Abstract
The small ubiquitin-related modifier, SUMO, post-translationally modifies a variety of proteins, affecting their interactions, localisation and stability. Sumoylation thereby influences numerous cellular functions, e.g. gene expression, chromosome segregation and the replication and repair of DNA. A mass spectrometry-based approach to identify sumoylated substrates in S. pombe is presented here. The approach provided a global overview of the S. pombe sumoylated proteome and identified sumoylation sites at the residue level in wild-type and mutant S. pombe cells. As expected, groups related to activities in the nucleus were highly represented. It was possible to locate the sumoylated lysines in crystal structures for some of the substrates, thereby revealing the tendency of sumoylation sites to be situated in flexible regions including near DNA binding domains for the structures for which DNA was present.
One of the SUMO-conjugates identified by mass spectrometry was the essential DNA binding protein Sap1. Sap1 is responsible for mating-type switching and for some replication fork blocks in S. pombe. Sap1 was determined to be sumoylated at 6 out of 29 lysines, primarily located in its two dimerization domains. The crystal structure of Sap1 revealed that at least one sumoylation site, K26, is in close proximity to the DNAbinding domain. A model of Sap1 bound to DNA located the site in such way that K26 could fit in the DNA minor groove. Sumoylation at this residue might affect DNA structure or Sap1-DNA interactions. In addition, the Sap1 function relationship was investigated in vivo by repeating a search for suppressors of the slow growth phenotype of abp1Δ cbh1Δ mutants. Autonomously replicating sequence binding protein 1 (Abp1) and cenp-B homologue 1 (Cbh1) co-localise with Sap1 in some genomic regions including retrotransposons LTRs where they have been proposed to exert antagonistic effects on DNA replication. A novel suppressor mutation identified in Sap1 as well as the Sap1-c suppressor reported in the literature are both situated at the dimer interface observed in the crystal, pointing to the mechanistic relevance of the interface in the control of DNA replication by Sap1. Finally, the structure reveals the existence of a deep pocket possibly involved in base-flipping.
One of the SUMO-conjugates identified by mass spectrometry was the essential DNA binding protein Sap1. Sap1 is responsible for mating-type switching and for some replication fork blocks in S. pombe. Sap1 was determined to be sumoylated at 6 out of 29 lysines, primarily located in its two dimerization domains. The crystal structure of Sap1 revealed that at least one sumoylation site, K26, is in close proximity to the DNAbinding domain. A model of Sap1 bound to DNA located the site in such way that K26 could fit in the DNA minor groove. Sumoylation at this residue might affect DNA structure or Sap1-DNA interactions. In addition, the Sap1 function relationship was investigated in vivo by repeating a search for suppressors of the slow growth phenotype of abp1Δ cbh1Δ mutants. Autonomously replicating sequence binding protein 1 (Abp1) and cenp-B homologue 1 (Cbh1) co-localise with Sap1 in some genomic regions including retrotransposons LTRs where they have been proposed to exert antagonistic effects on DNA replication. A novel suppressor mutation identified in Sap1 as well as the Sap1-c suppressor reported in the literature are both situated at the dimer interface observed in the crystal, pointing to the mechanistic relevance of the interface in the control of DNA replication by Sap1. Finally, the structure reveals the existence of a deep pocket possibly involved in base-flipping.
| Original language | English |
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| Publisher | Department of Biology, Faculty of Science, University of Copenhagen |
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| Publication status | Published - 2016 |