| Research Abstract |
DNA elongation reaction during eukaryotic DNA replication requires a set of replication proteins, PCNA and RFC, which function as a clamp and its loader protein, respectively. So far, their related proteins have been identified, for example, Rad9-1-1,rad17,Chl12,Mgs1, and expected to function as a novel clamp and loader proteins. Due to their similarities with PCNA and RFC, they will have central roles as connectors of various DNA metabolic pathways originated from DNA replication. To elucidate a whole feature of this network of multiple clamp and clamp loader proteins to maintain genome stability during DNA replication, we have studied on their structures and functions.
First, we have reconstituted a novel clamp complex (9-1-1) and a loader complex (Rad17-RFC), both of which are required for the human checkpoint pathway. We further demonstrated that they have indistinguishable structures from those of PCNA and RFC, respectively. Thus, a specific clamp and loader proteins will have a particular role in the checkpoint response pathway. Second, we have reconstituted a loader complex with chromosome cohesion factor, Chl12, and demonstrated that this complex functions as the second PCNA loader. Third, we 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. Furthermore, since it interacts specifically with DNA polymerase δ and stimulates the activity, it will be involved in regulation of DNA synthesis during DNA replication.
These results strongly suggest that multiple PCNA- and RFC-related proteins would organize a huge protein network to coordinate DNA replication and various pathways for precise DNA replication and chromosome segregation.
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