Abstract
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.
Highlights
The faithful replication of the genome by the DNA replication machinery is hindered by a range of exogenous and endogenous factors that are capable of disrupting replication forks
We find that the far C terminus is required to resolve DNA topological stress but not for fork pausing, replisome coupling, CPT resistance or DNA replication checkpoint (DRC) activation signalling
Defining how the Timeless protein functions to maintain genome stability, both generally and following replication stress (RS) has been complicated by its multiple functions
Summary
The faithful replication of the genome by the DNA replication machinery is hindered by a range of exogenous and endogenous factors that are capable of disrupting replication forks. Such cellular events are a prominent feature of the early stages of cancer and have been collectively referred to as replication stress (RS) [1]. On encountering RS, the replisome is thought to be stabilised at the replication fork until the impeding context is removed or bypassed and replication restarted [4]. A failure to stabilise the replisome is associated with erroneous processing of the replication fork, leading to either toxic recombination intermediates or disruption of replication restart
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