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

Fork rotation and DNA precatenation are restricted during DNA replication to prevent chromosomal instability

Faithful genome duplication and inheritance require the complete resolution of all intertwines within the parental DNA duplex. This is achieved by topoisomerase action ahead of the replication fork or by fork rotation and subsequent resolution of the DNA precatenation formed. Although fork rotation predominates at replication termination, in vitro studies have suggested that it also occurs frequently during elongation. However, the factors that influence fork rotation and how rotation and precatenation may influence other replication-associated processes are unknown. Here we analyze the causes and consequences of fork rotation in budding yeast. We find that fork rotation and precatenation preferentially occur in contexts that inhibit topoisomerase action ahead of the fork, including stable protein–DNA fragile sites and termination. However, generally, fork rotation and precatenation are actively inhibited by Timeless/Tof1 and Tipin/Csm3. In the absence of Tof1/Timeless, excessive fork rotation and precatenation cause extensive DNA damage following DNA replication. With Tof1, damage related to precatenation is focused on the fragile protein–DNA sites where fork rotation is induced. We conclude that although fork rotation and precatenation facilitate unwinding in hard-to-replicate contexts, they intrinsically disrupt normal chromosome duplication and are therefore restricted by Timeless/Tipin

Tpz1TPP1 SUMOylation reveals evolutionary conservation of SUMO-dependent Stn1 telomere association

Elongation of the telomeric overhang by telomerase is counteracted by synthesis of the complementary strand by the CST complex, CTC1(Cdc13)/Stn1/Ten1. Interaction of budding yeast Stn1 with overhang-binding Cdc13 is increased by Cdc13 SUMOylation. Human and fission yeast CST instead interact with overhang-binding TPP1/POT1. We show that the fission yeast TPP1 ortholog, Tpz1, is SUMOylated. Tpz1 SUMOylation restricts telomere elongation and promotes Stn1/Ten1 telomere association,and a SUMO-Tpz1 fusion protein has increased affinity for Stn1. Our data suggest that SUMO inhibits telomerase through stimulation of Stn1/Ten1 action by Tpz1, highlighting the evolutionary conservation of the regulation of CST function by SUMOylation