Project description:Eco1 is the acetyltransferase that establishes sister-chromatid cohesion during DNA replication. Budding yeast with an eco1 mutation that genocopies Roberts syndrome displaysreduced ribosomal DNA (rDNA) transcription and a transcriptional signature of starvation. Weshow that deleting FOB1, a gene encoding a specific replication fork blocking protein for therDNA region, rescues rRNA production and partially rescues transcription genome-wide. This experiment examines the effect of eco1 mutation on replication genome-wide. Furtherstudies show that deletion of FOB1 corrects the genome-wide replication defects, nucleolarstructure, and rDNA segregation in an eco1 mutant. Our study highlights cohesin's central role atthe rDNA for global control of DNA replication and gene expression. DNA content in eco1-W216G mutant and wt yeast is measured in duplicate by sequencing at 0, 20, and 40 minutes following release from G1 arrest.
Project description:Eco1 is the acetyltransferase that establishes sister-chromatid cohesion during DNA replication. Budding yeast with an eco1 mutation that genocopies Roberts syndrome displaysreduced ribosomal DNA (rDNA) transcription and a transcriptional signature of starvation. Weshow that deleting FOB1, a gene encoding a specific replication fork blocking protein for therDNA region, rescues rRNA production and partially rescues transcription genome-wide. This experiment examines the effect of eco1 mutation on replication genome-wide. Furtherstudies show that deletion of FOB1 corrects the genome-wide replication defects, nucleolarstructure, and rDNA segregation in an eco1 mutant. Our study highlights cohesin's central role atthe rDNA for global control of DNA replication and gene expression.
Project description:Eco1 is an acetyltransferase subunit of the cohesin complex and acts during DNA replication to establish cohesion between sister chromatids. However, cohesin has additional functions in gene expression, DNA damage repair, and higher-order organization of chromosomes. The eco1 mutant W216G disrupts acetyltansferase activity, and causes genome-wide transcriptional defects which can be suppressed by deletion of FOB1, a gene also involved in DNA replication. This experiment investigates gene expression differences between the eco1-W216G mutant, and mutants in FOB1, and RAD61 a gene involved in inhibition of cohesion establishment but mutation of which is able to suppress temperature sensitivity of the eco1-W216G mutant. Wt and mutant strains of yeast were grown to mid log phase in liquid culture in triplicate and harvested for comparison on Affymetrix microarrays. The following strains were compared: 1) eco1-W216G, 2) eco1-W216G fob1Δ, 3) eco1-W216G rad61Δ, 4) fob1Δ, 5) rad61Δ, and 6) WT.
Project description:Eco1 is an acetyltransferase subunit of the cohesin complex and acts during DNA replication to establish cohesion between sister chromatids. However, cohesin has additional functions in gene expression, DNA damage repair, and higher-order organization of chromosomes. The eco1 mutant W216G disrupts acetyltansferase activity, and causes genome-wide transcriptional defects which can be suppressed by deletion of FOB1, a gene also involved in DNA replication. This experiment investigates gene expression differences between the eco1-W216G mutant, and mutants in FOB1, and RAD61 a gene involved in inhibition of cohesion establishment but mutation of which is able to suppress temperature sensitivity of the eco1-W216G mutant.
Project description:DNA replication during S-phase is accompanied by establishment of sister chromatid cohesion to ensure faithful chromosome segregation. The Eco1 acetyltransferase, helped by factors including Ctf4 and Chl1, concomitantly acetylates the chromosomal cohesin complex to stabilize its cohesive links. Here we show that Ctf4 recruits the Chl1 helicase to the replisome via a conserved interaction motif that Chl1 shares with GINS and polymerase α. We visualize recruitment by EM analysis of a reconstituted Chl1-Ctf4-GINS assembly. The Chl1 helicase facilitates replication fork progression under conditions of nucleotide depletion, however this function does not require Ctf4 interaction. Conversely, Ctf4 interaction, but not helicase activity, is required for Chl1’s role in sister chromatid cohesion. A physical interaction between Chl1 and the cohesin complex during S-phase suggests that Chl1 contacts cohesin to facilitate its acetylation. Our results reveal how Ctf4 forms a replisomal interaction hub that coordinates replication fork progression and sister chromatid cohesion establishment.
Project description:Cohesion between sister chromatids depends on the chromosomal cohesin complex and allows the spindle apparatus in mitosis to recognize replicated chromosomes for segregation into daughter cells. Sister chromatid cohesion is established concomitant with DNA replication, and requires the essential Eco1 protein, a replication fork-associated acetyl transferase. The mechanism by which Eco1 establishes sister chromatid cohesion is not known. Here, we show that the cohesin subunit Smc3 is acetylated in an Eco1-dependent manner during S phase to establish sister chromatid cohesion. We isolated spontaneous suppressors of the thermosensitive eco1-1 allele in budding yeast, and identified the suppressor mutations from the hybridization pattern of genomic DNA on oligonucleotide tiling arrays. An acetylation mimicking mutation of a conserved lysine in Smc3 to asparagine (K113N) makes Eco1 dispensable for cell growth, indicating that Smc3 acetylation is Eco1’s only essential function. We identified a second set of eco1-1 suppressor mutations in the budding yeast ortholog of the cohesin regulator Wapl (Wpl1/Rad61). Wapl destabilizes cohesin on chromosomes, and Eco1-dependent Smc3 acetylation during S-phase might render cohesin resistant to Wapl. Our results clarify the role of Eco1 in the establishment of sister chromatid cohesion, and suggest that Eco1 modifies cohesin to stabilize an Eco1-independent cohesion establishment reaction.
Project description:Cohesin stably holds together the sister chromatids from S phase until mitosis. To do so, cohesin must be protected against its cellular antagonist Wapl. Eco1 acetylates cohesinâs Smc3 subunit, which locks together the sister DNAs. We used yeast genetics to dissect how Wapl drives cohesin from chromatin and identified mutants of cohesin that are impaired in ATPase activity but remarkably confer robust cohesion that bypasses the need for the cohesin protectors Eco1 in yeast and Sororin in human cells. We uncover an unexpected functional asymmetry within the heart of cohesinâs highly conserved ABC-like ATPase machinery and show that an activity associated with one of cohesinâs two ATPase sites drives DNA release from cohesin rings. This key mechanism is conserved from yeast to humans. We propose that Eco1 locks cohesin rings around the sister chromatids by counteracting an asymmetric cohesin-associated ATPase activity. Effect of mutations in Smc1 and Smc3 on cohesin loading onto chromosomes
Project description:Cohesin acetylation by Eco1 during DNA replication establishes sister chromatid cohesion. We show that acetylation makes cohesin resistant to Wapl activity from S-phase until mitosis. Wapl turns out to be a key regulator of cohesin dynamics on chromosomes by controling cohesin maintenance following its establishment in S-phase and its role in chromosome condensation.
Project description:Cohesin acetylation by Eco1 during DNA replication establishes sister chromatid cohesion. We show that acetylation makes cohesin resistant to Wapl activity from S-phase until mitosis. Wapl turns out to be a key regulator of cohesin dynamics on chromosomes by controling cohesin maintenance following its establishment in S-phase and its role in chromosome condensation.
Project description:Cohesin acetylation by Eco1 during DNA replication establishes sister chromatid cohesion. We show that acetylation makes cohesin resistant to Wapl activity from S-phase until mitosis. Wapl turns out to be a key regulator of cohesin dynamics on chromosomes by controling cohesin maintenance following its establishment in S-phase and its role in chromosome condensation.