Replisome progression complex links DNA replication to sister chromatid cohesion in Xenopus egg extracts

Abstract

Cohesin-mediated sister chromatid cohesion is established during the S-phase, and recent studies demonstrate that a cohesin protein ring concatenates sister DNA molecules. However, little is known about how DNA replication is linked to the establishment of sister chromatid cohesion. Here, we used Xenopus egg extracts to show that AND-1 and Tim1-Tipin, homologues of Saccharomyces cerevisiae Ctf4 and Tof1-Csm3, respectively, are associated with the replisome and are required for proper establishment of the cohesion observed in the M-phase extracts. Immunodepletion of both AND-1 and Tim1-Tipin from the extracts leads to aberrant sister chromatid cohesion, which is similarly induced by the depletion of cohesin. These results demonstrate that AND-1 and Tim1-Tipin are key factors linking DNA replication and establishment of sister chromatid cohesion. On the basis of the physical interactions between AND-1 and DNA polymerases, we discuss a model to describe how replisome progression complex establishes sister chromatid cohesion.

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

“…30,36 Some models of replication-coupled cohesion suggest that fork uncoupling may take place during interactions between replisomes and cohesin rings, an event that would activate Chk1. 19,20,28,30 Experiments described herein have replicated in genetically stable human fibroblast lines the observation that Tim-Tipin and Claspin contribute to ATR-dependent activation of Chk1 in response to fork uncoupling by DNA damage, namely UVC-induced photoproducts (Fig. 1A and B).…”

“…30 Reduction of Tim-Tipin and Claspin orthologs/analogs in various systems results in premature separation of sister chromatids in metaphase, a phenotype that could arise from incomplete establishment of cohesion during S phase or precocious removal of cohesins in metaphase. Studies using the Xenopus laevis egg extract system demonstrated that Tim1 specifically was required during replication for full paring of sister chromatids in metaphase, 19 which, taken together with other observations, indicates a role for Timeless orthologs in the establishment of SCC.…”

“…In various models, Timeless and Tipin orthologs have been shown not only to interact with cohesin ring subunits and to promote their association with chromatin, but also to interact with other replisome-associated proteins that contribute to normal SCC. These proteins include AND-1, 18,19 which has similarities to Tim-Tipin, including interaction with replicative DNA polymerases and promotion of normal DNA synthesis, 40 and ChlR1, 9 a helicase involved in lagging strand maturation, 41 that is associated with Warsaw breakage syndrome, a cohesinopathy. 24 During the course of DNA replication, replisomes are remodeled when polymerization is stalled or blocked by exogenous and endogenous challenges.…”

“…30 Some models posit that the association of cohesin rings with replisomes may require pausing of replication forks 23,30 an event that would require replisome stabilization through Tim-Tipin and Claspin. 19,20,31 The studies described here examined whether Timeless, Tipin and Claspin contribute to SCC in genetically stable, diploid human fibroblasts as part of, or independently of, their interactions with the checkpoint kinases ATR and Chk1. Even though Timeless and Tipin are known to form a heterodimeric complex, 3 targeting Timeless by siRNA-mediated knockdown produced a 10-fold greater increase in defective SCC compared to targeting Tipin.…”

“…3,6,17 Timeless, Tipin and Claspin orthologs/analogs also contribute to sister chromatid cohesion (SCC) in various organisms. 9,[18][19][20][21][22] SCC is required for identical partitioning of duplicated genomes to daughter cells. The cohesin complex, a multi-subunit ring, creates physical linkages between sister chromatids.…”