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Title: Premature Silencing of the Spindle Assembly Checkpoint Is Prevented by the Bub1-H2A-Sgo1-PP2A Axis in .
Creator: Jin, Fengzhi, Bokros, Michael, Wang, Yanchang
Abstract/Description: The spindle assembly checkpoint (SAC) monitors mistakes in kinetochore-microtubule interaction and its activation prevents anaphase entry. The SAC remains active until all chromosomes have achieved bipolar attachment which applies tension on kinetochores. Our previous data in budding yeast show that Ipl1/Aurora B kinase and a centromere-associated protein, Sgo1, are required to prevent SAC silencing prior to tension generation, but we believe that this regulatory network is incomplete. Bub1...
Show moreThe spindle assembly checkpoint (SAC) monitors mistakes in kinetochore-microtubule interaction and its activation prevents anaphase entry. The SAC remains active until all chromosomes have achieved bipolar attachment which applies tension on kinetochores. Our previous data in budding yeast show that Ipl1/Aurora B kinase and a centromere-associated protein, Sgo1, are required to prevent SAC silencing prior to tension generation, but we believe that this regulatory network is incomplete. Bub1 kinase is one of the SAC components, and Bub1-dependent H2A phosphorylation triggers centromere recruitment of Sgo1 by H2A in yeast and human cells. Although yeast cells lacking the kinase domain of Bub1 show competent SAC activation, we found that the mutant cells fail to maintain a prolonged checkpoint arrest in the presence of tensionless attachment. Mutation of the Bub1 phosphorylation site in H2A also results in premature SAC silencing in yeast cells. Previous data indicate that Sgo1 protein binds to PP2A, and we found that Δ mutants exhibited premature SAC silencing as well. We further revealed that mutants with abolished binding to H2A or PP2A displayed premature SAC silencing. Together, our results suggest that, in budding yeast , the Bub1-H2A-Sgo1-PP2A axis prevents SAC silencing and helps prolonged checkpoint arrest prior to tension establishment at kinetochores.
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Date Issued: 2017-03-01
Identifier: FSU_pmch_28040741, 10.1534/genetics.116.195727, PMC5340331, 28040741, 28040741, genetics.116.195727
Format: Citation

Title: An Mcm10 Mutant Defective in ssDNA Binding Shows Defects in DNA Replication Initiation.
Creator: Perez-Arnaiz, Patricia, Kaplan, Daniel L
Abstract/Description: Mcm10 is an essential protein that functions to initiate DNA replication after the formation of the replication fork helicase. In this manuscript, we identified a budding yeast Mcm10 mutant (Mcm10-m2,3,4) that is defective in DNA binding in vitro. Moreover, this Mcm10-m2,3,4 mutant does not stimulate the phosphorylation of Mcm2 by Dbf4-dependent kinase (DDK) in vitro. When we expressed wild-type levels of mcm10-m2,3,4 in budding yeast cells, we observed a severe growth defect and a...
Show moreMcm10 is an essential protein that functions to initiate DNA replication after the formation of the replication fork helicase. In this manuscript, we identified a budding yeast Mcm10 mutant (Mcm10-m2,3,4) that is defective in DNA binding in vitro. Moreover, this Mcm10-m2,3,4 mutant does not stimulate the phosphorylation of Mcm2 by Dbf4-dependent kinase (DDK) in vitro. When we expressed wild-type levels of mcm10-m2,3,4 in budding yeast cells, we observed a severe growth defect and a substantially decreased DNA replication. We also observed a substantially reduced replication protein A- chromatin immunoprecipitation signal at origins of replication, reduced levels of DDK-phosphorylated Mcm2, and diminished Go, Ichi, Ni, and San (GINS) association with Mcm2-7 in vivo. mcm5-bob1 bypasses the growth defect conferred by DDK-phosphodead Mcm2 in budding yeast. However, the growth defect observed by expressing mcm10-m2,3,4 is not bypassed by the mcm5-bob1 mutation. Furthermore, origin melting and GINS association with Mcm2-7 are substantially decreased for cells expressing mcm10-m2,3,4 in the mcm5-bob1 background. Thus, the origin melting and GINS-Mcm2-7 interaction defects we observed for mcm10-m2,3,4 are not explained by decreased Mcm2 phosphorylation by DDK, since the defects persist in an mcm5-bob1 background. These data suggest that DNA binding by Mcm10 is essential for the initiation of DNA replication.
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Date Issued: 2016-11-20
Identifier: FSU_pmch_27751725, 10.1016/j.jmb.2016.10.014, PMC5115986, 27751725, 27751725, S0022-2836(16)30429-6
Format: Citation

