Triple-Helix Formation Induces Recombination in Mammalian Cells via a Nucleotide Excision Repair-Dependent Pathway

Abstract

The ability to stimulate recombination in a site-specific manner in mammalian cells may provide a useful tool for gene knockout and a valuable strategy for gene therapy. We previously demonstrated that psoralen adducts targeted by triple-helix-forming oligonucleotides (TFOs) could induce recombination between tandem repeats of a supF reporter gene in a simian virus 40 vector in monkey COS cells. Based on work showing that triple helices, even in the absence of associated psoralen adducts, are able to provoke DNA repair and cause mutations, we asked whether intermolecular triplexes could stimulate recombination. Here, we report that triple-helix formation itself is capable of promoting recombination and that this effect is dependent on a functional nucleotide excision repair (NER) pathway. Transfection of COS cells carrying the dual supF vector with a purine-rich TFO, AG30, designed to bind as a third strand to a region between the two mutant supF genes yielded recombinants at a frequency of 0.37%, fivefold above background, whereas a scrambled sequence control oligomer was ineffective. In human cells deficient in the NER factor XPA, the ability of AG30 to induce recombination was eliminated, but it was restored in a corrected subline expressing the XPA cDNA. In comparison, the ability of triplex-directed psoralen cross-links to induce recombination was only partially reduced in XPA-deficient cells, suggesting that NER is not the only pathway that can metabolize targeted psoralen photoadducts into recombinagenic intermediates. Interestingly, the triplex-induced recombination was unaffected in cells deficient in DNA mismatch repair, challenging our previous model of a heteroduplex intermediate and supporting a model based on end joining. This work demonstrates that oligonucleotidemediated triplex formation can be recombinagenic, providing the basis for a potential strategy to direct genome modification by using high-affinity DNA binding ligands.

DNA triple helices: Biological consequences and therapeutic potential

“…These features, along with facile chemistries to covalently couple DNA damaging agents to the TFOs, make triplexes attractive probes for directing site-specific DNA damage [36][37][38]. Targeting DNA damage to a specific site via triplex formation can be used to induce mutations [32,39,40] and/or recombination in vitro and in vivo [41][42][43], presumably through recognition of the triplex structures as damage by the repair machinery of the cell [31,32,[44][45][46]. Thus, triplex technology provides a mechanism to modify gene structure and function in living organisms [39,47].…”

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

“…[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.…”

Human XPA and RPA DNA repair proteins participate in specific recognition of triplex-induced helical distortions

“…To address this question we tested a triplex formed on a different TFO-recognition site, the supFG1 triplex site, which has been well characterized (27,30,34,35). Triplexes formed at this site are thought to be substrates for NER (28)(29)(30). We used a radiolabeled 188-bp plasmid fragment containing a 5Ј AT psoralen crosslinking site at the triplex-duplex junction (Fig.…”

“…In addition, intermolecular triplex formation has been found to stimulate recombination in mammalian cells and cell-free extracts (28,29). Evidence suggests that the ability of triplexes to induce mutagenesis and recombination depends on the capacity of triplex structures to provoke DNA repair (28)(29)(30). Moreover, it has been suggested that intramolecular triplex DNA structures may exist transiently in vivo and play a role in gene expression and genomic instability (31,32).…”

“…Recently, we have found that systemically administered TFOs can induce mutagenesis of a targeted gene in somatic cells of mice (27). In addition, intermolecular triplex formation has been found to stimulate recombination in mammalian cells and cell-free extracts (28,29). Evidence suggests that the ability of triplexes to induce mutagenesis and recombination depends on the capacity of This paper was submitted directly (Track II) to the PNAS office.…”