Nuclear Extracts Promote Gene Correction and Strand Pairing of Oligonucleotides to the Homologous Plasmid

Abstract

We compared strand pairing and gene correction activities between different constructs of oligonucleotides, using homologous supercoiled DNA and eukaryotic nuclear extracts. The RNA-DNA chimeric oligonucleotide was more efficient in strand pairing and gene correction than its DNA-DNA homolog. Single-stranded deoxyoligonucleotides showed similar strand pairing and correction activity to the modified RNA-DNA chimeric oligonucleotides, whereas single-stranded ribooligonucleotides did not show either activity. However, the correlations were not always linear, suggesting that only a fraction of the joint molecules may be processed to cause the final gene correction. Several mammalian extracts with markedly different in vitro activity showed the similar amounts of the joint molecules. These results led us to conclude that strand pairing is a necessary event in gene correction but may not be the rate-limiting step. Furthermore, depletion of HsRad51 protein caused large decreases in both strand-pairing and functional activities, whereas supplementation of HsRad51 produced only a slight increase in the repair activity, indicating that HsRad51 participates in the strand pairing, but subsequent steps define the frequency of gene correction. In addition, we found that the structure and stability of intermediates formed by single-stranded deoxyoligonucleotides and RNA-DNA chimeric oligonucleotides were different, suggesting that they differ in their mechanisms of gene repair.