In vitro and in vivo nucleotide exchange directed by chimeric RNA/DNA oligonucleotides in Saccharomyces cerevisae

Targeted gene repair directed by chimeric RNA/DNA oligonucleotides has prov en successful in eukaryotic cells including animal and plant models, In man y cases, however, there has been a disparity in the levels of gene correcti on or frequency, While the delivery of these chimera into the nucleus and t he long-term stability or purity of these molecules may contribute to this variability, understanding the molecular regulation of conversion is the ke y to improving or stabilizing frequency. To this end, we have identified ge nes that control targeted repair, using the genetically tractable organism, Saccharomyces cerevisae and a bank of yeast mutants. Results from experime nts in cell-free extracts focused our attention on RAD52, RAD1 and RAD59 as central regulatory factors, RAD1 and RAD59 appear to be required for high levels of conversion whereas RAD52 appears to act, surprisingly, in a suppr essive fashion. Results from the in vitro experiments were translated into targeting experiments in vivo. Here, mutations in a fusion construct, conta ining a marker gene, were converted to wild type, evidenced by the expressi on of green fluorescence in converted cells. Because the repaired fusion ge ne contains a corrected neomycin sequence, cells were subsequently placed u nder G418 selection and conversion confirmed at the genetic level, Taken to gether, these results establish, for the first time, genes that participate in the regulation of targeted gene repair and provide a novel system for e valuating true frequencies of correction, Importantly, this system enables visualization of corrected (green) and uncorrected (clear) cells enabling m easurements of conversion in real time.