Title: The Replication Initiation Protein Sld3/Treslin Orchestrates the Assembly of the Replication Fork Helicase during S Phase.
Creator: Bruck, Irina, Kaplan, Daniel L
Abstract/Description: The initiation of DNA replication is a highly regulated process in eukaryotic cells, and central to the process of initiation is the assembly and activation of the replication fork helicase. The replication fork helicase is comprised of CMG (Cdc45, Mcm2-7, and GINS) in eukaryotic cells, and the mechanism underlying assembly of the CMG during S phase was studied in this article. We identified a point mutation of Sld3 that is specifically defective for Mcm3 and Mcm5 interaction (sld3-m10), and...
Show moreThe initiation of DNA replication is a highly regulated process in eukaryotic cells, and central to the process of initiation is the assembly and activation of the replication fork helicase. The replication fork helicase is comprised of CMG (Cdc45, Mcm2-7, and GINS) in eukaryotic cells, and the mechanism underlying assembly of the CMG during S phase was studied in this article. We identified a point mutation of Sld3 that is specifically defective for Mcm3 and Mcm5 interaction (sld3-m10), and also identified a point mutation of Sld3 that is specifically defective for single-stranded DNA (ssDNA) interaction (sld3-m9). Expression of wild-type levels of sld3-m9 resulted in a severe DNA replication defect with no recruitment of GINS to Mcm2-7, whereas expression of wild-type levels of sld3-m10 resulted in a severe replication defect with no Cdc45 recruitment to Mcm2-7. We propose a model for Sld3-mediated control of replication initiation, wherein Sld3 manages the proper assembly of the CMG during S phase. We also find that the biochemical functions identified for Sld3 are conserved in human Treslin, suggesting that Treslin orchestrates assembly of the CMG in human cells.
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Date Issued: 2015-11-06
Identifier: FSU_pmch_26405041, 10.1074/jbc.M115.688424, PMC4646389, 26405041, 26405041, M115.688424
Format: Citation

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

Title: Replicative stress induces intragenic transcription of the ASE1 gene that negatively regulates Ase1 activity.
Creator: McKnight, Kelly, Liu, Hong, Wang, Yanchang
Abstract/Description: Intragenic transcripts initiate within the coding region of a gene, thereby producing shorter mRNAs and proteins. Although intragenic transcripts are widely expressed [1], their role in the functional regulation of genes remains largely unknown. In budding yeast, DNA replication stress activates the S phase checkpoint that stabilizes replication forks and arrests cells in S phase with a short spindle [2-4]. When yeast cells were treated with hydroxyurea (HU) to block DNA synthesis and induce...
Show moreIntragenic transcripts initiate within the coding region of a gene, thereby producing shorter mRNAs and proteins. Although intragenic transcripts are widely expressed [1], their role in the functional regulation of genes remains largely unknown. In budding yeast, DNA replication stress activates the S phase checkpoint that stabilizes replication forks and arrests cells in S phase with a short spindle [2-4]. When yeast cells were treated with hydroxyurea (HU) to block DNA synthesis and induce replication stress, we found that Ase1, a conserved spindle midzone protein [5], appeared as two short protein isoforms in addition to the full-length protein. We further demonstrated that the short isoforms result from intragenic transcription of ASE1, which depends on the S phase checkpoint. Blocking generation of the short isoforms leads to a destabilized S phase spindle, characterized by increased spindle dynamics and frequent spindle collapse. Because the short Ase1 isoforms localize at the spindle in HU-treated cells and overexpression of the short Ase1 isoforms impairs the spindle midzone localization of full-length Ase1, it is likely that the presence of short Ase1 isoforms stabilizes the spindle by antagonizing full-length Ase1. Together, our results reveal intragenic transcription as a unique mechanism to downregulate gene functions in response to DNA replication stress.
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Date Issued: 2014-05-19
Identifier: FSU_pmch_24768052, 10.1016/j.cub.2014.03.040, PMC4031280, 24768052, 24768052, S0960-9822(14)00332-7
Format: Citation

