The overall goal of our research remains the elucidation of the mechanism by which chromosomes replicate in eukaryotic cells using yeast as a model system. The progress made during the last two years of this project has defined our goals and our research is now centered on the multimeric DNA polymerase I and an ARS binding factor. The program has become progressively molecular to take advantage of our progress in the purification of a highly processive multimeric DNA polymerase I, purifying and cloning the DNA primase, and in cloning the ARS binding factor I, the putative factor involved in the initiation of yeast DNA replication. With regard to the multimeric polymerase I our goals are: completion of the purification and characterization of the multimeric primase-polymerase complex, (2) completion of the characterization of the primase gene, (3) analysis of the effect of a newly-discovered yeast single stranded DNA binding protein (yeast SSB) on the functions of the multimeric DNA polymerase I, (4) characterization of the multimeric primase-polymerase associated enzymatic activities, individual subunits, and reconstitution of the multimeric polymerase activity, (5) characterization of the exonuclease activity associated with the complex and purification of the polypeptide. ARS Binding Factor I is a unique DNA binding protein that binds with high specificity to homologous sequences at various ARSs and appears to be involved in the initiation of DNA replication. Thus, elucidation of the role of this factor in ARSs is important for overall understanding of the mechanism of eukaryotic DNA replication. We have isolated a single copy gene for this factor and our goals for further elucidation of its functions are as follows: (1) determination of the primary structure of ABFI by DNA sequencing, (2) isolation of mutants and analysis of the functions of ABFI in vivo and in vitro.
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