Processing of targeted psoralen cross-links in Xenopus oocytes.
Processing of targeted psoralen cross-links in Xenopus oocytes.
复制标题
非洲爪蟾卵母细胞中靶向补骨脂素交联的处理。
DOI：
10.1128/mcb.17.11.6645
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发表时间：
1997
影响因子：
5.3
通讯作者：
Carroll,D
中科院分区：
文献类型：
--
作者：
Segal,DJ;Faruqi,AF;Glazer,PM;Carroll,D
补骨脂素交联已被证明在细菌、酵母和哺乳动物细胞中具有诱变性和重组性。双链断裂（DSB）被认为是消除补骨脂素交联的中间体。最近的工作表明，可以通过使用靶向补骨脂素加合物来实现位点特异性诱变和重组。含有补骨脂素加合物的质粒的命运在爪蟾细胞中进行了评估，爪蟾细胞是一种实验系统，具有良好的重组能力和在 DNA 代谢中间体分析中的优势。补骨脂素加合物通过形成三链体的寡核苷酸递送至特定位点。由于在目标位点观察到诱变，因此这些损伤在卵母细胞中被清楚地识别和处理。将诱导突变谱与哺乳动物细胞中类似研究中发现的突变谱进行比较。携带多个随机加合物的质粒优先被降解，可能是由于 DSB 的引入。然而，当检查携带位点特异性加合物的 DNA 时，没有观察到质粒损失，并且发现交联的去除非常缓慢。使用具有一或两个交联位点的底物进行 DSB 依赖性同源重组的灵敏测定。在单个损伤中没有观察到加合物刺激的重组，并且在成对损伤中仅观察到非常低的水平，即使当卵母细胞去除大部分交联时也是如此。我们得出的结论是，爪蟾细胞中的补骨脂素加合物不能有效地形成 DSB 或其他重组结构。虽然补骨脂素不是一种有前途的刺激位点特异性重组的试剂，但它可以有效诱导靶向突变。
Psoralen cross-links have been shown to be both mutagenic and recombinagenic in bacterial, yeast, and mammalian cells. Double-strand breaks (DSBs) have been implicated as intermediates in the removal of psoralen cross-links. Recent work has suggested that site-specific mutagenesis and recombination might be achieved through the use of targeted psoralen adducts. The fate of plasmids containing psoralen adducts was evaluated inXenopusoocytes, an experimental system that has well-characterized recombination capabilities and advantages in the analysis of intermediates in DNA metabolism. Psoralen adducts were delivered to a specific site by a triplex-forming oligonucleotide. These lesions are clearly recognized and processed in oocytes, since mutagenesis was observed at the target site. The spectrum of induced mutations was compared with that found in similar studies in mammalian cells. Plasmids carrying multiple random adducts were preferentially degraded, perhaps due to the introduction of DSBs. However, when DNAs carrying site-specific adducts were examined, no plasmid loss was observed and removal of cross-links was found to be very slow. Sensitive assays for DSB-dependent homologous recombination were performed with substrates with one or two cross-link sites. No adduct-stimulated recombination was observed with a single lesion, and only very low levels were observed with paired lesions, even when a large proportion of the cross-links was removed by the oocytes. We conclude that DSBs or other recombinagenic structures are not efficiently formed at psoralen adducts inXenopusoocytes. While psoralen is not a promising reagent for stimulating site-specific recombination, it is effective in inducing targeted mutations.