The puzzling smc5/6 complex : : piecing together functions in segregation and repair

Sammanfattning: Genome stability is essential in order for cells to survive. During their life cycle, parental cells must divide and properly allocate their genetic material to daughter cells. To ensure correct distribution, multiple proteins are involved in replicating and segregating the genome. Even under unchallenged conditions, replication and segregation errors can occur frequently but are in most cases efficiently repaired. Replication, segregation and repair are all fundamental processes that are under tight control. Failure to correctly execute these processes leads to genomic instability, which can drive tumor development. The structural maintenance of chromosomes 5/6 (Smc5/6) protein complex is involved in all of these fundamental processes and is therefore considered to be an important guardian of genome stability. However, the mechanism of Smc5/6 function in these processes remains to be determined. The diversity of Smc5/6 functions in chromosome replication, segregation and repair has puzzled researchers since the discovery of the complex in the 1990’s. The aim of this thesis was to explore novel functions of the Smc5/6 complex in DNA repair and chromosome segregation in the budding yeast Saccharomyces cerevisiae. We first studied the Smc5/6 complex role in DNA repair, the results of which are presented in paper I. We drew the conclusion that modification of the Smc5/6 subunit Mms21 by phosphorylation was needed for its full SUMO ligase activity. Phosphorylation of Mms21 was dependent on the Mec1 kinase, a checkpoint protein sensor that phosphorylate substrates in response to DNA damage. Two targets of Mms21 SUMOylation were investigated, and both were SUMOylated at a reduced level without Mms21 phosphorylation. In the presence of DNA damage, Mms21 phosphorylation was important for maintaining genome stability. In paper II, we focused on chromosomal association of the Smc5/6 complex. Smc5/6 associated to DNA in a cohesion-dependent manner during S-phase, followed replication fork progression, and accumulated in the G2/M-phase. Moreover, Smc5/6 binding along chromosome arms increased in the absence of Topoisomerase II (Top2) activity, the lack of which is known to accumulate sister chromatid intertwinings (SCIs). The level of Smc5/6 enrichment predicted the missegregation pattern of Top2 mutants, suggesting that Smc5/6 binds SCIs. In addition, we observed that Smc5/6 promoted segregation of chromosomes that accumulated SCIs in the absence of functional Top2. This led to the conclusion that the Smc5/6 complex is likely to bind SCIs and to facilitate their resolution. In paper III, we further investigated the role of Smc5/6 in chromosome segregation. This study revealed that Smc5/6 mutants activated the spindle assembly checkpoint (SAC) in a Mad2-dependent pathway. Together with earlier and present investigations in our lab, we speculate that Smc5/6 mutants delay replication of especially longer chromosomes, which hinders proper kinetochore-microtubule attachment. This in turn activates the SAC and halts the cell cycle. These results will add more pieces to the puzzle to build a more comprehensive picture of Smc5/6 function.