Triplex-induced Recombination in Human Cell-free Extracts

Abstract

Triple helix-forming oligonucleotides (TFOs) can bind to polypurine/polypyrimidine regions in DNA in a sequence-specific manner. Triple helix formation has been shown to stimulate recombination in mammalian cells in both episomal and chromosomal targets containing direct repeat sequences. Bifunctional oligonucleotides consisting of a recombination donor domain tethered to a TFO domain were found to mediate site-specific recombination in an intracellular SV40 vector target. To elucidate the mechanism of triplex-induced recombination, we have examined the ability of intermolecular triplexes to provoke recombination within plasmid substrates in human cell-free extracts. An assay for reversion of a point mutation in the supFG1 gene in the plasmid pSupFG1/G144C was established in which recombination in the extracts was detected upon transformation into indicator bacteria. A bifunctional oligonucleotide containing a 30-nucleotide TFO domain linked to a 40-nucleotide donor domain was found to mediate gene correction in vitro at a frequency of 46 ؋ 10 ؊5, at least 20-fold above background and over 4-fold greater than the donor segment alone. Physical linkage of the TFO to the donor was unnecessary, as co-mixture of separate TFO and donor segments also yielded elevated gene correction frequencies. When the recombination and repair proteins HsRad51 and XPA were depleted from the extracts using specific antibodies, the triplex-induced recombination was diminished, but was either partially or completely restored upon supplementation with the purified HsRad51 or XPA proteins, respectively. These results establish that triplex-induced, intermolecular recombination between plasmid targets and short fragments of homologous DNA can be detected in human cell extracts and that this process is dependent on both XPA and HsRad51.

Branched oligonucleotides induce in vivo gene conversion of a mutated EGFP reporter

“…39 The importance of Rad51 for oligonucleotide-induced gene conversion in cell lines has also been documented for both short DNA fragments 8 and bifunctional, triple helix containing oligonucleotides. 20 b-oligonucleotides that contain more than one mismatch produce consistently higher sequence conversion rates than b-oligonucleotides that contain only a single mismatch. Mismatches between homologous strands strongly reduce the rates of homologous recombination.…”

“…[6][7][8][9] Furthermore, bifunctional oligonucleotides that consist of a triple helix forming DNA recognition sequence, coupled either to DNA intercalating agents like psoralen [10][11][12][13][14][15][16][17][18] or to relatively short DNA domains that should serve as a template for the cell's own recombination or repair machinery, show some promise. [19][20][21][22] Triple helix forming oligonucleotides (TFOs) recognize and require extensive polypurine stretches that are rare in the genome. 23 Attempts have been made to modify bases in the TFOs to tolerate pyrimidine interruptions [24][25][26][27][28] and to increase binding kinetics.…”

“…19,20,30,31 hRad51 is the human homologue of the bacterial RecA protein that plays an important role in homologous recombination and recombinational repair. 32 Both RecA and hRad51 proteins bind readily to single-stranded (ss) DNA and promote DNA pairing and strand exchange with homologous duplex DNA in vitro.…”

Implications of cell cycle progression on functional sequence correction by short single-stranded DNA oligonucleotides

“…Various strategies have been developed to achieve therapeutic targeted gene repair, including small fragment homologous sequence 1,2 ssODN, [3][4][5][6] triple helixforming oligonucleotides containing a reactive group, [7][8][9] bifunctional oligonucleotides based on a triple helixforming DNA recognition domain 10,11 and branched oligonucleotides. 12 Although a general proof of principle for oligonucleotide-based sequence alteration appears to be established, the paradigm for oligonucleotide-based genome alteration remain largely unknown.…”

Genome medicine: gene therapy for the millennium, 30 September–3 October 2001, Rome, Italy

“…One area that has seen significant progress has been gene targeting via oligonucleotides. [13][14][15] These advances include the use small DNA fragments (SDF), [16][17][18][19] chimeric DNA/RNA oligonucleotides (RDOs) 20,21 or triplex-forming oligonucleotides (TFOs) 22,23 to effect sequence-specific modification of genomic DNA. The strengths of the individual gene targeting strategies, when combined, have the potential to develop therapeutic approaches that overcome the limitations of individual strategies into an efficaciously synergistic therapy.…”