The equal distribution of the replicated genome to daughter cells is essential for cell survival. Failure to accomplish this step results in aneuploidy leading to decreased viability in yeast and chromosomal loss, developmental defects and malignancies in humans. In order to prevent such chromosomal aberrations, cells have evolved a conserved mechanism to regulate this process. Chromatids are bound together from the time of their duplication in S-phase until their equal distribution between daughter cells during anaphase. Though sister chromatid pairing and partition to daughter cells are under intensive investigation in many laboratories, little is known how sister chromatid cohesion is established. Genetic studies in yeast have identified a number of gene products essential for sister chromatid cohesion. These genes are divided into three groups: structural factors, made up of the four-subunit cohesin complex (Smc1, Scc1, Smc3 and Scc3), which holds sister chromatids together after replication. The cohesin connection to chromatids is severed through the cleavage of the Scc1 subunit by the protease separase during cell division;deposition factors, which include the proteins Scc2 and Scc4 that load cohesin onto DNA and Pds5 which plays a role in maintaining the chromatin-bound cohesin and establishment factors, Ctf7, Ctf4, Ctf18, Dcc1, Ctf8 and ChlR1, which interact with many replication components and function during S-phase. We plan to: A) develop an in vitro system capable of loading cohesin onto DNA. For this purpose, we will isolate the deposition factors Scc2p and Scc4p, examine their interactions with cohesin and determine whether this results in the opening of the cohesin ring and its loading on DNA. The putative role of the pre- replication complex in this reaction will be studied;B) further characterize the in vitro properties of the establishment proteins Ctf18-Dcc1-Ctf8-RFC2-5 (a PCNA clamp loader), Ctf7p (a PCNA interacting protein and acetylase), ChlR1 p (a 5'-3'DNA helicase) and Ctf4p (interacts with Pol alpha). We plan to explore their action in supporting DNA synthesis with DNA substrates that mimic fork-like structures. These studies are directed at exploring a model in which these proteins, all of which appear to be associated with the replication fork, act jointly with other replication factors to remodel the fork and allow its passage through the large cohesin ring.