Title: The replication initiation protein Sld2 regulates helicase assembly.
Creator: Bruck, Irina, Kaplan, Daniel L
Abstract/Description: Assembly of the Cdc45-Mcm2-7-GINS (CMG) replicative helicase complex must be regulated to ensure that DNA unwinding is coupled with DNA synthesis. Sld2 is required for the initiation of DNA replication in budding yeast. We identified a mutant of Sld2, Sld2-m1,4, that is specifically defective in Mcm2-7 binding. When this sld2-m1,4 mutant is expressed, cells exhibit severe inhibition of DNA replication. Furthermore, the CMG complex assembles prematurely in G1 in mutant cells, but not wild-type...
Show moreAssembly of the Cdc45-Mcm2-7-GINS (CMG) replicative helicase complex must be regulated to ensure that DNA unwinding is coupled with DNA synthesis. Sld2 is required for the initiation of DNA replication in budding yeast. We identified a mutant of Sld2, Sld2-m1,4, that is specifically defective in Mcm2-7 binding. When this sld2-m1,4 mutant is expressed, cells exhibit severe inhibition of DNA replication. Furthermore, the CMG complex assembles prematurely in G1 in mutant cells, but not wild-type cells. These data suggest that Sld2 binding to Mcm2-7 is essential to block the inappropriate formation of a CMG helicase complex in G1. We also study a mutant of Sld2 that is defective in binding DNA, sld2-DNA, and find that sld2-DNA cells exhibit no GINS-Mcm2-7 interaction. These data suggest that Sld2 association with DNA is required for CMG assembly in S phase.
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Date Issued: 2014-01-24
Identifier: FSU_pmch_24307213, 10.1074/jbc.M113.532085, PMC3900945, 24307213, 24307213, M113.532085
Format: Citation

Title: Slk19 clusters kinetochores and facilitates chromosome bipolar attachment.
Creator: Richmond, Daniel, Rizkallah, Raed, Liang, Fengshan, Hurt, Myra M, Wang, Yanchang
Abstract/Description: In all eukaryotic cells, DNA is packaged into multiple chromosomes that are linked to microtubules through a large protein complex called a kinetochore. Previous data show that the kinetochores are clustered together during most of the cell cycle, but the mechanism and the biological significance of kinetochore clustering are unknown. As a kinetochore protein in budding yeast, the role of Slk19 in the stability of the anaphase spindle has been well studied, but its function in chromosome...
Show moreIn all eukaryotic cells, DNA is packaged into multiple chromosomes that are linked to microtubules through a large protein complex called a kinetochore. Previous data show that the kinetochores are clustered together during most of the cell cycle, but the mechanism and the biological significance of kinetochore clustering are unknown. As a kinetochore protein in budding yeast, the role of Slk19 in the stability of the anaphase spindle has been well studied, but its function in chromosome segregation has remained elusive. Here we show that Slk19 is required for kinetochore clustering when yeast cells are treated with the microtubule-depolymerizing agent nocodazole. We further find that slk19Δ mutant cells exhibit delayed kinetochore capture and chromosome bipolar attachment after the disruption of the kinetochore-microtubule interaction by nocodazole, which is likely attributed to defective kinetochore clustering. In addition, we show that Slk19 interacts with itself, suggesting that the dimerization of Slk19 may mediate the interaction between kinetochores for clustering. Therefore Slk19 likely acts as kinetochore glue that clusters kinetochores to facilitate efficient and faithful chromosome segregation.
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Date Issued: 2013-03-01
Identifier: FSU_pmch_23283988, 10.1091/mbc.E12-07-0552, PMC3583661, 23283988, 23283988, mbc.E12-07-0552
Format: Citation

