Site-directed inhibition of DNA replication by triple helix formation
Site-directed inhibition of DNA replication by triple helix formation
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DOI：
10.1096/fj.01-0440com
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发表时间：
2001-12-01
期刊：
影响因子：
4.8
通讯作者：
Giovannangeli, C
中科院分区：
文献类型：
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作者：
Diviacco, S;Rapozzi, V;Giovannangeli, C
序列特异性DNA识别可以通过使用三链体形成分子，即寡核苷酸（TFO）和肽核酸（PNA）来实现。它们已被用于调节转录或诱导细胞中选定位点的基因组DNA修饰，最近，在体内。我们已经确定了三链体结构可以抑制细胞中DNA复制的条件。将适合于三链体形成的寡嘧啶、寡嘌呤序列插入质粒中SV 40复制起点的两侧。将该含有插入物的质粒与亲本质粒一起在COS-1细胞中复制，并定量相应复制DNA之间的比率。含有插入片段的质粒复制的选择性抑制可归因于配体与寡嘧啶、寡嘌呤序列的结合。使用三链体形成分子观察到DNA复制的抑制，所述三链体形成分子诱导在双链靶序列处的共价结合（用TFO-peptien缀合物和照射）或在链置换后的非共价三链体形成（用bis-PNA）。相反，在没有共价交联的情况下，TFO（已被证明可以阻止转录延伸）不会对复制起作用。这些结果为未来设计和使用细胞内DNA信息处理的特异性抑制剂开辟了新的前景。
Sequence-specific DNA recognition can be achieved by the use of triplex-forming molecules, namely, oligonucleotides (TFO) and peptide nucleic acids (PNAs). They have been used to regulate transcription or induce genomic DNA modifications at a selected site in cells and, recently, in vivo. We have determined the conditions under which a triplex structure can inhibit DNA replication in cells. An oligopyrimidine.oligopurine sequence suitable for triplex formation was inserted in a plasmid on both sides of the SV40 origin of replication. This insert-containing plasmid was replicated in COS-1 cells together with the parent plasmid, and the ratio between the corresponding replicated DNAs was quantitated. Selective inhibition of replication of the insert-containing plasmid can be ascribed to ligand binding to the oligopyrimidine.oligopurine sequence. Inhibition of DNA replication was observed using triplex-forming molecules that induce either covalent binding at the double-stranded target sequence (with TFO-psoralen conjugate and irradiation) or noncovalent triplex formation after strand displacement (with bis-PNA). In contrast, in the absence of covalent cross-linking, TFOs (which have been shown to arrest transcription elongation) did not act on replication. These results open new perspectives for future design and use of specific inhibitors of intracellular DNA information processing.