Determination of significance of SUMOylation of the Smc5/6 complex

Abstract

The fascination of scientists to the Smc5/6 complex began in the 1990s, with the characterization of UV and γ radiation-sensitive rad18 mutant and its identification as a component of a novel Structural Maintenance of Chromosome (SMC) complex in the fission yeast. A major impetus to this field of study came with the finding that this novel SMC complex had an intriguing set of components, with both a SUMO ligase and a putative Ubiquitin ligase, in addition to four other Non-SMC-Elements (NSE). Detailed structural analyses of the Smc5/6 complex opened a plethora of questions about the possible roles of the complex, ranging from DNA damage to protein homeostasis and transport. Several subunits of the Smc5/6 complex are known to be SUMOylated but despite commendable attempts by several groups, the essential role of SUMOylation of the complex remains a mystery. In the present study, we have attempted to understand the significance of SUMOylation of the complex by generating putative non-SUMOylatable (ns) forms of Smc5 and Nse4 proteins within the complex. Analysis of the phenotypic defects of the putative ns mutants revealed involvement of the complex in DNA damage and cell morphology. Interestingly, in the presence of oxidative stress, Nse4 SUMOylation was found to be enhanced especially 30-50 minute post treatment with H2O2. Thus, the present study suggests a novel role of Nse4 SUMOylation in the oxidative stress response. In addition to SUMOylation, Nse4 is also known to be phosphorylated. Putative phosho-mimetic and phospho-ablative forms of Nse4 were generated to assess the physiological significance of this post-translational modification. While Nse4 phopho-ablative mutant showed only mild DNA damage sensitivity, the temperature sensitive phospho-mimetic mutant showed marked sensitivity to genotoxic agents and 1mM H2O2. These findings hint towards a novel role of Nse4 phosphorylation in both DNA damage and oxidative stress response.