DNA damage provokes cohesin sumoylation in Saccharomyces cerevisiae

Abstract

Cohesin tethers sister chromatids together from their creation during S-phase until their separation at the metaphase to anaphase transition. Cohesion is essential for correct segregation and therefore genomic stability. Cohesin is also involved the regulation of transcription and in DNA repair. Cohesin affects these processes by its ability to hold two pieces of DNA together. Cohesin is subject to a variety of post-translational modifications. In this study, the modification of cohesin, more specifically the Scc1 subunit, by Small ubiquitin-like modifier (SUMO) was analysed. It was found that cohesin is sumoylated during an unperturbed cell cycle, but is hyper-modified after DNA damage. To determine the function Scc1 modification by SUMO, attempts were made to identify the modified residues. Mass spectrometry techniques for SUMO site identification were developed in parallel to site-directed mutagenesis studies. Potential sumoylation sites were identified on Scc1, but when these were mutated there was little effect on either viability or the sumoylation pattern obtained. Mutants were then constructed in which only one lysine was present. Surprisingly, several of these were viable, not sensitive to methyl methanesulfonate and were not sumoylated, suggesting that either sumoylation of Scc1 is not essential and not required after DNA damage, or that sumoylation of the rest of the cohesin complex can compensate. To investigate this, Scc1 fusions with the SUMO E2 ligase, Ubc9, and a SUMO isopeptidase, Ulp1, to mimic constitutively sumoylated and unsumoylatable cohesin respectively, were constructed. Unexpectedly, it was found that both fusions relocalized to a double strand break, and that the Ulp1 fusion was recruited to a greater extent. Data from our laboratory suggests that the Ubc9 fusion is not competent for cohesion after damage, although it is sufficient for viability. Sumoylation may therefore be involved in cohesin unloading or turnover, which might be required after DNA damage.