TARGETED MUTAGENESIS OF DNA VIA TRIPLE HELIX FORMATION

在哺乳动物细胞中选择性地突变和灭活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，并可能最终具有治疗应用。寡核苷酸可以与双链DNA结合并以序列特异性的方式形成三螺旋。由该基金资助的研究表明，通过将三螺旋形成的寡核苷酸（TFO）连接到诱变原上，三螺旋形成的序列特异性可以归因于诱变原的作用，因此DNA损伤和突变可以定向到特定位点。发现与补骨脂素结合的tfo在细胞SV40载体中产生supF报告基因的位点特异性突变，频率高达2%。研究还发现，即使在没有栓系诱变原的情况下，三联体的形成也可能是诱变的，三联体既可以刺激也可以抑制细胞修复途径。最近，利用DNA TFOs或由肽核酸（PNAs）组成的寡聚物，观察到染色体基因的靶向诱变，成功地靶向了可恢复的、染色体整合的lambda载体中的supF基因和hprt基因。在这个更新的应用程序中，建议使用新的lambda载体进一步研究tfo的染色体基因靶向性，该载体含有修饰的supF报告基因，用于研究三重定向诱变。实验将重点测试三重基序、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介导的靶向性的影响。一系列新的TFO修饰，包括碱基、糖和主干的改变，将由PI组织的一个合作者团队提供，并将测试它们对三联体形成、稳定性和诱变的影响，以确定细胞中基因靶向的最佳试剂。其他实验将进一步测试PNAs的染色体靶向性，并探索补骨脂素- pna偶联物的效用。DNA损伤和修复在三螺旋体介导的突变中的作用将被研究，以确定有效的基因组修饰试剂和探索三螺旋DNA代谢的细胞途径。一系列新的TFO-诱变剂偶联物将被测试，包括烷基化剂、喜树碱和三联体特异性插入剂。三联体相关DNA损伤的修复和三联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，包括修复内切酶切口、受损片段的切除和修复相关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.