Investigating the functions of Tof1/Timeless in Saccharomyces cerevisiae and human cells

Aberrant DNA replication is a major source of genome instability in cells. The evolutionarily conserved Fork Protection Complex (FPC) consists of Tof1/Csm3 and Mrc1 in S. cerevisiae or Timeless/Tipin and Claspin in higher eukaryotes. FPC proteins travel with replication forks to mediate intra-S-checkpoint signalling and replisome stability in response to DNA replication stress (RS). However, the exact mechanisms by which the FPC mediates these functions remain unclear. This thesis has addressed this problem in the budding yeast S. cerevisiae through the generation and characterisation of a series of Tof1 mutants. Specifically, using C-terminal Tof1 truncation mutants this thesis provides novel insights into the structure-function relationship of Tof1 in S. cerevisiae and the various roles this protein plays in responding to RS. I have demonstrated that Tof1 plays a specific role in responding to topological stress that is distinct from its other functions, and identified a second domain within the C-terminus of the protein that is required for suppression of camptothecin (CPT) induced lethality of tof1? cells, for replication fork pausing and for coupling helicase and polymerase activities in the presence of hydroxyurea (HU). I have also shown that the aforementioned functions of Tof1 are not dependent on intra-S-checkpoint activation, as an N-terminal segment alone of Tof1 is capable of checkpoint activation in response to HU. Together these results give a novel insight into how the Tof1 protein responds to different types of RS, and show that it operates through distinct domains capable of dealing with different obstacles to DNA replication. Lastly, in addition to this work in S. cerevisiae I have targeted the endogenous TIMELESS gene in human cells with an inducible degron tag, in attempt to generate conditional Timeless cell lines. This work has further supported the evidence for an essential role for Timeless in mammals.