Title: Coordination of chromatid separation and spindle elongation by antagonistic activities of mitotic and S-phase CDKs.
Creator: Liang, Fengshan, Richmond, Daniel, Wang, Yanchang
Abstract/Description: Because cohesion prevents sister-chromatid separation and spindle elongation, cohesion dissolution may trigger these two events simultaneously. However, the relatively normal spindle elongation kinetics in yeast cohesin mutants indicates an additional mechanism for the temporal control of spindle elongation. Here we show evidence indicating that S-phase CDK (cyclin dependent kinase) negatively regulates spindle elongation. In contrast, mitotic CDK promotes spindle elongation by activating...
Show moreBecause cohesion prevents sister-chromatid separation and spindle elongation, cohesion dissolution may trigger these two events simultaneously. However, the relatively normal spindle elongation kinetics in yeast cohesin mutants indicates an additional mechanism for the temporal control of spindle elongation. Here we show evidence indicating that S-phase CDK (cyclin dependent kinase) negatively regulates spindle elongation. In contrast, mitotic CDK promotes spindle elongation by activating Cdc14 phosphatase, which reverses the protein phosphorylation imposed by S-phase CDK. Our data suggest that S-phase CDK negatively regulates spindle elongation partly through its phosphorylation of a spindle pole body (SPB) protein Spc110. We also show that hyperactive S-phase CDK compromises the microtubule localization of Stu2, a processive microtubule polymerase essential for spindle elongation. Strikingly, we found that hyperactive mitotic CDK induces uncoupled spindle elongation and sister-chromatid separation in securin mutants (pds1Δ), and we speculate that asynchronous chromosome segregation in pds1Δ cells contributes to this phenotype. Therefore, the tight temporal control of spindle elongation and cohesin cleavage assure orchestrated chromosome separation and spindle elongation.
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Date Issued: 2013-01-01
Identifier: FSU_pmch_23468650, 10.1371/journal.pgen.1003319, PMC3584997, 23468650, 23468650, PGENETICS-D-12-02045
Format: Citation

Title: Loss of function of the Cik1/Kar3 motor complex results in chromosomes with syntelic attachment that are sensed by the tension checkpoint.
Creator: Jin, Fengzhi, Liu, Hong, Li, Ping, Yu, Hong-Guo, Wang, Yanchang
Abstract/Description: The attachment of sister kinetochores by microtubules emanating from opposite spindle poles establishes chromosome bipolar attachment, which generates tension on chromosomes and is essential for sister-chromatid segregation. Syntelic attachment occurs when both sister kinetochores are attached by microtubules from the same spindle pole and this attachment is unable to generate tension on chromosomes, but a reliable method to induce syntelic attachments is not available in budding yeast. The...
Show moreThe attachment of sister kinetochores by microtubules emanating from opposite spindle poles establishes chromosome bipolar attachment, which generates tension on chromosomes and is essential for sister-chromatid segregation. Syntelic attachment occurs when both sister kinetochores are attached by microtubules from the same spindle pole and this attachment is unable to generate tension on chromosomes, but a reliable method to induce syntelic attachments is not available in budding yeast. The spindle checkpoint can sense the lack of tension on chromosomes as well as detached kinetochores to prevent anaphase onset. In budding yeast Saccharomyces cerevisiae, tension checkpoint proteins Aurora/Ipl1 kinase and centromere-localized Sgo1 are required to sense the absence of tension but are dispensable for the checkpoint response to detached kinetochores. We have found that the loss of function of a motor protein complex Cik1/Kar3 in budding yeast leads to syntelic attachments. Inactivation of either the spindle or tension checkpoint enables premature anaphase entry in cells with dysfunctional Cik1/Kar3, resulting in co-segregation of sister chromatids. Moreover, the abolished Kar3-kinetochore interaction in cik1 mutants suggests that the Cik1/Kar3 complex mediates chromosome movement along microtubules, which could facilitate bipolar attachment. Therefore, we can induce syntelic attachments in budding yeast by inactivating the Cik1/Kar3 complex, and this approach will be very useful to study the checkpoint response to syntelic attachments.
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Date Issued: 2012-02-01
Identifier: FSU_pmch_22319456, 10.1371/journal.pgen.1002492, PMC3271067, 22319456, 22319456, PGENETICS-D-11-01695
Format: Citation

