TARGETED MUTAGENESIS OF DNA VIA TRIPLE HELIX FORMATION

选择性突变和灭活哺乳动物细胞中特定基因的能力将是探索与癌症和其他疾病相关的关键途径的有价值的研究工具，并可能最终应用于治疗。寡核苷酸可以与双链DNA结合，以序列特异性的方式形成三股螺旋。由这笔赠款资助的工作表明，通过将三螺旋形成的寡核苷酸(TFO)连接到诱变剂，可以将三链形成的序列特异性赋予诱变剂的作用，因此DNA损伤和突变可以定向到特定的位置。与补骨脂素结合的TFOS被发现在细胞中的SV40载体中产生位点特异性突变的supF报告基因，频率高达2%。还发现，即使在没有栓系诱变剂的情况下，三联体的形成也可能是突变的，并且三联体既可以刺激细胞修复途径，也可以抑制细胞修复途径。最近，利用DNA TFOS或由肽核酸组成的寡聚体(PNAS)对染色体基因进行了定向突变，并成功地将supF基因和hprt基因靶向于可恢复的、染色体整合的lambda载体。在这一新的应用中，建议进一步研究TFOS的染色体基因靶向，使用包含为研究三重定向突变而优化的修改的supF报告基因的新的lambda载体。实验将集中在测试三链基序、靶点转录、细胞周期阶段和染色质结构对染色体可及性和TFO介导的靶向的影响。一系列新颖的TFO修饰，包括碱基、糖和骨架的变化，将由PI组装的合作者团队提供，并将测试它们对三链形成、稳定性和突变的影响，以确定在细胞中进行基因靶向的最佳试剂。其他实验将进一步测试PNAS的染色体靶向，并将探索补骨脂素-PNA结合物的用途。将研究DNA损伤和修复在三链介导的突变中的作用，以寻找有效的基因组修饰试剂，并探索三螺旋DNA代谢的细胞途径。将测试一系列新型TFO-诱变剂结合物，包括烷化剂、喜树碱和三链特异性嵌入剂。三链相关DNA损伤的修复和三链本身将在人类细胞提取物中进行研究，重点是检测核苷酸切除修复(NER)途径的特定方面，包括修复核酸内切酶伤口、切除受损片段和修复相关DNA合成。

The ability to selectively mutate and inactivate specific genes in mammalian cells would be a valuable research tool to probe critical pathways associated with cancer and other diseases and might eventually have therapeutic applications. Oligonucleotides can bind to duplex DNA and form triple helices in a sequence- specific manner. Work funded by this grant has shown that by linking a triple helix-forming oligonucleotide (TFO) to a mutagen, the sequence specificity of triplex formation can be imparted to the action of the mutagen, and so DNA damage and thereby mutations can be directed to a specific site. TFOs conjugated to psoralen were found to produce site-specific mutations in supF reporter genes in an SV40 vector in cells, at frequencies up to 2 percent. It was also found that triplex formation, even in the absence of a tethered mutagen, can be mutagenic and that triplexes can both stimulate and inhibit cellular repair pathways. Recently, targeted mutagenesis of chromosomal genes was observed, using either DNA TFOs or oligomers composed of peptide nucleic acids (PNAs), with successful targeting of a supF gene in a recoverable, chromosomally integrated lambda vector and of the hprt gene. In this renewal application, work is proposed to further investigate chromosomal gene targeting by TFOs, using new lambda vectors containing modified supF reporter genes optimized for studying triplex-directed mutagenesis. Experiments will focus on testing the influence of triplex motif, target site transcription, cell cycle phase, and chromatin structure on chromosome accessibility and TFO-mediated targeting. A series of novel TFO modifications, including base, sugar, and backbone changes, will be provided by a team of collaborators assembled by the PI and will be tested for their effect on triplex formation, stability, and mutagenesis to identify optimal reagents for gene targeting in cells. Additional experiments will further test chromosome targeting by PNAs and will explore the utility of psoralen-PNA conjugates. The roles of DNA damage and repair in the triplex-mediated mutagenesis will be examined, to identify effective reagents for genome modification and to probe cellular pathways of triple helix DNA metabolism. A series of novel TFO- mutagen conjugates will be tested, including alkylating agents, camptothecin, and triplex-specific intercalators. The repair of triplex-associated DNA damage and of triplexes, themselves, will be investigated in human cell extracts, with an emphasis on assays to examine specific aspects of the nucleotide excision repair (NER) pathway, including repair endonuclease incisions, excision of damaged fragments, and repair-associated DNA synthesis.