The mechanics of DNA replication have been extensively studied in prokaryotic systems, but comparatively little is known about this process in eukaryotic organisms. Understanding the biochemistry of how genetic information is faithfully replicated and accurately segregated to daughter cells is an important part of understanding both genetic disorders and normal development processes. The objective of this proposal is to isolate components of the DNA replication complex from a eukaryotic source in order to reveal the architecture of this protein machine in eukaryotic cells. DNA polymerase accessory proteins will be isolated from the yeast S. cerevisiae by using DNA polymerase protein affinity chromatography to take advantage of the protein-protein interactions required to assemble and maintain a polymerase holoenzyme. Proteins that interact directly with the polymerases will be retained and partially purified by insoluble matrices containing covalently attached yeast DNA polymerases. The effects these accessory proteins have on the rate, processivity, accuracy and template specificity of the polymerases will be measured in vitro. The roles played by these proteins in vivo will be studied after isolating the genes that encode them and replacing the normal copies of these genes with mutant versions. The viability, rates of chromosome loss and recombination, and sensitivity to DNA damage in the absence of functional DNA polymerase accessory proteins will be determined. Information from the properties of the accessory proteins in both biochemical and genetic assays will be combined to reveal the roles played by these proteins in copying and maintaining the integrity of the yeast genome.
| Miles, J; Formosa, T (1992) Evidence that POB1, a Saccharomyces cerevisiae protein that binds to DNA polymerase alpha, acts in DNA metabolism in vivo. Mol Cell Biol 12:5724-35 |
| Miles, J; Formosa, T (1992) Protein affinity chromatography with purified yeast DNA polymerase alpha detects proteins that bind to DNA polymerase. Proc Natl Acad Sci U S A 89:1276-80 |