Title: Novel E3 ubiquitin ligases that regulate histone protein levels in the budding yeast Saccharomyces cerevisiae.
Creator: Singh, Rakesh Kumar, Gonzalez, Melanie, Kabbaj, Marie-Helene Miquel, Gunjan, Akash
Abstract/Description: Core histone proteins are essential for packaging the genomic DNA into chromatin in all eukaryotes. Since multiple genes encode these histone proteins, there is potential for generating more histones than what is required for chromatin assembly. The positively charged histones have a very high affinity for negatively charged molecules such as DNA, and any excess of histone proteins results in deleterious effects on genomic stability and cell viability. Hence, histone levels are known to be...
Show moreCore histone proteins are essential for packaging the genomic DNA into chromatin in all eukaryotes. Since multiple genes encode these histone proteins, there is potential for generating more histones than what is required for chromatin assembly. The positively charged histones have a very high affinity for negatively charged molecules such as DNA, and any excess of histone proteins results in deleterious effects on genomic stability and cell viability. Hence, histone levels are known to be tightly regulated via transcriptional, posttranscriptional and posttranslational mechanisms. We have previously elucidated the posttranslational regulation of histone protein levels by the ubiquitin-proteasome pathway involving the E2 ubiquitin conjugating enzymes Ubc4/5 and the HECT (Homologous to E6-AP C-Terminus) domain containing E3 ligase Tom1 in the budding yeast. Here we report the identification of four additional E3 ligases containing the RING (Really Interesting New Gene) finger domains that are involved in the ubiquitylation and subsequent degradation of excess histones in yeast. These E3 ligases are Pep5, Snt2 as well as two previously uncharacterized Open Reading Frames (ORFs) YKR017C and YDR266C that we have named Hel1 and Hel2 (for Histone E3 Ligases) respectively. Mutants lacking these E3 ligases are sensitive to histone overexpression as they fail to degrade excess histones and accumulate high levels of endogenous histones on histone chaperones. Co-immunoprecipitation assays showed that these E3 ligases interact with the major E2 enzyme Ubc4 that is involved in the degradation related ubiquitylation of histones. Using mutagenesis we further demonstrate that the RING domains of Hel1, Hel2 and Snt2 are required for histone regulation. Lastly, mutants corresponding to Hel1, Hel2 and Pep5 are sensitive to replication inhibitors. Overall, our results highlight the importance of posttranslational histone regulatory mechanisms that employ multiple E3 ubiquitin ligases to ensure excess histone degradation and thus contribute to the maintenance of genomic stability.
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Date Issued: 2012-01-01
Identifier: FSU_pmch_22570702, 10.1371/journal.pone.0036295, PMC3343073, 22570702, 22570702, PONE-D-11-24652
Format: Citation

Title: Histone levels are regulated by phosphorylation and ubiquitylation-dependent proteolysis.
Creator: Singh, Rakesh Kumar, Kabbaj, Marie-Helene Miquel, Paik, Johanna, Gunjan, Akash
Abstract/Description: Histone levels are tightly regulated to prevent harmful effects such as genomic instability and hypersensitivity to DNA-damaging agents due to the accumulation of these highly basic proteins when DNA replication slows down or stops. Although chromosomal histones are stable, excess (non-chromatin bound) histones are rapidly degraded in a Rad53 (radiation sensitive 53) kinase-dependent manner in Saccharomyces cerevisiae. Here we demonstrate that excess histones associate with Rad53 in vivo and...
Show moreHistone levels are tightly regulated to prevent harmful effects such as genomic instability and hypersensitivity to DNA-damaging agents due to the accumulation of these highly basic proteins when DNA replication slows down or stops. Although chromosomal histones are stable, excess (non-chromatin bound) histones are rapidly degraded in a Rad53 (radiation sensitive 53) kinase-dependent manner in Saccharomyces cerevisiae. Here we demonstrate that excess histones associate with Rad53 in vivo and seem to undergo modifications such as tyrosine phosphorylation and polyubiquitylation, before their proteolysis by the proteasome. We have identified the Tyr 99 residue of histone H3 as being critical for the efficient ubiquitylation and degradation of this histone. We have also identified the ubiquitin conjugating enzymes (E2) Ubc4 and Ubc5, as well as the ubiquitin ligase (E3) Tom1 (temperature dependent organization in mitotic nucleus 1), as enzymes involved in the ubiquitylation of excess histones. Regulated histone proteolysis has major implications for the maintenance of epigenetic marks on chromatin, genomic stability and the packaging of sperm DNA.
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Date Issued: 2009-08-01
Identifier: FSU_pmch_19578373, 10.1038/ncb1903, PMC2720428, 19578373, 19578373, ncb1903
Format: Citation

