Researches on functional link between DNA replication and chromosomal cohesion through PCNA clamp

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1. Functional interaction of clamp loader complexes with DNA polymerase etaOne of clamp loader complexes, Ctf18-RFC, which consists of the chromosome cohesion factors Ctf18, Dccl, and Ctf8 and four small subunits of RFC, functions as a second PCNA loader. We identified Pol eta as a DNA polymerase activity stimulated by Ctf18-RFC but not by RFC. Ctf18-RFC stimulated Pol eta on its own, and the addition of PCNA cooperatively increased that stimulation. Furthermore, Ctf18-RFC interacted physically with pol eta. In addition, we observed the similar physical interaction of RFC with pol eta, but affecting its DNA synthesis activity differently from Ctf18-RFC. We propose that the novel loader-DNA polymerase interactions can switch the activity of a DNA replication fork to adapt to various template structures.2. Molecular interaction between loader complexes and replication fork componentsUsing human cell lines ectopically expressing tagged loader complexes, FLAG-RFC, FLAG-(Rad17-RFC), FLAG-(Ctf18-RFC), we have identified Pol epsilon and RPA as common loader binding proteins. Further studies with recombinant proteins have indicated that their interactions are direct and specific. All loaders can associate with these proteins through their common small subunits, and also through specific subunits, suggesting roles to maintain functional protein assemblies at the replication fork.3. Functions of WRNIP1 proteinWe purified one of RFC-related proteins, WRNIP1, which has been identified as a Werner DNA helicase binding protein. This protein forms a self-oligomerized complex and exhibits ATPase activity. It also interacts specifically with replicative DNA polymerase delta and stimulates the activity, suggesting its role in regulation of DNA synthesis by Pol delta.