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 triplex-directed 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.
Highlights
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)
Using human cell extracts to study the repair of the shuttle vector-psoralen-TFO substrate in vitro, we show that a triple helix of sufficient length and binding affinity can inhibit excision of the damaged DNA
We have shown that pso-AG10 and pso-AG30 bind to the target site in Oligonucleotide Irradiation Mutants/totala
Summary
Of triplex-forming oligonucleotides (TFOs) as a method to deliver a tethered mutagen to a selected gene for the site-specific introduction of DNA damage (9 –12). Release of the damaged DNA fragment via helicase activity is followed by gap-filling repair synthesis [19] Based on this model of NER, we have designed experiments to examine the effect of a triple helix on DNA repair and mutagenesis. We have developed a strategy to deliver targeted psoralen adducts using an oligonucleotide that can be subsequently detached from the adduct by disulfide bond reduction In this way, psoralen mutagenesis at the same site of adduct formation can be directly compared in the presence of triple helices of differing lengths as well as in the absence of an associated third strand. Using human cell extracts to study the repair of the shuttle vector-psoralen-TFO substrate in vitro, we show that a triple helix of sufficient length and binding affinity can inhibit excision of the damaged DNA. We propose a model correlating these results and suggesting that the incision steps in NER can be blocked by the presence of a third strand
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