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Conserved mechanism for coordinating replication fork helicase assembly with phosphorylation of the helicase.
Title
Conserved mechanism for coordinating replication fork helicase assembly with phosphorylation of the helicase.
Creator
Bruck, Irina, Kaplan, Daniel L
Abstract/Description
Dbf4-dependent kinase (DDK) phosphorylates minichromosome maintenance 2 (Mcm2) during S phase in yeast, and Sld3 recruits cell division cycle 45 (Cdc45) to minichromosome maintenance 2-7 (Mcm2-7). We show here DDK-phosphoryled Mcm2 preferentially interacts with Cdc45 in vivo, and that Sld3 stimulates DDK phosphorylation of Mcm2 by 11-fold. We identified a mutation of the replication initiation factor Sld3, Sld3-m16, that is specifically defective in stimulating DDK phosphorylation of Mcm2....
Show moreDbf4-dependent kinase (DDK) phosphorylates minichromosome maintenance 2 (Mcm2) during S phase in yeast, and Sld3 recruits cell division cycle 45 (Cdc45) to minichromosome maintenance 2-7 (Mcm2-7). We show here DDK-phosphoryled Mcm2 preferentially interacts with Cdc45 in vivo, and that Sld3 stimulates DDK phosphorylation of Mcm2 by 11-fold. We identified a mutation of the replication initiation factor Sld3, Sld3-m16, that is specifically defective in stimulating DDK phosphorylation of Mcm2. Wild-type expression levels of sld3-m16 result in severe growth and DNA replication defects. Cells expressing sld3-m16 exhibit no detectable Mcm2 phosphorylation in vivo, reduced replication protein A-ChIP signal at an origin, and diminished Go, Ichi, Ni, and San association with Mcm2-7. Treslin, the human homolog of Sld3, stimulates human DDK phosphorylation of human Mcm2 by 15-fold. DDK phosphorylation of human Mcm2 decreases the affinity of Mcm5 for Mcm2, suggesting a potential mechanism for helicase ring opening. These data suggest a conserved mechanism for replication initiation: Sld3/Treslin coordinates Cdc45 recruitment to Mcm2-7 with DDK phosphorylation of Mcm2 during S phase.
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Date Issued
2015-09-08
Identifier
FSU_pmch_26305950, 10.1073/pnas.1509608112, PMC4568703, 26305950, 26305950, 1509608112
Format
Citation
signaling network that silences the spindle assembly checkpoint upon the establishment of chromosome bipolar attachment.
Title
The signaling network that silences the spindle assembly checkpoint upon the establishment of chromosome bipolar attachment.
Creator
Jin, Fengzhi, Wang, Yanchang
Abstract/Description
Improper kinetochore attachments activate the spindle assembly checkpoint (SAC) to prevent anaphase onset, but it is poorly understood how this checkpoint is silenced to allow anaphase onset. Chromosome bipolar attachment applies tension on sister kinetochores, and the lack of tension delays anaphase onset. In budding yeast, the delay induced by tension defects depends on the intact SAC as well as increase in ploidy (Ipl1)/Aurora kinase and a centromere-associated protein ShuGOshin (Sgo1)....
Show moreImproper kinetochore attachments activate the spindle assembly checkpoint (SAC) to prevent anaphase onset, but it is poorly understood how this checkpoint is silenced to allow anaphase onset. Chromosome bipolar attachment applies tension on sister kinetochores, and the lack of tension delays anaphase onset. In budding yeast, the delay induced by tension defects depends on the intact SAC as well as increase in ploidy (Ipl1)/Aurora kinase and a centromere-associated protein ShuGOshin (Sgo1). Here we provide evidence indicating that Ipl1-dependent phosphorylation of the kinetochore protein Duo1 and Mps1 interacting (Dam1) prevents SAC silencing when tension is absent. The nonphosphorylatable dam1 mutant cells, as well as sgo1 mutant cells, are competent in SAC activation but unable to prevent SAC silencing in response to tension defects. We further found that phosphomimetic dam1 mutants exhibited delayed anaphase onset mainly due to the failure in SAC silencing, but destabilized kinetochore attachment likely plays a minor role in this delay. Because the tension resulting from bipolar attachment triggers the dephosphorylation of Dam1 by protein phosphatase 1, this dephosphorylation likely coordinates SAC silencing with chromosome bipolar attachment. Therefore, Sgo1, Ipl1 kinase, Dam1, and protein phosphatase 1 comprise the SAC silencing network that ensures the correct timing for anaphase onset.
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Date Issued
2013-12-24
Identifier
FSU_pmch_24324173, 10.1073/pnas.1307595111, PMC3876237, 24324173, 24324173, 1307595111
Format
Citation
Simplified protein design biased for prebiotic amino acids yields a foldable, halophilic protein.
Title
Simplified protein design biased for prebiotic amino acids yields a foldable, halophilic protein.
