Chimeric DNAJRNA double hairpins are synthetic oligonucleotides that direct targeted nucleotide substitutions in bacteria, yeast, plants and animals. We have postulated that cellular recombinases catalyze joint molecule formation between these oligonucleotides and homologous dsDNA. According to our model, resolution of these joints can be accompanied by point mutation of the target DNA when the oligonucleotide is mismatched within the intermediate joint. Since chimeric hairpins are effective gene repair agents in yeast, we propose to validate our model in this microorganism. Experiments will be carried out with whole cells, cell-free extracts, and purified protein with the goal of delineating the mechanism of joint molecule formation and gene substitution or frameshift mutations in yeast will be monitored by targeting episomal or chromosomal genes that express a fluorescent signal or an antibiotic resistance marker. Targeting of the episomal target in different knock out strains will drive the discovery of formation between chimeric hairpins and dsDNA will be optimized using gene products from the RAD52 epistasis group either alone or in combination with other related enzymes. Nucleoprotein intermediates formed during strand exchange will be probed to elucidate how a chimeric DNAIRNA backbone enhances recombination. Performed double D-loop joints that contain a specific mismatch or unpaired base will be used as substrates to study targeted nucleotides exchange. The mechanism which emerges from this study should provide guidance in the use of chimeric hairpin oligonucleotides in other systems including plant, animal and human cells.
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