TARGETED MUTAGENESIS OF DNA VIA TRIPLE HELIX FORMATION

在哺乳动物细胞中选择性突变和复制特定基因的能力将是一种有价值的研究工具，可以探测与癌症和其他疾病相关的关键途径，并最终可能具有治疗应用。 寡核苷酸可以以序列特异性方式结合双链DNA并形成三螺旋。 由该基金资助的工作表明，通过将三螺旋形成寡核苷酸（TFO）与诱变剂连接，可以将三螺旋形成的序列特异性赋予诱变剂的作用，因此DNA损伤和突变可以指向特定位点。 研究发现，与peptide结合的TFO在细胞中的SV 40载体中的supF报告基因中产生位点特异性突变，频率高达2%。 还发现，即使在不存在系留诱变剂的情况下，三链体形成也可以是诱变性的，并且三链体可以刺激和抑制细胞修复途径。 最近，观察到染色体基因的靶向诱变，使用DNA TFO或由肽核酸（PNA）组成的寡聚体，成功靶向可回收的染色体整合λ载体中的supF基因和hprt基因。在此更新申请中，提出了使用新的λ载体进一步研究TFO的染色体基因靶向，所述载体含有经修饰的supF报告基因，所述报告基因经优化用于研究三重定向诱变。 实验将集中于测试三链体基序，靶位点转录，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。 一系列新的TFO修饰，包括碱基、糖和骨架变化，将由PI组装的合作者团队提供，并将测试其对三链体形成、稳定性和诱变的影响，以确定用于细胞基因靶向的最佳试剂。 另外的实验将进一步测试PNA的染色体靶向，并将探索peptiden-PNA缀合物的效用。 DNA损伤和修复在三螺旋介导的诱变中的作用将被检查，以确定用于基因组修饰的有效试剂，并探测三螺旋DNA代谢的细胞途径。 将测试一系列新型TFO-诱变剂缀合物，包括烷化剂、喜树碱和三链体特异性嵌入剂。 三链体相关的DNA损伤和三链体本身的修复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切口，切除受损片段和修复相关的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.