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

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. (150 words) Abbreviations: aa amino acids CPT Camptothecin DDC DNA damage checkpoint DPC DNA protein cross-link DRC DNA replication checkpoint HU Hydroxyurea RFB Replication fork barrier RS Replication stress

TIMELESS‐TIPIN and UBXN‐3 promote replisome disassembly during DNA replication termination in Caenorhabditis elegans

“…In both yeast and metazoa, the TIMELESS‐TIPIN complex governs multiple aspects of DNA replication fork biology. For example, TIMELESS and TIPIN are important for the rate of fork progression and are required for activation of the S‐phase checkpoint pathway that helps cells to survive DNA replication stress (Tourriere et al , 2005; Unsal‐Kacmaz et al , 2007; Smith et al , 2009; Westhorpe et al , 2020). In addition, the TIMELESS‐TIPIN complex recruits additional factors to forks, such as the DDX11 helicase that unwinds G4 motifs that can form on the parental DNA template during the course of replication (Lerner et al , 2020).…”

Timeless in animal circadian clocks and beyond

“…4B,C). Similar to its yeast homolog topoisomerase 1-associated factor 1 (tof1) [144], mTIM and its evolutionally conserved partner Tim-interacting protein (TIPIN) maintain replisome stability [145,146] and promote fork progression through hard-to-replicate regions [147][148][149][150][151]. In response to DNA damage, mTIM collaborates with cardinal signaling kinases ataxia telangiectasia-mutated checkpoint kinase 1 (ATR-CHK1) [140,152], ataxia telangiectasia and Rad3-related checkpoint kinase 2 (ATM-CHK2) [153], and poly [ADP-ribose] polymerase 1 (PARP1) [154,155] to facilitate proper checkpoint control and DNA repair [156][157][158].…”