Title: The molecular function of the yeast polo-like kinase Cdc5 in Cdc14 release during early anaphase.
Creator: Liang, Fengshan, Jin, Fengzhi, Liu, Hong, Wang, Yanchang
Abstract/Description: In the budding yeast Saccharomyces cerevisiae, Cdc14 is sequestered within the nucleolus before anaphase entry through its association with Net1/Cfi1, a nucleolar protein. Protein phosphatase PP2A(Cdc55) dephosphorylates Net1 and keeps it as a hypophosphorylated form before anaphase. Activation of the Cdc fourteen early anaphase release (FEAR) pathway after anaphase entry induces a brief Cdc14 release from the nucleolus. Some of the components in the FEAR pathway, including Esp1, Slk19, and...
Show moreIn the budding yeast Saccharomyces cerevisiae, Cdc14 is sequestered within the nucleolus before anaphase entry through its association with Net1/Cfi1, a nucleolar protein. Protein phosphatase PP2A(Cdc55) dephosphorylates Net1 and keeps it as a hypophosphorylated form before anaphase. Activation of the Cdc fourteen early anaphase release (FEAR) pathway after anaphase entry induces a brief Cdc14 release from the nucleolus. Some of the components in the FEAR pathway, including Esp1, Slk19, and Spo12, inactivate PP2A(Cdc55), allowing the phosphorylation of Net1 by mitotic cyclin-dependent kinase (Cdk) (Clb2-Cdk1). However, the function of another FEAR component, the Polo-like kinase Cdc5, remains elusive. Here, we show evidence indicating that Cdc5 promotes Cdc14 release primarily by stimulating the degradation of Swe1, the inhibitory kinase for mitotic Cdk. First, we found that deletion of SWE1 partially suppresses the FEAR defects in cdc5 mutants. In contrast, high levels of Swe1 impair FEAR activation. We also demonstrated that the accumulation of Swe1 in cdc5 mutants is responsible for the decreased Net1 phosphorylation. Therefore, we conclude that the down-regulation of Swe1 protein levels by Cdc5 promotes FEAR activation by relieving the inhibition on Clb2-Cdk1, the kinase for Net1 protein.
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Date Issued: 2009-08-01
Identifier: FSU_pmch_19570916, 10.1091/mbc.E08-10-1049, PMC2777927, 19570916, 19570916, E08-10-1049
Format: Citation

Title: DNA damage checkpoints inhibit mitotic exit by two different mechanisms.
Creator: Liang, Fengshan, Wang, Yanchang
Abstract/Description: Cyclin-dependent kinase (CDK) governs cell cycle progression, and its kinase activity fluctuates during the cell cycle. Mitotic exit pathways are responsible for the inactivation of CDK after chromosome segregation by promoting the release of a nucleolus-sequestered phosphatase, Cdc14, which antagonizes CDK. In the budding yeast Saccharomyces cerevisiae, mitotic exit is controlled by the FEAR (for "Cdc-fourteen early anaphase release") and mitotic exit network (MEN) pathways. In response to...
Show moreCyclin-dependent kinase (CDK) governs cell cycle progression, and its kinase activity fluctuates during the cell cycle. Mitotic exit pathways are responsible for the inactivation of CDK after chromosome segregation by promoting the release of a nucleolus-sequestered phosphatase, Cdc14, which antagonizes CDK. In the budding yeast Saccharomyces cerevisiae, mitotic exit is controlled by the FEAR (for "Cdc-fourteen early anaphase release") and mitotic exit network (MEN) pathways. In response to DNA damage, two branches of the DNA damage checkpoint, Chk1 and Rad53, are activated in budding yeast to prevent anaphase entry and mitotic exit, allowing cells more time to repair damaged DNA. Here we present evidence indicating that yeast cells negatively regulate mitotic exit through two distinct pathways in response to DNA damage. Rad53 prevents mitotic exit by inhibiting the MEN pathway, whereas the Chk1 pathway prevents FEAR pathway-dependent Cdc14 release in the presence of DNA damage. In contrast to previous data, the Rad53 pathway negatively regulates MEN independently of Cdc5, a Polo-like kinase essential for mitotic exit. Instead, a defective Rad53 pathway alleviates the inhibition of MEN by Bfa1.
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Date Issued: 2007-07-01
Identifier: FSU_pmch_17485442, 10.1128/MCB.00095-07, PMC1951953, 17485442, 17485442, MCB.00095-07
Format: Citation

