Chloroplast lysates support directed mutagenesis via modified DNA and chimeric RNA/DNA oligonucleotides

Chimeric RNA/DNA and modified DNA oligonucleotides have been shown to direc t gene-conversion events in vitro through a process involving proteins from several DNA-repair pathways. Recent experiments have extended the utility of these molecules to plants, and we previously demonstrated that plant cel l-free extracts are competent to support oligonucleotide-directed genetic r epair. Using this system, we are studying Arabidopsis DNA-repair mutants an d the role of plant proteins in the DNA-repair process. Here we describe a method for investigating mechanisms of plastid DNA-repair pathways. Using a genetic readout system in bacteria and chimeric or modified DNA oligonucle otides designed to direct the conversion of mutations in antibiotic resista nce genes, we have developed an assay for genetic repair of mutations in a spinach chloroplast lysate system. We report genetic repair of point and fr ameshift mutations directed by both types of modified oligonucleotides. Thi s system enables the mechanistic study of plastid gene repair and facilitat es the direct comparison between plant nuclear and organelle DNA-repair pat hways.