Triplex-induced recombination in human cell-free extracts - Dependence on XPA and HsRad51

Triple helix-forming oligonucleotides (TFOs) can bind to polypurine/polypyr imidine regions in DNA in a sequence-specific manner. Triple helix formatio n has been shown to stimulate recombination in mammalian cells in both epis omal and chromosomal targets containing direct repeat sequences. Bifunction al oligonucleotides consisting of a recombination donor domain tethered to a TFO domain were found to mediate site-specific recombination in an intrac ellular SV40 vector target. To elucidate the mechanism of tripler-induced r ecombination, we have examined the ability of intermolecular triplexes to p rovoke recombination within plasmid substrates in human cell-free extracts. An assay for reversion of a point mutation in the supFG1 gene in the plasm id pSupFG1/G144C was established in which recombination in the extracts was detected upon transformation into indicator bacteria. A bifunctional oligo nucleotide containing a 30-nucleotide TFO domain linked to a 40-nucleotide donor domain was found to mediate gene correction in vitro at a frequency o f 46 x 10(-5), at least 20-fold above background and over 4-fold greater th an the donor segment alone. Physical linkage of the TFO to the donor was un necessary, as co-mixture of separate TFO and donor segments also yielded el evated gene correction frequencies. When the recombination and repair prote ins HsRad51 and XPA were depleted from the extracts using specific antibodi es, the tripler-induced recombination was diminished, but was either partia lly or completely restored upon supplementation with the purified HsRad51 o r XPA proteins, respectively. These results establish that tripler-induced, intermolecular recombination between plasmid targets and short fragments o f homologous DNA can be detected in human cell extracts and that this proce ss is dependent on both XPA and HsRadB1.