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

Title: Phosphatase 2A negatively regulates mitotic exit in Saccharomyces cerevisiae.
Creator: Wang, Yanchang, Ng, Tuen-Yung
Abstract/Description: In budding yeast Saccharomyces cerevisiae, Cdc5 kinase is a component of mitotic exit network (MEN), which inactivates cyclin-dependent kinase (CDK) after chromosome segregation. cdc5-1 mutants arrest at telophase at the nonpermissive temperature due to the failure of CDK inactivation. To identify more negative regulators of MEN, we carried out a genetic screen for genes that are toxic to cdc5-1 mutants when overexpressed. Genes that encode the B-regulatory subunit (Cdc55) and the three...
Show moreIn budding yeast Saccharomyces cerevisiae, Cdc5 kinase is a component of mitotic exit network (MEN), which inactivates cyclin-dependent kinase (CDK) after chromosome segregation. cdc5-1 mutants arrest at telophase at the nonpermissive temperature due to the failure of CDK inactivation. To identify more negative regulators of MEN, we carried out a genetic screen for genes that are toxic to cdc5-1 mutants when overexpressed. Genes that encode the B-regulatory subunit (Cdc55) and the three catalytic subunits (Pph21, Pph22, and Pph3) of phosphatase 2A (PP2A) were isolated. In addition to cdc5-1, overexpression of CDC55, PPH21, or PPH22 is also toxic to other temperature-sensitive mutants that display defects in mitotic exit. Consistently, deletion of CDC55 partially suppresses the temperature sensitivity of these mutants. Moreover, in the presence of spindle damage, PP2A mutants display nuclear localized Cdc14, the key player in MEN pathway, indicative of MEN activation. All the evidence suggests the negative role of PP2A in mitotic exit. Finally, our genetic and biochemical data suggest that PP2A regulates the phosphorylation of Tem1, which acts at the very top of MEN pathway.
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Date Issued: 2006-01-01
Identifier: FSU_pmch_16079183, 10.1091/mbc.E04-12-1109, PMC1345648, 16079183, 16079183, E04-12-1109
Format: Citation

Saccharomyces cerevisiae Proteins/genetics (13)	+ -
Saccharomyces cerevisiae Proteins/metabolism (12)	+ -
Cell Cycle Proteins/metabolism (10)	+ -
Cell Cycle Proteins/genetics (9)	+ -
Saccharomyces cerevisiae/metabolism (9)	+ -

Mutation (7)	+ -
Nuclear Proteins/genetics (7)	+ -
Phosphorylation (7)	+ -
Nuclear Proteins/metabolism (6)	+ -
Chromosome Segregation/genetics (4)	+ -
Microtubule-Associated Proteins/genetics (4)	+ -
Protein Binding (4)	+ -
Protein-Serine-Threonine Kinases/genetics (4)	+ -
Protein-Serine-Threonine Kinases/metabolism (4)	+ -
Saccharomyces cerevisiae/cytology (4)	+ -
Anaphase/genetics (3)	+ -
DNA-Binding Proteins/genetics (3)	+ -
DNA-Binding Proteins/metabolism (3)	+ -
Histones/metabolism (3)	+ -
Microtubules/genetics (3)	+ -
Minichromosome Maintenance Proteins/metabolism (3)	+ -
Mitosis/genetics (3)	+ -
Mutation/genetics (3)	+ -
Protein Kinases/metabolism (3)	+ -
Protein Tyrosine Phosphatases/genetics (3)	+ -
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