Understanding The Role of Bir1 Sumoylation in Mitotic Regulation

Abstract

To ensure the integrity of genome over generations, organisms have evolved multiple mechanisms to respond to DNA damage or chromosome instability. Protein sumoylation is one such mechanism for genome maintenance. Sumoylation describes a process in which concerted action of E1, E2 and E3 ligases facilitates the covalent linkage of small ubiquitin-like modifiers (SUMO) peptide to target proteins. In Saccharomyces cerevisiae, Siz1, Siz2 and Mms21 are three E3 SUMO ligases. Each of these ligases has preferred targets and it is generally accepted that they contribute to genome stability. Understanding specific SUMO targets will reveal mechanistic insights into the role of sumoylation in genome maintenance. In S. cerevisiae, Bir1, a subunit of chromosomal passenger complex (CPC), is a target of Mms21. During mitosis, CPC, a four-subunit complex composed of Bir1, Ipl1, Sli15 and Nbl1, mediates the spindle assembly checkpoint (SAC) to establish chromosome bi-orientation. Appropriate tension between two sister chromatids serves as a molecular signal for successful bi-orientation. Lack of tension may be a sign of incorrect attachment of mitotic spindles to the chromosome or unresolved replication intermediates resulting from DNA damage accumulation. Bir1, along with the CPC complex, plays a pivotal role in this process and the goal of our research is to understand the functional significance of Bir1 sumoylation. We hypothesize that sumoylation contributes to Bir1's regulation of mitotic progression. An existing piece of evidence supports this model: a decrease in sumoylation of Bir1 or Sli15 correlates with SAC deactivation when cells were treated with a genotoxic agent, methyl methanesulfonate (MMS). We genetically modified BIR1 to mimic conditions in which Bir1 is constantly sumoylated (BIR1-SuON) or cannot be sumoylated (BIR1-SuOff). These genetically modified strains are not sensitive to DNA damaging agents such as MMS or ultraviolet radiation similar to previously reported temperature sensitive mutants of Bir1 (bir1-107, bir1-110 and bir1-111) or Ipl1 (ipl1-321). This observation is different from the phenotype of mms21 mutants, which are known to be sensitive to genotoxic agents, such as MMS. These data together suggest that sumoylation of Bir1 by Mms21 is not responsible for resisting cell death when genome stability is challenged. Consistent with this idea, we observed that BIR1-SuOn allele did not rescue mms21 mutants’ sensitivity to MMS treatment. Intriguingly, we observed that both ipl1-321 and BIR1-SuOff mutants grew better than the control when cells were treated with high concentration of MMS. Similarly, it has been reported that hydroxyurea or benomylcan suppress ipl1-321 mutants’ temperature sensitivity. These observations suggest two possibilities: 1) sumoylated Bir1 shares some functions with Ipl1 in regulating chromosome segregation or 2) both BIR1-SuOff and ipl1-321 mutants have relatively unstable genome that allows them to generate revertants in the presence of genotoxic agents. To further test the idea that sumoylated Bir1 has a role in chromosome segregation, we are currently studying the phenotype of bir1 SUMO mutants in genetic background that disrupts mitotic regulation.