Wang G, Glazer P M
Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut 06520-8040, USA.
J Biol Chem. 1995 Sep 22;270(38):22595-601. doi: 10.1074/jbc.270.38.22595.
Oligonucleotides can bind as third strands of DNA in a sequence-specific manner to form triple helices. Psoralen-conjugated, triplex-forming oligonucleotides (TFOs) have been used for the site-specific modification of DNA to inhibit transcription and to target mutations to selected genes. Such strategies, however, must take into account the ability of the cell to repair the triplex-directed lesion. We report experiments showing that the pattern of mutations produced by triplex-targeted psoralen adducts in an SV40 shuttle vector in monkey COS cells can be influenced by the associated third strand. Mutations induced by psoralen adducts in the context of a TFO of length 10 were the same as those generated by isolated adducts but were found to be different from those generated in the presence of a TFO of length 30 at the same target site. In complementary experiments, HeLa whole cell extracts were used to directly assess repair of the TFO-directed psoralen adducts in vitro. Excision of the damaged DNA was inhibited in the context of the 30-mer TFO, but not the 10-mer. These results suggest that an extended triple helix of length 30, which exceeds the typical size of the nucleotide excision repair patch in mammalian cells, can alter repair of an associated psoralen adduct. We present a model correlating these results and proposing that the incision steps in nucleotide excision repair in mammalian cells can be blocked by the presence of a third strand of sufficient length and binding affinity, thereby changing the pattern of mutations. These results may have implications for the use of triplex-forming oligonucleotides for genetic manipulation, and they may lead to the use of such oligonucleotides as tools to probe DNA repair pathways.
寡核苷酸可以以序列特异性方式作为DNA的第三条链结合，形成三链螺旋。补骨脂素缀合的三链形成寡核苷酸（TFOs）已被用于DNA的位点特异性修饰，以抑制转录并将突变靶向选定基因。然而，此类策略必须考虑细胞修复三链定向损伤的能力。我们报告的实验表明，猴COS细胞中SV40穿梭载体中三链靶向补骨脂素加合物产生的突变模式可受相关第三条链的影响。长度为10的TFO背景下补骨脂素加合物诱导的突变与分离加合物产生的突变相同，但发现在相同靶位点存在长度为30的TFO时产生的突变与之不同。在互补实验中，使用HeLa全细胞提取物直接评估体外TFO定向补骨脂素加合物的修复情况。在30聚体TFO背景下，受损DNA的切除受到抑制，但10聚体TFO则不然。这些结果表明，长度为30的延伸三链螺旋超过了哺乳动物细胞中核苷酸切除修复片段的典型大小，可改变相关补骨脂素加合物的修复。我们提出了一个将这些结果关联起来的模型，并提出哺乳动物细胞中核苷酸切除修复的切口步骤可被足够长度和结合亲和力的第三条链的存在所阻断，从而改变突变模式。这些结果可能对使用三链形成寡核苷酸进行基因操作有影响，并且可能导致将此类寡核苷酸用作探测DNA修复途径的工具。
