Investigating fork rotation and DNA pre-catenation in Saccharomyces cerevisiae

During DNA replication, the intertwining between the two strands of the parental DNA double helix needs to be resolved. This is achieved in two ways: by the action of topoisomerases ahead of the replication fork or by fork rotation and precatenation of the newly replicated DNA helixes. However, the factors that influence fork rotation and pre-catenation remain unknown. In this thesis, I used classical genetics and high-resolution two-dimensional agarose gel electrophoresis to identify the replisome-associated factors important for fork rotation and DNA pre-catenation during DNA replication in the yeast Saccharomyces cerevisiae. The results indicate that fork rotation and precatenation are impeded by two non-essential evolutionarily conserved replisome components: the Timeless and Tipin homologs, Tof1 and Csm3. Tof1/Csm3 are required for maintaining genome integrity during unperturbed and perturbed DNA replication. Similarly, checkpoint activation is also thought to stabilize the replisome in both unchallenged and challenged cells. However, none of the checkpoint kinases were found to alter the frequency of fork rotation during DNA replication in our study. Finally, constitutive DNA damage was found to be dramatically increased on newly replicated chromatids in the absence of Top2 and/or Tof1 as a consequence of excessive fork rotation and DNA pre-catenation during DNA replication in both western blot and ChIp-Seq experiments. This led to the activation of the DNA damage checkpoint and extensive DNA repair. These results suggest that although fork rotation and pre-catenation facilitate DNA unwinding under certain chromosomal contexts, excessive fork rotation and precatenation lead to defects on the newly replicated chromatids and therefore must be inhibited by Tof1/Csm3. In conclusion, I showed that Tof1/Timeless and Csm3/Tipin proteins regulate DNA replication and prevent chronic genome instability by minimizing pre-catenation during DNA replication.