Accurate DNA replication is essential for long-term survival. While the core catalytic machinery needed to synthesize DNA has been well- characterized in prokaryotes and to a lesser extent in eukaryotes, components that promote efficiency of replication have received less attention. While such factors are necessary for faithful genome duplication, they are difficult to study using conventional methods since they would have only subtle effects on cell-free assays and genetic perturbations would be hard to interpret mechanistically. This proposal explores the usefulness of methods that exploit associations between proteins to identify and characterize the components of the replication complex in the yeast Saccharomyces cerevisiae. Yeast DNA polymerase alpha has been overexpressed, purified, and used as the ligand in protein affinity chromatography experiments in which proteins that interact with polymerase are retained from an extract by a matrix containing covalently attached Polalpha molecules. Binding proteins are sequenced, the genes encoding them are isolated, and cells carrying mutations in these genes are produced. The properties of one such binding protein has been studied both by in vitro assays and by examining the phenotypic consequences of mutations. At least four additional polymerase binding proteins have been identified and will be characterized. Other methods that exploit protein-protein interactions will also be used. Antibodies against replication proteins have been prepared and will be used to detect stable interactions by co-immunoprecipitation. Genetic suppressors of temperature-sensitive mutations in two essential DNA polymerase genes will be studied using a modification of an existing transposon-based mutagenesis procedure. Proteins that interact with DNA polymerase alpha in vivo will be identified and characterized using the """"""""two hybrid"""""""" method in which the protein-protein interaction links a DNA recognition element to a transcriptional activator. Methods that rely only on the interactions between proteins to reconstruct complexes can reveal information about essential accessory factors and about important fidelity factors. These methods will be used to characterize aspects of DNA replication in a eukaryotic cell that have been refractory to traditional methods.
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