The smc5/6 complex : linking DNA replication with chromosome segregation

Abstract: In order to faithfully propagate the genetic material from one generation to the next, cells need to properly replicate and segregate their chromosomes. The three well-conserved eukaryotic Structural Maintenance of Chromosomes (SMC) protein complexes, cohesin, condensin and the Smc5/6 complex (Smc5/6) organize chromosomes to ensure that the daughter cells receive a full complement of chromosomes. Cohesin holds sister chromatids, which are the products of replication, together to allow chromosome biorientation prior to segregation. Condensin promotes the condensation of chromosomes to allow them to segregate away from each other during anaphase. The least well-characterized SMC complex, Smc5/6, promotes proper DNA replication, and correct segregation of the ribosomal DNA. Another group of proteins that organizes chromosomes are the topoisomerases. These enzymes cut and paste chromosomes to allow the unwinding of the DNA double helix during replication, and the untangling of chromosomes during segregation. Failure to correctly execute these fundamental processes often leads to cell death. However, it can also lead to cells acquiring the wrong number of chromosomes, i.e. aneuploidy, which is a hallmark of cancer cells. Knowledge of how chromosomes are organized and maintained is therefore important not only to understand the basic principles of life, but also to understand cancerous cells. With the projects presented in this thesis, we aimed to extend our knowledge about the functions of Smc5/6 and topoisomerases during DNA replication and chromosome segregation, using the model organism Saccharomyces cerevisiae (S. cerevisiae). Since the SMC complexes perform their functions by directly associating with chromosomes, an important focus of our studies has been to characterize the chromosomal association pattern of Smc5/6 in detail, in order to reveal new clues about its functions. The main findings of the four projects are introduced below. In Paper I, we presented new functions of Smc5/6 and type I topoisomerases in the timely replication of long S. cerevisiae chromosomes. We also showed that the chromosomal association of Smc5/6 is regulated by chromosome length and topoisomerase II. The data allowed us to propose a model in which Smc5/6 promotes replication by stimulating fork rotation to reduce topological stress ahead of the fork. In Paper II, we showed that Smc5/6 requires sister chromatids to be held together in order to associate with chromosomes. Smc5/6 was also shown to promote correct segregation of short entangled chromosomes. Our extensive characterization of the chromosomal association of Smc5/6 led us to the hypothesis that Smc5/6 associates to chromosomal loci where the sister chromatids are entangled, and that topological stress during replication affect the level of chromosome entanglement. In Paper III, we created a hard-to-replicate region of DNA by artificially inducing high convergent RNA polymerase II-driven transcription. This caused the replication fork to pause, which was dependent on the highly expressed gene that opposed the direction of replication. The paused fork was assisted past this obstacle by the Rrm3 helicase. In addition, Smc5/6 associated to chromatin behind the paused fork, where it remained also after replication. Our results strengthened the hypothesis that topological stress is a factor that contributes to the recruitment of Smc5/6 to chromosomes. In Paper IV, we dissected the role of the Nse5 subunit of Smc5/6 during replication stress induced by hydroxyurea, which inhibits the production of nucleotides. We showed that Nse5 is required for the sumoylation of Smc5, and the recruitment of the complex to stalled forks. The results also indicated that the former of these functions is dispensable, while the latter is important, for Smc5/6 to stabilize stalled replication forks and prevent aberrant recombination at these forks. The results of this thesis increase our understanding of how chromosomes are replicated and segregated, and highlight the importance of analyzing the topological status of chromosomes to fully understand the processes that maintain genome stability.