Separable functions of Tof1/Timeless in intra-S-checkpoint signalling, replisome stability and DNA topological stress

The highly conserved Tof1/Timeless proteins minimise replication stress and promote normal DNA replication. They are required to mediate the DNA replication checkpoint (DRC), the stable pausing of forks at protein fork blocks, the coupling of DNA helicase and polymerase functions during replication stress (RS) and the preferential resolution of DNA topological stress ahead of the fork. Here we demonstrate that the roles of the Saccharomyces cerevisiae Timeless protein Tof1 in DRC signalling and resolution of DNA topological stress require distinct N and C terminal regions of the protein, whereas the other functions of Tof1 are closely linked to the stable interaction between Tof1 and its constitutive binding partner Csm3/Tipin. By separating the role of Tof1 in DRC from fork stabilisation and coupling, we show that Tof1 has distinct activities in checkpoint activation and replisome stability to ensure the viable completion of DNA replication following replication stress. ChIP-seq experiments: In the absence of Tof1 or Mrc1 the helicase advances beyond the point of nascent DNA incorporation, indicating uncoupling of helicase and polymerase activities in the replisome. This uncoupling is predicted to lead to increased binding of the single stranded DNA binding protein, RPA1, to the single stranded regions generated by uncoupled helicase action. We have experimentally confirmed this prediction using RPA1 ChIP-SEQ in HU treated wildtype, tof1 and mrc1 cells and compared to various tof1 mutants. Sync-seq experiments: We used sync-seq technique follow DNA replciation restart after HU arrest in various tof1 mutants by comparing relative DNA copy numbers from cells in G1, 60 min HU treated and 80 min after HU release.