Altered Repair of Targeted Psoralen Photoadducts in the Context of an Oligonucleotide-mediated Triple Helix

Abstract

Oligonucleotides can bind as third strands of DNA in a sequence-specific manner to form triple helices. Psoralen-conjugated, triplex-forming oligonucleotides (TFOs) have been used for the site-specific modification of DNA to inhibit transcription and to target mutations to selected genes. Such strategies, however, must take into account the ability of the cell to repair the triplexdirected lesion. We report experiments showing that the pattern of mutations produced by triplex-targeted psoralen adducts in an SV40 shuttle vector in monkey COS cells can be influenced by the associated third strand. Mutations induced by psoralen adducts in the context of a TFO of length 10 were the same as those generated by isolated adducts but were found to be different from those generated in the presence of a TFO of length 30 at the same target site. In complementary experiments, HeLa whole cell extracts were used to directly assess repair of the TFO-directed psoralen adducts in vitro. Excision of the damaged DNA was inhibited in the context of the 30-mer TFO, but not the 10-mer. These results suggest that an extended triple helix of length 30, which exceeds the typical size of the nucleotide excision repair patch in mammalian cells, can alter repair of an associated psoralen adduct. We present a model correlating these results and proposing that the incision steps in nucleotide excision repair in mammalian cells can be blocked by the presence of a third strand of sufficient length and binding affinity, thereby changing the pattern of mutations. These results may have implications for the use of triplex-forming oligonucleotides for genetic manipulation, and they may lead to the use of such oligonucleotides as tools to probe DNA repair pathways.Oligonucleotides can bind in the major groove of duplex DNA to form triple helices in a sequence-specific manner (1-4). Progress in elucidating the third strand binding code has raised the possibility of developing nucleic acids as sequencespecific reagents for research and possibly therapeutic applications. Oligonucleotide-mediated triplex formation has been shown to inhibit transcription factor binding to promoter sites and to block transcription in vitro and in vivo (5). Strategies to enhance transcription inhibition using oligonucleotide-intercalator conjugates have also been employed (6, 7). Triplex formation has further been used as a tool to generate unique cleavage sites in DNA in vitro (8). We and others have explored the use of triplex-forming oligonucleotides (TFOs) 1 as a method to deliver a tethered mutagen to a selected gene for the site-specific introduction of DNA damage (9 -12). Psoralen-conjugated oligonucleotides directed to a site in a mutation reporter gene were found to induce targeted mutations in (10) and SV40 (9) DNA in experiments in which the triplex formation was carried out in vitro. In recent work, conditions were determined under which targeted mutagenesis of an SV40 vector could be achieved in vivo via intracellular triplex formation using psoralen-linked TF...

Targeted gene correction using psoralen, chlorambucil and camptothecin conjugates of triplex forming peptide nucleic acid (PNA)

“…The apparent absence of psoralen-PNA processing may relate to the findings of Wang et al 35 describing the repair of psoralenTFOs in HeLa cell extract. These authors reported that whereas a 10mer psoralen-conjugated TFO could be efficiently removed augment the correction frequency.…”

Third strand-mediated psoralen-induced correction of the sickle cell mutation on a plasmid transfected into COS-7 cells

“…Psoraleninduced mutagenesis has also been found to depend on whether the photoproduct is a monoadduct or a crosslink and even on the size of the psoralen-delivery strand. 11,12,30,31 Bound but not covalently attached third strands are also recognized as DNA damage and are mutagenic in these cells. 32 Particularly appropriate for our effort to correct the sickle cell A ÁT-T ÁA transversion, psoralen adducts at T residues preferentially induce T ÁA-A ÁT transversions.…”

“…[12][13][14] Thus, nucleotide excision repair has been shown to be responsible for repairing bulky DNA adducts. Psoraleninduced mutagenesis has also been found to depend on whether the photoproduct is a monoadduct or a crosslink and even on the size of the psoralen-delivery strand.…”

“…This approach uses selective chemical modification of the mutant base pair to trigger error prone DNA repair mechanisms that tend in some cases to insert the desired wild-type base pair. [11][12][13][14][15] The demonstration target we selected for this approach is the mutation responsible for sickle cell anemia, a heritable hematological disease which results from an A ÁT-T ÁA transversion in the sixth codon of the b-globin gene. This transversion changes a GAG codon to GTG, resulting in a single amino-acid change from Glu to Val.…”

“…18,19,26,27 The psoralen photoadduct is recognized as DNA damage and is removed by the DNA repair mechanisms of the cell, which often replace the psoralen-T adduct with an A residue, resulting in the desired mutation correction. 12,14 To explore the feasibility of the triplex-mediated approach for the desired mutation correction, experiments were undertaken under tractable circumstances with a plasmid bearing a fragment of the sickle cell b-globin gene sequence ( Figure 3). A plasmid photomodified in vitro and purified from nonmodified plasmid before transfection was used for this purpose.…”