Division of labor of the replication fork protection complex subunits in sister chromatid cohesion and Chk1 activation

Sister chromatid cohesion (SCC) is established during S phase near the replication fork. The cohesin complex, which has a major role in holding two sisters, consists of the Smc1, Smc3, Scc1/Mcd1 and Scc3 subunits (Fig. 1). It has been proposed that the cohesin complex forms a ring-like structure that is designed to entrap two sister chromatids. Since cohesin is loaded onto chromatin before DNA replication, the replication fork is thought to pass through cohesin rings as cells replicate chromosomes.1-3 Considering that the replication fork contains the large replication machinery, cohesin rings may represent a significant obstacle for replication fork progression, leading to fork arrest at cohesin-bound chromosomal sites (Fig. 1). Accordingly, recent studies have focused on understanding how DNA replication is coordinated with the establishment of SCC. Figure 1. Separation of functions between Timeless (Tim) and Tipin (Tip). The replication fork may stall at cohesin-bound sites. ssDNA accumulated at stalled forks is coated by RPA bound by Tipin and ATRIP, which recruit Timeless and ATR, respectively. ... It is widely understood that stalled forks activate the S-phase checkpoint. In response to stalled forks, the ATR kinase transduces a signal to phosphorylate the effector kinase Chk1 in a manner dependent on mediator proteins such as Claspin, Timeless and Tipin, resulting in cell cycle arrest to allow time for DNA repair (Fig. 1).4 It has been proposed that S-phase checkpoint genes also safeguard sister chromatid cohesion in yeast.5 However, how this checkpoint controls SCC is not known. In a recent report, Smith-Roe et al.6 carefully analyzed cohesion defects in the absence of Timeless and Tipin, which are known to form the replication fork protection complex (FPC).7 Previous studies showed that the Timeless-Tipin FPC is involved in a variety of genome maintenance processes, including Chk1 activation, replication fork stabilization and SCC.8 However, how the FPC coordinates such multiple mechanisms is enigmatic. Smith-Roe et al.6 found that Timeless depletion causes a strong defect in SCC, whereas depletion of its partner Tipin has only minor effects on SCC. They also tested the involvement of other S-phase checkpoint factors, including ATR, Chk1 and Claspin. They found that Chk1 is not required for cohesion, while ATR and Claspin depletion only cause minor cohesion problems, similar to Tipin depletion. What are these results telling us? Why do these S-phase checkpoint factors have different influences on SCC? It has been reported that Tipin binds replication protein A (RPA) and recruits Timeless to single-stranded DNA (ssDNA), an intermediate generated at stalled forks in response to replication stress.9 Importantly, Tipin also recruits Claspin to ssDNA,9 and Claspin is essential for the phosphorylation of Chk1 by ATR.4 Since ATR is also recruited to ssDNA via interaction of its partner ATRIP and RPA,4 it is suggested that Tipin-mediated Claspin recruitment to the fork promotes the phosphorylation of Chk1 by ATR, resulting in activation of Chk1 followed by cell cycle arrest (Fig. 1).9 Then what is the role of Timeless? Timeless is also required for Chk1 activation.8,9 However, its role in Chk1 activation may be stabilization of Tipin, as Timeless downregulation leads to the reduced level of Tipin.10 These observations suggest that Timeless and Tipin have separate roles at the fork. Tipin seems to play an important role as a mediator of Chk1 activation by recruiting Claspin to ssDNA. In contrast, once recruited to the fork, Timeless appears to play a critical role in the establishment of SCC, which is independent of Tipin. Therefore, the work by Smith-Roe et al.6 strongly suggests that there is a division of labor between Timeless and Tipin at the replication fork. What would be the possible mechanisms for Timeless-dependent SCC? Interestingly, Leman et al. showed that Timeless interacts with cohesin subunits, whereas Tipin-cohesin interaction is weak. They also showed that cohesin subunits are dissociated from chromatin in Timeless-depleted cells.7 It has been suggested that Ctf7/EcoI-dependent acetylation of cohesin loosens the ring to allow fast progression of the replication fork.3 This situation could lead to temporal dissociation of cohesin from the chromatin, unless there is a mechanism to sustain cohesin subunits. Timeless seems to be in a perfect position to carry out this job, holding cohesin subunits to prevent their dissociation from chromosomes when the fork passes through the ring. Since Timeless is also important for replication recovery after fork arrest,7 it is also possible that Timeless regulates replisome assembly to allow for resumption of replication fork progression every time the fork stalls at cohesin sites. Further research would answer these questions and reveal the sophisticated mechanisms by which Timeless coordinates replication fork progression and SCC.