Targeted mutagenesis of DNA via triple helix formation

Yale University, New Haven CT

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Abstract

DESCRIPTION (provided by applicant): Molecules that bind DNA in a sequence-specific manner offer the potential to regulate gene structure and function. Triplex-forming oligonucleotides (TFOs) bind in the major groove of duplex DNA with high sequence specificity at polypurine sites. Initial work funded by this grant demonstrated that TFOs conjugated to psoralen could confer sequence specificity on the mutagenic effects of the psoralen in both episomal and chromosomal targets in mammalian cells. It was also found that triple helix formation, itself, constitutes a DNA lesion that can provoke DNA repair, leading to mutagenesis and stimulating recombination. The ability of TFOs to mediate targeted modification at chromosomal sites was demonstrated both in cultured mammalian cells and in mice following systemic administration of the TFOs. In this renewal application, we propose to further examine the repair of triplex structures, with an emphasis on aspects of the nucleotide excision repair (NER) pathway, including recognition by the transcription coupled repair (TCR) and global genome repair (GGR) sub-pathways and processing by repair endonuclease incisions. We will also test the roles of certain DNA mismatch repair (MMR) factors, the Werners's and Bloom's helicases, and selected polymerases in the metabolism of triplex DNA. In addition, we will continue to examine cellular processes that may affect gene targeting by TFOs, such as cell cycle phase, histone acetylation, and transcriptional activity. We will also examine selected TFO chemical modifications, including backbone, sugar, and base substitutions, for improved gene targeting in cells and for the ability of the associated triplexes to induce repair. Peptide nucleic acids (PNAs), a special class of DNA analogs with advantageous DNA binding properties, will also be investigated. These studies will lead to a greater understanding of the utility of TFOs and related molecules as gene targeting reagents for both research and possible therapeutic applications. In addition, an increased understanding of how triple helices and other unusual DNA structures disrupt genome integrity may provide clues to mechanisms of endogenous genomic instability associated with cancer and other diseases.