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 oligonucleotide-mediated triplex formation can be recombinagenic, providing the basis for a potential strategy to direct genome modification by using high-affinity DNA binding ligands.

FIG. 1

Schematic representation of the pSupFAR vector. The SV40-based shuttle vector contains two mutant supF genes in the form of a tandem dimer. The upstream mutant supF gene, supF1, contains a C-to-G point mutation at nucleotide position 163; the downstream mutant supF gene, supF2, contains a G-to-A point mutation at nucleotide position 115. The two supF genes are separated by a 9-bp sequence that contains an EagI restriction site used for cloning. At the 3′ end of supF1 is an engineered polypurine sequence (bp 167 to 196), creating a high-affinity third-strand binding site. A purine-rich oligonucleotide with a length of 30 nucleotides (AG30) was designed to form a triple helix in the anti-parallel triplex motif at this site, as shown. As a control, SCR30, containing the same base composition but a scrambled sequence, was used. In some experiments, the AG30 and the SCR30 oligonucleotides were conjugated at their 5′ end to 4′-hydroxymethyl-4,5′,8-trimethylpsoralen via the 4′ hydroxymethyl position, as shown in the case of pso-AG30. In this case, by formation of the triple helix, psoralen intercalation is targeted to the duplex-triplex junction at bp 166 to 167 of supF1. Upon photoactivation with UVA, both monoadducts and interstrand cross-links are generated at the thymidines in these base pairs.

FIG. 2

Triplex-induced recombination in COS cells. Cells pretransfected with the pSupFAR vector were subsequently transfected with the indicated oligonucleotides. After 48 h, shuttle vector DNA was isolated from the cells for analysis of supF gene function and quantification of recombination events. The number of blue colonies (representing recombinants) out of the total number of colonies is presented to the right of each bar, with the bars indicating recombination frequency. One sample received only irradiation with UVA light (1.8 J/cm² of broad band long wavelength UV light centered at 365 nm) and no oligonucleotide, as indicated. In the case of pso-AG30 plus UVA, the irradiation was administered 2 h after TFO transfection.

FIG. 3

Triplex-induced recombination in human repair-proficient and repair-deficient cells. The cells, either XPA (XP20S, deficient in the damage recognition factor XPA) or XPA corrected [XP20S(pCAH19WS) cells, a subline of XP2OS expressing wild-type XPA cDNA], were pretransfected with the pSupFAR vector and subsequently transfected with the indicated oligonucleotides. After 48 h, shuttle vector DNA was recovered and analyzed as described for Fig. 2.

FIG. 4

Recombination induced by triplex-directed psoralen adducts in human repair-proficient and repair-deficient cells. Cells included either XPA (XP2OS, deficient in the NER damage recognition factor XPA) or XPA corrected [XP2OS(pCAH19WS) cells, a subline of XP2OS expressing wild-type XPA cDNA], along with cells from patients with xeroderma pigmentosum groups F and G, both of which are deficient in specific endonuclease activities required for NER. As indicated, the pSupFAR vector DNA was incubated in vitro with pso-AG30, followed by UVA irradiation to generate triplex-directed, psoralen photoadducts. The vector-triplex-psoralen adduct complexes were transfected into the indicated cells. Control samples included vector DNA irradiated with UVA in the absence of any oligonucleotide and then transfected directly into the various cell lines, as indicated. After 48 h, shuttle vector DNA was recovered and analyzed for recombination as described for Fig. 2.

FIG. 5

Recombination induced by triplex-directed psoralen adducts in human DNA MMR-deficient and -proficient cells. Cells included the human colon cancer cell line, HCT116, deficient in the MMR factor MLH1, along with a subline corrected by whole-chromosome [chr.] transfer to restore MMR function (HCT116.3-6, corrected with chromosome 3) and a control sub-line (HCT116.2-3, with chromosome 2). A similar set of MSH2-deficient and corrected cell lines, HC (MSH2 deficient), HC.2-4 (corrected with chromosome 2), and HC.7-2 (control with chromosome 7) were also compared. The pSupFAR vector DNA was incubated in vitro with pso-AG30, followed by UVA irradiation to generate triplex-directed, psoralen photoadducts. The vector-triplex-psoralen adduct complexes were transfected into the indicated cells. Control samples included vector DNA irradiated with UVA in the absence of any oligonucleotide and then transfected directly into the various cell lines, as indicated. After 48 h, shuttle vector DNA was recovered and analyzed for recombination as described for Fig. 2.

FIG. 6

Sequence analysis of deletion mutations generated in the supF gene in COS cells by triplex-associated psoralen adducts (A) and by triple helix formation alone (B). The deleted sequences are indicated within the brackets. Underlined nucleotides indicate stretches of microhomology at the deletion end points.

FIG. 7

Model for intramolecular recombination induced by triplex-directed DNA damage. The model illustrates a proposed end-joining pathway in which the third strand-directed psoralen interstrand cross-link is processed into strand breaks, either by NER or by an NER-independent mechanism. (The potential ability of a triple helix to block repair endonuclease activity is also indicated as a complicating factor [61].) The resulting ends are subject to exonuclease digestion, eliminating the mutant sequences and exposing regions of homology capable of joint formation to reconstruct a functional supF gene.