Targeted mutagenesis of DNA via triple helix formation

通过三螺旋形成进行 DNA 定向诱变

• 批准号: 7281053
• 负责人: PETER M GLAZER
• 金额: $ 9.59万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7281053
• 关键词: DNA binding protein; DNA damage; DNA repair; HeLa cells; acetylation; autoradiography; biotechnology; camptothecin; cell cycle; chemical structure function; chromatin; enzyme activity; functional /structural genomics; gel electrophoresis; gene mutation; gene targeting; genetic mapping; genetic transcription; nucleotide analog; oligonucleotides; phosphoester ligase; polymerase chain reaction; reporter genes; site directed mutagenesis; southern blotting; triple helix

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.

描述（由申请人提供）：以序列特异性结合DNA的分子提供了调节基因结构和功能的潜力。三核苷酸（TFOS）在双链DNA的主要凹槽中结合，在息肉素位点具有高序列特异性。该赠款资助的最初作品表明，与牛皮兰共轭的TFO可以在哺乳动物细胞中赋予牛cor体和染色体靶标的coralen的诱变作用的序列特异性。还发现，三重螺旋形成本身构成可以引起DNA修复的DNA病变，从而导致诱变和刺激重组。在培养的哺乳动物细胞和全身给药TFOS培养的哺乳动物细胞和小鼠中，TFO在染色体位点介导靶向修饰的能力都得到了证明。 在此续签应用中，我们建议进一步检查三重结构的修复，重点是核苷酸切除修复（NER）途径的各个方面，包括通过转录耦合耦合修复（TCR）和全球基因组修复（GGR）子道的识别，并通过修复内核酸内核酸内核酸内核酸内切开式切口进行处理。我们还将测试某些DNA不匹配修复（MMR）因子，湿透者和Bloom的解旋酶的作用，以及在Triplex DNA代谢中选择的聚合酶。此外，我们将继续检查可能影响TFO基因靶向基因的细胞过程，例如细胞周期阶段，组蛋白乙酰化和转录活性。我们还将检查选定的TFO化学修饰，包括主链，糖和碱取代，以改善细胞中的基因靶向以及相关的三环诱导修复的能力。还将研究肽核酸（PNAS），这是具有有利DNA结合特性的特殊类型类似物。 这些研究将导致对TFO和相关分子的效用，作为研究和可能的治疗应用的基因靶向试剂。此外，对三螺旋和其他异常DNA结构如何破坏基因组完整性如何为内源性基因组不稳定性与癌症和其他疾病相关的机制提供了线索。