Creator
Longo, Liam M, Lee, Jihun, Blaber, Michael
Abstract/Description
A compendium of different types of abiotic chemical syntheses identifies a consensus set of 10 "prebiotic" α-amino acids. Before the emergence of biosynthetic pathways, this set is the most plausible resource for protein formation (i.e., proteogenesis) within the overall process of abiogenesis. An essential unsolved question regarding this prebiotic set is whether it defines a "foldable set"--that is, does it contain sufficient chemical information to permit cooperatively folding polypeptides...
Show moreA compendium of different types of abiotic chemical syntheses identifies a consensus set of 10 "prebiotic" α-amino acids. Before the emergence of biosynthetic pathways, this set is the most plausible resource for protein formation (i.e., proteogenesis) within the overall process of abiogenesis. An essential unsolved question regarding this prebiotic set is whether it defines a "foldable set"--that is, does it contain sufficient chemical information to permit cooperatively folding polypeptides? If so, what (if any) characteristic properties might such polypeptides exhibit? To investigate these questions, two "primitive" versions of an extant protein fold (the β-trefoil) were produced by top-down symmetric deconstruction, resulting in a reduced alphabet size of 12 or 13 amino acids and a percentage of prebiotic amino acids approaching 80%. These proteins show a substantial acidification of pI and require high salt concentrations for cooperative folding. The results suggest that the prebiotic amino acids do comprise a foldable set within the halophile environment.
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Date Issued
2013-02-05
Identifier
FSU_pmch_23341608, 10.1073/pnas.1219530110, PMC3568330, 23341608, 23341608, 1219530110
Format
Citation
period of the circadian oscillator is primarily determined by the balance between casein kinase 1 and protein phosphatase 1.
Title
The period of the circadian oscillator is primarily determined by the balance between casein kinase 1 and protein phosphatase 1.
Creator
Lee, Hyeong-min, Chen, Rongmin, Kim, Hyukmin, Etchegaray, Jean-Pierre, Weaver, David R, Lee, Choogon
Abstract/Description
Mounting evidence suggests that PERIOD (PER) proteins play a central role in setting the speed (period) and phase of the circadian clock. Pharmacological and genetic studies have shown that changes in PER phosphorylation kinetics are associated with changes in circadian rhythm period and phase, which can lead to sleep disorders such as Familial Advanced Sleep Phase Syndrome in humans. We and others have shown that casein kinase 1δ and ε (CK1δ/ε) are essential PER kinases, but it is clear that...
Show moreMounting evidence suggests that PERIOD (PER) proteins play a central role in setting the speed (period) and phase of the circadian clock. Pharmacological and genetic studies have shown that changes in PER phosphorylation kinetics are associated with changes in circadian rhythm period and phase, which can lead to sleep disorders such as Familial Advanced Sleep Phase Syndrome in humans. We and others have shown that casein kinase 1δ and ε (CK1δ/ε) are essential PER kinases, but it is clear that additional, unknown mechanisms are also crucial for regulating the kinetics of PER phosphorylation. Here we report that circadian periodicity is determined primarily through PER phosphorylation kinetics set by the balance between CK1δ/ε and protein phosphatase 1 (PP1). In CK1δ/ε-deficient cells, PER phosphorylation is severely compromised and nonrhythmic, and the PER proteins are constitutively cytoplasmic. However, when PP1 is disrupted, PER phosphorylation is dramatically accelerated; the same effect is not seen when PP2A is disrupted. Our work demonstrates that the speed and rhythmicity of PER phosphorylation are controlled by the balance between CK1δ/ε and PP1, which in turn determines the period of the circadian oscillator. Thus, our findings provide clear insights into the molecular basis of how the period and phase of our daily rhythms are determined.
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Date Issued
2011-09-27
Identifier
FSU_pmch_21930935, 10.1073/pnas.1107178108, PMC3182690, 21930935, 21930935, 1107178108
Format
Citation
Experimental Support for the Evolution of Symmetric Protein Architecture from a Simple Peptide Motif
Title
Experimental Support for the Evolution of Symmetric Protein Architecture from a Simple Peptide Motif.
Creator
Lee, Jihun, Blaber, Michael
Abstract/Description
The majority of protein architectures exhibit elements of structural symmetry, and "gene duplication and fusion" is the evolutionary mechanism generally hypothesized to be responsible for their emergence from simple peptide motifs. Despite the central importance of the gene duplication and fusion hypothesis, experimental support for a plausible evolutionary pathway for a specific protein architecture has yet to be effectively demonstrated. To address this question, a unique "top-down...
