SUMO Chain Formation Is Required for Response to Replication Arrest in S. pombe

Abstract

SUMO is a ubiquitin-like protein that is post-translationally attached to one or more lysine residues on target proteins. Despite having only 18% sequence identity with ubiquitin, SUMO contains the conserved ββαββαβ fold present in ubiquitin. However, SUMO differs from ubiquitin in having an extended N-terminus. In S. pombe the N-terminus of SUMO/Pmt3 is significantly longer than those of SUMO in S. cerevisiae, human and Drosophila. Here we investigate the role of this N-terminal region. We have used two dimensional gel electrophoresis to demonstrate that S. pombe SUMO/Pmt3 is phosphorylated, and that this occurs on serine residues at the extreme N-terminus of the protein. Mutation of these residues (in pmt3-1) results in a dramatic reduction in both the levels of high Mr SUMO-containing species and of total SUMO/Pmt3, indicating that phosphorylation of SUMO/Pmt3 is required for its stability. Despite the significant reduction in high Mr SUMO-containing species, pmt3-1 cells do not display an aberrant cell morphology or sensitivity to genotoxins or stress. Additionally, we demonstrate that two lysine residues in the N-terminus of S. pombe SUMO/Pmt3 (K14 and K30) can act as acceptor sites for SUMO chain formation in vitro. Inability to form SUMO chains results in aberrant cell and nuclear morphologies, including stretched and fragmented chromatin. SUMO chain mutants are sensitive to the DNA synthesis inhibitor, hydroxyurea (HU), but not to other genotoxins, such as UV, MMS or CPT. This implies a role for SUMO chains in the response to replication arrest in S. pombe.

DNA Repair and Global Sumoylation Are Regulated by Distinct Ubc9 Noncovalent Complexes

“…Following purification of abundant SUMO chain species and mass spectrometry-based analysis of tryptic digests, we identified several lysine residues that were modified with the LGG tripeptide, including the amino terminal K14 and K30 residues (Table 3). These residues were also recently identified by comparison to budding yeast SUMO chain modification sites and site-directed mutagenesis (43). Fission yeast expressing SUMO K14/30R from the genomic locus appeared to be wild type for growth but, like SUMO D81R and pli1⌬ cells, showed reduced levels of highmolecular-weight SUMO conjugates (Fig.…”

Starting and stopping SUMOylation

“…SUMO itself has also been demonstrated to be phosphorylated (Matic et al 2008;Skilton et al 2009). However, the role of this modification remains to be fully determined.…”

SUMO in the mammalian response to DNA damage

“…The SUMO-2 and SUMO-3 families (which differ by only three amino acids) contain within their sequence a ψKXE consensus and are therefore able to form poly-SUMO chains, whereas SUMO-1, like the Saccharomyces cerevisiae SUMO, lacks this consensus and is conjugated as a single moiety to substrate proteins. (Schizosaccharomyces pombe has a single SUMO with an N-terminal extension containing lysine residues that enable SUMO-chain extensions [1].) Specificity of targeting is apparently achieved with relatively few E3s and just one E2.…”