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1.
Nat Cell Biol ; 13(12): 1450-6, 2011 Oct 23.
Article in English | MEDLINE | ID: mdl-22020438

ABSTRACT

Kinases and phosphatases regulate messenger RNA synthesis through post-translational modification of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (ref. 1). In yeast, the phosphatase Cdc14 is required for mitotic exit(2,3) and for segregation of repetitive regions(4). Cdc14 is also a subunit of the silencing complex RENT (refs 5,6), but no roles in transcriptional repression have been described. Here we report that inactivation of Cdc14 causes silencing defects at the intergenic spacer sequences of ribosomal genes during interphase and at Y' repeats in subtelomeric regions during mitosis. We show that the role of Cdc14 in silencing is independent of the RENT deacetylase subunit Sir2. Instead, Cdc14 acts directly on RNA polymerase II by targeting CTD phosphorylation at Ser 2 and Ser 5. We also find that the role of Cdc14 as a CTD phosphatase is conserved in humans. Finally, telomere segregation defects in cdc14 mutants(4) correlate with the presence of subtelomeric Y' elements and can be rescued by transcriptional inhibition of RNA polymerase II.


Subject(s)
Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Gene Silencing/physiology , Protein Tyrosine Phosphatases/genetics , Protein Tyrosine Phosphatases/metabolism , RNA Polymerase II/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Telomere/metabolism , Transcription, Genetic/physiology , Cell Cycle Proteins/antagonists & inhibitors , DNA, Ribosomal Spacer/genetics , Interphase/genetics , Mitosis/genetics , Phosphorylation/genetics , Protein Tyrosine Phosphatases/antagonists & inhibitors , RNA Polymerase II/antagonists & inhibitors , Repetitive Sequences, Nucleic Acid , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Suppression, Genetic/physiology , Telomere/enzymology
2.
Nature ; 458(7235): 219-22, 2009 Mar 12.
Article in English | MEDLINE | ID: mdl-19158678

ABSTRACT

Chromosome condensation and the global repression of gene transcription are features of mitosis in most eukaryotes. The logic behind this phenomenon is that chromosome condensation prevents the activity of RNA polymerases. In budding yeast, however, transcription was proposed to be continuous during mitosis. Here we show that Cdc14, a protein phosphatase required for nucleolar segregation and mitotic exit, inhibits transcription of yeast ribosomal genes (rDNA) during anaphase. The phosphatase activity of Cdc14 is required for RNA polymerase I (Pol I) inhibition in vitro and in vivo. Moreover Cdc14-dependent inhibition involves nucleolar exclusion of Pol I subunits. We demonstrate that transcription inhibition is necessary for complete chromosome disjunction, because ribosomal RNA (rRNA) transcripts block condensin binding to rDNA, and show that bypassing the role of Cdc14 in nucleolar segregation requires in vivo degradation of nascent transcripts. Our results show that transcription interferes with chromosome condensation, not the reverse. We conclude that budding yeast, like most eukaryotes, inhibit Pol I transcription before segregation as a prerequisite for chromosome condensation and faithful genome separation.


Subject(s)
Anaphase/physiology , Cell Cycle Proteins/metabolism , Protein Tyrosine Phosphatases/metabolism , RNA Polymerase I/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/physiology , Transcription, Genetic/physiology , Adenosine Triphosphatases/metabolism , Chromosome Segregation , DNA-Binding Proteins/metabolism , Multiprotein Complexes/metabolism , Protein Binding/physiology , RNA, Fungal/metabolism , RNA, Ribosomal/metabolism , Saccharomyces cerevisiae/metabolism
3.
Electrophoresis ; 28(21): 3845-53, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17922509

ABSTRACT

A series of circular shuttle vectors were constructed that could replicate and transcribe in the cells of both Escherichia coli and Saccharomyces cerevisiae. 2-D agarose gel electrophoresis run without or in the presence of different concentrations of chloroquine (CHL) revealed that bacterial plasmids were more negatively (-) supercoiled than minichromosomes isolated from budding yeast. Attempts to increase (-) supercoiling in S. cerevisiae or to reduce it in E. coli have deleterious biological consequences. These observations indicate that DNA supercoiling can vary in different species but cells are exquisitely sensitive to sudden changes in supercoiling. In E. coli, the observation that cell growth as well as ColE1 plasmid copy number decrease when DNA relaxes suggests that supercoiling could affect cell viability by regulating the initiation of both transcription and replication.


Subject(s)
Chloroquine/chemistry , DNA, Bacterial/chemistry , DNA, Fungal/chemistry , DNA, Superhelical/chemistry , Escherichia coli/genetics , Saccharomyces cerevisiae/genetics , Cell Survival/drug effects , Chloroquine/pharmacology , DNA Replication/drug effects , Electrophoresis, Agar Gel/methods , Electrophoresis, Gel, Two-Dimensional/methods , Plasmids , Transcription, Genetic
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