Investigation into the role of the SUMO-like domains of the Schizosaccharomyces pombe DNA repair protein Rad60

Ubiquitin and ubiquitin-like (UBLs) proteins are post-translational modifiers that share a characteristic ßßaßßaß fold. SUMO (small ubiquitin-like modifier) is one of a number of ubiquitin-like proteins. Unlike ubiquitin, SUMO does not appear to have a role in protein degradation. Instead it has been shown to have roles in facilitating protein-protein interactions, altering protein localisation and modulating protein activity. Analysis of protein databases indicates the existence of ubiquitin-fusion proteins, which act to functionally mimic ubiquitination by interacting with the proteosome. During the course of this project a family of SUMO-like domain (SLD) proteins has been identified and termed the RENi family after its best-studied members S. pombe Rad60, S. cerevisiae Esc2 and M. musculus Nip45. I have initiated an investigation into the importance of the two SUMO-like domains for S. pombe Rad60 function. A rad60 mutant deleted for SLD1 (rad60-SLD1?) is not viable suggesting that SLD1 is required for the essential role of Rad60. A rad60 mutant deleted for SLD2 (rad60-ct) is viable but cells are sensitive to DNA damaging agents. This implies that SLD2 is not required for the essential function of Rad60 but is required for the response to DNA damage. The C-terminally truncated Rad60 protein (Rad60-ct) is mis-localised in rad60-ct cells. Provision of an NLS to the C-terminus of the Rad60-ct protein restores nuclear localisation but does not rescue the HU and MMS sensitivity of rad60-ct cells. Instead, expression of the Rad60-ctNLS protein has a dominant-negative effect in both wild-type and rad60-ct cells. The same phenomenon was observed when SLD2 was replaced with SUMO. This suggests that SLD2 is required not only to localise Rad60 to the nucleus, but also for the DNA damage response itself. Molecular modelling suggests that SLD1 and SLD2 can adopt the characteristic ßßaßßaß fold. A novel ‘recombinase-mediated cassette-exchange’ system was used to initiate a structure/function study of Rad60 SLD2 by mutating residues predicted to help maintain the hydrophobic core. The DNA damage sensitive phenotype of L348G, L338G, L346G and I334G substitutions support the hypothesis that the SLD2 adopts a SUMO-like fold. Sumoylation of Rad60, in vitro, can be enhanced by the SUMO E3 ligase, Pli1 but not Nse2. Rad60 is sumoylated in a manner dependent on the C-terminus, which has is required to interact with the Hus5 conjugator, in vitro. This suggests that SLD2 may act to recruit Hus5 for sumoylation of itself and/or other proteins.