Show moreThe majority of protein architectures exhibit elements of structural symmetry, and "gene duplication and fusion" is the evolutionary mechanism generally hypothesized to be responsible for their emergence from simple peptide motifs. Despite the central importance of the gene duplication and fusion hypothesis, experimental support for a plausible evolutionary pathway for a specific protein architecture has yet to be effectively demonstrated. To address this question, a unique "top-down symmetric deconstruction" strategy was utilized to successfully identify a simple peptide motif capable of recapitulating, via gene duplication and fusion processes, a symmetric protein architecture (the threefold symmetric β-trefoil fold). The folding properties of intermediary forms in this deconstruction agree precisely with a previously proposed "conserved architecture" model for symmetric protein evolution. Furthermore, a route through foldable sequence-space between the simple peptide motif and extant protein fold is demonstrated. These results provide compelling experimental support for a plausible evolutionary pathway of symmetric protein architecture via gene duplication and fusion processes.
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Date Issued
2011
Identifier
FSU_migr_biomed_faculty_publications-0020, 10.1073/pnas.1015032108, PMC3017207
Format
Citation
Temporal control of the dephosphorylation of Cdk substrates by mitotic exit pathways in budding yeast.
Title
Temporal control of the dephosphorylation of Cdk substrates by mitotic exit pathways in budding yeast.
Creator
Jin, Fengzhi, Liu, Hong, Liang, Fengshan, Rizkallah, Raed, Hurt, Myra M, Wang, Yanchang
Abstract/Description
The temporal phosphorylation of cell cycle-related proteins by cyclin-dependent kinases (Cdks) is critical for the correct order of cell cycle events. In budding yeast, CDC28 encodes the only Cdk and its association with various cyclins governs the temporal phosphorylation of Cdk substrates. S-phase Cdk substrates are phosphorylated earlier than mitotic Cdk substrates, which ensures the sequential order of DNA synthesis and mitosis. However, it remains unclear whether Cdk substrates are...
Show moreThe temporal phosphorylation of cell cycle-related proteins by cyclin-dependent kinases (Cdks) is critical for the correct order of cell cycle events. In budding yeast, CDC28 encodes the only Cdk and its association with various cyclins governs the temporal phosphorylation of Cdk substrates. S-phase Cdk substrates are phosphorylated earlier than mitotic Cdk substrates, which ensures the sequential order of DNA synthesis and mitosis. However, it remains unclear whether Cdk substrates are dephosphorylated in temporally distinct windows. Cdc14 is a conserved protein phosphatase responsible for the dephosphorylation of Cdk substrates. In budding yeast, FEAR (Cdc14 early anaphase release) and MEN (mitotic exit network) activate phosphatase Cdc14 by promoting its release from the nucleolus in early and late anaphase, respectively. Here, we show that the sequential Cdc14 release and the distinct degradation timing of different cyclins provides the molecular basis for the differential dephosphorylation windows of S-phase and mitotic cyclin substrates. Our data also indicate that FEAR-induced dephosphorylation of S-phase Cdk substrates facilitates anaphase progression, revealing an extra layer of mitotic regulation.
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Date Issued
2008-10-21
Identifier
FSU_pmch_18845678, 10.1073/pnas.0808719105, PMC2570984, 18845678, 18845678, 0808719105
Format
Citation
Pds1/Esp1-dependent and -independent sister chromatid separation in mutants defective for protein phosphatase 2A.
Title
Pds1/Esp1-dependent and -independent sister chromatid separation in mutants defective for protein phosphatase 2A.
Creator
Tang, Xianying, Wang, Yanchang
Abstract/Description
Spindle disruption or DNA damage prevents sister chromatid separation through the activation of checkpoint pathways that inhibit anaphase entry by stabilizing the anaphase inhibitor Pds1. Mutation of CDC55, which encodes a B regulatory subunit of protein phosphatase 2A (PP2A), results in precocious sister chromatid separation when spindle is disrupted. Here we report that decreased Pds1 levels in Deltacdc55 mutants contribute to sister chromatid separation in the presence of nocodazole, a...
Show moreSpindle disruption or DNA damage prevents sister chromatid separation through the activation of checkpoint pathways that inhibit anaphase entry by stabilizing the anaphase inhibitor Pds1. Mutation of CDC55, which encodes a B regulatory subunit of protein phosphatase 2A (PP2A), results in precocious sister chromatid separation when spindle is disrupted. Here we report that decreased Pds1 levels in Deltacdc55 mutants contribute to sister chromatid separation in the presence of nocodazole, a microtubule-depolymerizing drug. However, in the presence of DNA damage, Deltacdc55 mutant cells separate sister chromatids without noticeable decrease of Pds1 or cohesin Mcd1/Scc1 levels. Further analysis demonstrates that Deltacdc55 mutants lose cohesion along the entire chromosomes when the spindle is disrupted. In contrast, separation of sister chromatids is limited to the centromeric regions in Deltacdc55 cells after DNA damage. Moreover, mutation of TPD3, which encodes the A regulatory subunit of PP2A, also results in sister chromatid separation in DNA- or spindle-damage-arrested cells. These data suggest that PP2A regulates sister chromatid cohesion in Pds1-dependent and -independent manners.
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Date Issued
2006-10-31
Identifier
FSU_pmch_17050679, 10.1073/pnas.0607856103, PMC1637575, 17050679, 17050679, 0607856103
Format
Citation