Genetic repair of mutations in plant cell-free extracts directed by specific chimeric oligonucleotides

Chimeric oligonucleotides are synthetic molecules comprised of RNA and DNA bases assembled in a double hairpin conformation. These molecules have been shown to direct gene conversion events in mammalian cells and animals thro ugh a process involving at least one protein from the DNA mismatch repair p athway. The mechanism of action for gene repair in mammalian cells has been partially elucidated through the use of a cell-free extract system. Recent experiments have expanded the utility of chimeric oligonucleotides to plan ts and have demonstrated genotypic and phenotypic conversion, as well as Me ndelian transmission. Although these experiments showed correction of point and frameshift mutations, the biochemical and mechanistic aspects of the p rocess were not addressed. In this paper, we describe the establishment of cell-free extract systems from maize (Zea mays), banana (Musa acuminata cv Rasthali), and;tobacco (Nicotiana tabacum). Using a genetic readout system in bacteria and chimeric oligonucleotides designed to direct the conversion of mutations in antibiotic-resistant genes, we demonstrate gene repair of point and frameshift mutations. Whereas extracts from banana and maize cata lyzed repair of mutations in a precise fashion, cell-free extracts prepared from tobacco exhibited either partial repair or non-targeted nucleotide co nversion. In addition, an all-DNA hairpin molecule also mediated repair alb eit in an imprecise fashion in all cell-free extracts tested. This system e nables the mechanistic study of gene repair in plants and may facilitate th e identification of DNA repair proteins operating in plant cells.