Sister chromatid cohesion establishment by acetylation

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. High density oligonucleotide tiling arrays were used to hybridize genomic DNA from the Saccharomyces cerevisiae eco1-1 mutant and from spontaneous suppressor mutants that rescue eco1-1 thermosensitive growth. Hybridization intensities at each oligonucleotide probe were compared using the algorithm discribed in [D. Gresham et al. Science 311, 1932 (2006)] to locate the nucleotide changes causing the suppression. One such mutation was in the SMC3 gene, introducing an acetylation mimicking asparagine mutation to an evolutionarily conserved lysine that serves as a substrate to Eco1 acetyltransferase activity. A separate complementation group had mutations in the RAD61 gene (here renamed WPL1), the ortholog of human and fission yeast Wapl. WPL1 inactivation allowed cell growth in the absence of Eco1. Tiling arrays were used again to analyze Wpl1 and cohesin chromatin immunoprecipitates, to demonstrate their colocalization along chromosomes in G2.