Timeless functions independently of the Tim-Tipin complex to promote sister chromatid cohesion in normal human fibroblasts

The Timeless-Tipin complex and Claspin are mediators of the ATR-dependent activation of Chk1 in the intra-S checkpoint response to stalled DNA replication forks. Tim-Tipin and Claspin also contribute to sister chromatid cohesion (SCC) in various organisms, likely through a replication-coupled process. Some models of the establishment of SCC posit that interactions between cohesin rings and replisomes could result in physiological replication stress requiring fork stabilization. The contributions of Timeless, Tipin, Claspin, Chk1 and ATR to SCC were investigated in genetically stable, human diploid fibroblast cell lines. Whereas Timeless, Tipin and Claspin showed similar contributions to UVC-induced activation of Chk1, siRNA-mediated knockdown of Timeless induced a 100-fold increase in sister chromatid discohesion, whereas the inductive effects of knocking down Tipin, Claspin and ATR were 4–20-fold. Knockdown of Chk1 did not significantly affect SCC. Consistent findings were obtained in two independently derived human diploid fibroblast lines and support a conclusion that SCC in human cells is strongly dependent on Timeless but independent of Chk1. Furthermore, the 10-fold difference in discohesion observed when depleting Timeless versus Tipin indicates that Timeless has a function in SCC that is independent of the Tim-Tipin complex, even though the abundance of Timeless is reduced when Tipin is targeted for depletion. A better understanding of how Timeless, Tipin and Claspin promote SCC will elucidate non-checkpoint functions of these proteins at DNA replication forks and inform models of the establishment of SCC.Key words: cohesion, intra-S checkpoint, Timeless, Tipin, Claspin, ATR, Chk1, human, fibroblast