Genetic Repair of Mutations in Plant Cell-Free Extracts Directed by Specific Chimeric Oligonucleotides

Abstract

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 through a process involving at least one protein from the DNA mismatch repair pathway. 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 plants and have demonstrated genotypic and phenotypic conversion, as well as Mendelian transmission. Although these experiments showed correction of point and frameshift mutations, the biochemical and mechanistic aspects of the process 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 catalyzed repair of mutations in a precise fashion, cell-free extracts prepared from tobacco exhibited either partial repair or non-targeted nucleotide conversion. In addition, an all-DNA hairpin molecule also mediated repair albeit in an imprecise fashion in all cell-free extracts tested. This system enables the mechanistic study of gene repair in plants and may facilitate the identification of DNA repair proteins operating in plant cells.Chimeric RNA/DNA oligonucleotides (chimeras) have been used to direct single base changes in episomal and chromosomal targets in mammalian cells Yoon et al., 1996; Alexeev and Yoon, 1998;Kren et al., 1998;Lai and Lien, 1999). These molecules have also been effective in mediating similar reactions in plant cells (Beetham et al., 1999;Zhu et al., 1999). Independently, these two groups demonstrated that mutations can be corrected in tobacco (Nicotiana tabacum) and maize (Zea mays) cells. Using a marker gene system, Zhu et al. (1999) corrected a mutation in vivo and showed that the targeted base was in fact altered as directed by the chimeric oligonucleotide. Although Beetham et al. (1999) were able to recover herbicide-resistant plant cells due to the action of the chimera, the targeted base in tobacco cells was not changed. Instead, the nucleotide located at the 5Ј side of the target base was mutated. Because such a change would also produce an amino acid alteration that would confer herbicide resistance, the plant cells were recoverable under the appropriate selection. These results were the first to demonstrate degeneracy in targeted gene repair because experiments in mammalian cells produced only precise nucleotide alterations.The process by which these nucleotide conversions are made is still undefined, but recent evidence suggests that mismatch repair plays a critical role in mammalian cells. Using c...

Re-engineering plant gene targeting

“…Whether this in vivo lack of precision, or slippage, is limited to certain plant species is unknown. However, these 'ectopic' conversions are not observed in plasmid targeting experiments involving maize or Arabidopsis cell free extracts (CFE) and have only been observed in vitro when using tobacco CFEs [71].…”

“…To help elucidate the factors involved in the conversion mechanism and as a system to study plant DNA repair mechanisms, a cell-free assay utilizing protein extracts from either plastids or whole cells has been established [71,72]. This work has also provided a mechanistic explanation of the in vivo plant results.…”

In vivo gene repair of point and frameshift mutations directed by chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides

“…These vectors have been used to repair point and frameshift mutations in mammalian cell culture (3)(4)(5)(6)(7)(8)(9), cell-free extracts (10)(11)(12) and animal models (13)(14)(15). In addition, gene repair using COs has been carried out successfully in plants ( [16][17][18] and plant cell-free extracts (19).…”

Repair of Damaged DNA by Arabidopsis Cell Extract

“…To the best of our knowledge, this approach has not been used to monitor DNA repair in plants. The only related assay was reported by Gamper et al (2000) and Rice et al (2000) for the estimation of mismatch repair proficiency of plant cell-free extracts. They used chimeric RNA/DNA oligonucleotides as a repair template to monitor the conversion of point and frameshift mutations on plasmid DNA.…”