In eukaryotic cells the regulation and mechanism of nuclear DNA replication is poorly understood. Our long-term objective is to gain better understanding of this process on the molecular level. The models systems used in these studies is a Saccharomyces cerevisiae origin of replication clone on plasmids. We have made the following major steps pointed towards achieving these goals: (i) we have delineated an yeast chromosomal origin of replication, the ARS121; (ii) we have purified and clone a protein, OBF1(also known as ABF1 and BAF1), that binds specifically to a DNA replication enhancer found in ARS121 and other yeast origins; (iii) we have now reconstituted in vitro an OBF1-dependent, putative multiprotein initiation complex at he ARS121 origin. These studies suggest that the function of OBF1 in initiation of DNA replication is to interact with another factor, OBF2, to promote the ATP-dependent binding of a third factor, CBF; (iv) we have shown that OBF1 is phosphorylated in vivo at multiple serine residues; (v) we have shown cell cycle-specific phosphorylation of a least three sites in the OBF1 protein; (vi) we have identified and purified a multiprotein complex kinase. These findings suggest a possible regulations of OBF1 functions by phosphorylation. The short-term objectives of this proposal are: (i) to elucidate the mechanism of the assembly of the putative initiation complex at the ARS121 origin. This will involve the purification and cloning of OBF2 and CBF and the analysis of protein-DNA and Protein-protein interactions at the ARS121 origin; (ii) to delineate the OBFI domains important for its function in replication; (iii) to understand the regulation of OBF1 function by phosphorylation. This will involve the identification, isolation and cloning of the kinases and phosphorylation. This will involve the identification, isolation and cloning of the kinases and phosphatases. It will also involve both in vitro and vivo analysis of the effect of ser/ala mutations in the OBFI phosphorylation sites on OBFI function replication.
