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的分子提供了调节基因结构和功能的可能性。三链形成寡核苷酸(TFOS)结合在双链DNA的主槽上，在多嘌呤位点具有高度的序列特异性。由这笔赠款资助的初步工作表明，与补骨脂素偶联的TFOS可以赋予补骨脂素在哺乳动物细胞的表观和染色体靶点上的序列特异性。还发现，三螺旋形成本身就构成了一种DNA损伤，可以引发DNA修复，导致突变和刺激重组。TFOS在培养的哺乳动物细胞和全身给药后的小鼠身上都证明了TFOS在染色体位置介导靶向修饰的能力。 在这一更新应用中，我们建议进一步研究三链结构的修复，重点放在核苷酸切除修复(NER)途径的各个方面，包括转录偶联修复(TCR)和全球基因组修复(GGR)亚途径的识别以及修复核酸内切酶切割的处理。我们还将测试某些DNA错配修复(MMR)因子、Werners‘s和Bloom’s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TFOS基因打靶的细胞过程，如细胞周期时相、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联体诱导修复的能力。多肽核酸(PNA)是一类特殊的DNA类似物，具有良好的DNA结合特性，也将被研究。 这些研究将使人们更好地了解TFOS及其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作用。此外，对三螺旋和其他不寻常的DNA结构如何破坏基因组完整性的了解的加深，可能为揭示与癌症和其他疾病相关的内源性基因组不稳定的机制提供线索。

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.