4A). "
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    ABSTRACT: The Saccharomyces cerevisiae cyclin Clb5 is required for premeiotic S phase, meiotic recombination, and successful progression through meiosis. Clb5 is not essential for mitotic proliferation because Clb1-Clb4 can support DNA replication in clb5 clb6 mutants. Clb1, Clb3, and Clb4 accumulate in clb5 clb6 cells during meiotic differentiation yet fail to promote premeiotic DNA replication. When expressed under the regulation of the CLB5 promoter, Clb1 and Clb3 accumulate and are active in the early stages of meiotic differentiation but cannot induce premeiotic DNA replication, suggesting that they do not target Cdk1 to the necessary substrates. The Clb5 hydrophobic patch (HP) residues are important for Clb5 function but this motif alone does not provide the specificity required for Clb5 to induce premeiotic S phase. Domain exchange experiments demonstrated that the amino terminus of Clb5 when fused to Clb3 confers upon Clb3 the ability to induce premeiotic S phase. Chimeric cyclins containing smaller regions of the Clb5 amino terminus displayed reduced ability to activate premeiotic DNA replication despite being more abundant and having greater associated histone H1 kinase activity than endogenous Clb5. These observations suggest that Clb5 has a unique ability to trigger premeiotic S phase and that the amino-terminal region of Clb5 contributes to its specificity and regulates the functions performed by the cyclin-Cdk complex.
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