Mutations that prevent methylation of cohesin render sensitivity to DNA damage in S. pombe

Abstract

The canonical role of cohesin is to mediate sister chromatid cohesion. In addition, cohesin plays important roles in processes such as DNA repair and regulation of gene expression. Mounting evidence suggests that various post-translational modifications, including phosphorylation, acetylation and sumoylation regulate cohesin functions. Our mass spectrometry analysis of cohesin purified from Schizosaccharomyces pombe cells revealed that the cohesin subunit Psm1 is methylated on two evolutionarily conserved lysine residues, K536 and K1200. We found that mutations that prevent methylation of Psm1 K536 and K1200 render sensitivity to DNA-damaging agents and show positive genetic interactions with mutations in genes encoding the Mus81–Eme1 endonuclease. Yeast two-hybrid and co-immunoprecipitation assays showed that there were interactions between subunits of the cohesin and Mus81–Eme1 complexes. We conclude that cohesin is methylated and that mutations that prevent methylation of Psm1 K536 and K1200 show synthetic phenotypes with mutants defective in the homologous recombination DNA repair pathway.

Sororin is an evolutionary conserved antagonist of WAPL

“…Anti-tubulin antibody (Sigma-Aldrich T5168; 1:10,000 dilution) and rabbit anti-mouse IgG-HRP secondary antibody (Jackson ImmunoResearch 115-035-033, 1:10,000 dilution) were used to detect alpha tubulin as a loading control. To measure the DNA content of the cells, flow cytometry was performed according to 98 using Sytox Green (S7020, Invitrogen) and FACS NovoCyte Penteon (Agilent).…”

Regulation of Structure-Specific Endonucleases in Replication Stress

“…mutants in fission yeast suggests that methylation of cohesin subunits may be important for Mus81 activity at the stalled replication fork. Alternatively, Mus81 may be required for recruitment of the cohesin to sites of DNA damage [102]. In human cells, depletion of SMC2, which is required for chromosome condensation, or WAPL (Wings apart protein-like), which is required for release of sister-chromatid arm cohesin, results in failure to recruit MUS81 to chromatin [101].…”