Targeted mutagenesis of DNA via triple helix formation

描述（由申请人提供）：以序列特异性方式结合DNA的分子具有调节基因结构和功能的潜力。三链体形成寡核苷酸（TFO）在多嘌呤位点以高序列特异性结合在双链体DNA的大沟中。由该基金资助的初步工作表明，与peptide缀合的TFO可以赋予peptide在哺乳动物细胞中的附加体和染色体靶点中的致突变作用的序列特异性。还发现，三螺旋形成本身构成DNA损伤，其可以引起DNA修复，导致诱变和刺激重组。在培养的哺乳动物细胞和全身给予TFO后的小鼠中均证明了TFO介导染色体位点靶向修饰的能力。 在此更新申请中，我们建议进一步研究三链体结构的修复，重点是核苷酸切除修复（NER）途径的各个方面，包括转录偶联修复（TCR）和全局基因组修复（GGR）子途径的识别以及修复核酸内切酶切口的加工。我们还将测试某些DNA错配修复（MMR）因子，Werners和Bloom的解旋酶以及选定的聚合酶在三链DNA代谢中的作用。此外，我们将继续研究可能影响TFO基因靶向的细胞过程，如细胞周期阶段，组蛋白乙酰化和转录活性。我们还将研究选定的TFO化学修饰，包括骨架，糖和碱基取代，以改善细胞中的基因靶向和相关的三链体诱导修复的能力。肽核酸（PNA），一类特殊的DNA类似物具有有利的DNA结合特性，也将进行研究。 这些研究将使人们更好地了解TFO和相关分子作为基因靶向试剂在研究和可能的治疗应用中的效用。此外，对三重螺旋和其他不寻常的DNA结构如何破坏基因组完整性的更多理解可能为与癌症和其他疾病相关的内源性基因组不稳定性机制提供线索。

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.