CTF4 and CTF18 are required for high-fidelity chromosome segregation. Both exhibit genetic and physical ties to replication fork constituents. We find that absence of either CTF4 or CTF18 causes sister chromatid cohesion failure and leads to a preanaphase accumulation of cells that depends on the spindle assembly checkpoint. The physical and genetic interactions between CTF4, CTF18, and core components of replication fork complexes observed in this study and others suggest that both gene products act in association with the replication fork to facilitate sister chromatid cohesion. We find that Ctf18p, an RFC1-like protein, directly interacts with Rfc2p, Rfc3p, Rfc4p, and Rfc5p. However, Ctf18p is not a component of biochemically purified proliferating cell nuclear antigen loading RF-C, suggesting the presence of a discrete complex containing Ctf18p, Rfc2p, Rfc3p, Rfc4p, and Rfc5p. Recent identification and characterization of the budding yeast polymerase , encoded by TRF4, strongly supports a hypothesis that the DNA replication machinery is required for proper sister chromatid cohesion. Analogous to the polymerase switching role of the bacterial and human RF-C complexes, we propose that budding yeast RF-C CTF18 may be involved in a polymerase switch event that facilities sister chromatid cohesion. The requirement for CTF4 and CTF18 in robust cohesion identifies novel roles for replication accessory proteins in this process.The establishment of sister chromatid cohesion during S phase is a critical step in the series of events leading to highfidelity cell division. By holding sisters together, cohesion proteins enable kinetochores to face opposite poles of the mitotic spindle, facilitating capture by microtubules from opposite poles (99). The sister chromatid association is sufficient to resist the separating force of the mitotic spindle until each kinetochore has been captured, at which time sister chromatid associations are released at the initiation of anaphase (reviewed in references 50, 72, 77, and 88). Because cohesion tightly binds sisters together from their synthesis to their separation, it must be properly established and maintained in a flexible environment supporting chromatin alterations that permit transcription, replication, repair, and condensation of the genome.Cohesion between sister chromatids is carried out by at least four classes of proteins. The core particle, cohesin, is composed of at least four subunits encoded in budding yeast by the SMC1, SMC3, MCD1 (SCC1), and SCC3 (IRR1) genes (33, 68). Fully assembled cohesin binds chromatin in vitro and in vivo (9, 68, 97, 101). Orthologs of cohesins have been identified in Xenopus laevis, Drosophila melanogaster, Schizosaccharomyces pombe, Arabidopsis thaliana, Mus musculus, and Homo sapiens (6,18,58,59,82,94,100,109,110). Interestingly, although Mcd1p is required for both cohesion and chromosome condensation in budding yeast, these processes are carried out by distinct protein complexes in the Xenopus experimental system (33,40,41,58). In additio...