A detailed analysis of the yeast replicative DNA polymerase, DNA pol I, is being undertaken at the molecular and genetic level. The objectives of this project are to map and clone the gene for DNA pol I, to identify subunits and accessory proteins that influence DNA pol I, activity and, ultimately, to identify the proteins that regulate its activity on native DNA templates and to determine the mechanism of their interactions. Using purified DNA pol I, we have identified several proteins that stimulate its synthetic activity. These include three different RNase H proteins, three different single-stranded DNA binding proteins and a DNA-dependent ATPase (ATPase III) that possesses a helicase activity. An aphidicolin-sensitive (aph-s) strain of yeast has been used to identify several possible DNA pol I clones. Acquisition of the pol I gene in a high copy number plasmid would presumably confer an aphidicolin-resistant phenotype to the transformed aph-s cell. Analysis of aphidicolin-resistant transformants showed that three different DNA sequences transformed cells to aphidicolin resistant. All three produced slightly increased levels of pol I activity in crude extracts. These DNA sequences are being subcloned to determine the ends of the aphidicolin-resistance genes by deletion mapping. An internal fragment of the aphidicolin-resistance gene will then be cloned into the yeast integration vector YIp5 for a gene disruption experiment to determine the requirement of the intact gene for cell viability. The pol I gene is expected to be essential for viability.
