Psoralen interstrand cross-link repair is specifically altered by an adjacent triple-stranded structure
Psoralen interstrand cross-link repair is specifically altered by an adjacent triple-stranded structure
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DOI：
10.1093/nar/gkh267
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发表时间：
2004-02-01
影响因子：
14.9
通讯作者：
Praseuth, D
中科院分区：
文献类型：
--
作者：
Guillonneau, F;Guieysse, AL;Praseuth, D
通过使用序列特异性DNA配体将DNA损伤剂靶向到特定的DNA位点，已经成功地指导了基因组修饰。对这种靶向损伤的修复过程以及邻近复合体的影响在很大程度上尚不清楚。通过这种方式，补骨脂素靶向已经产生了定向链间交联链(ICL)。在哺乳动物细胞中，负责ICL去除的机制还远未被理解，建议涉及突变和重组途径。在这里，通过使用补骨脂素结合物的三链形成寡核苷酸的光激活补骨脂素部分，在选定的位置引入了独特的ICL。对补骨脂素ICL的体外和细胞内两种类型的交联物进行了评价，要么只含有补骨脂素ICL，要么具有相邻的三链结构。我们发现，邻近的三链结构的存在干扰了补骨脂素ICL处理的不同阶段：(I)ICL诱导的HeLa细胞提取物中的DNA修复合成受到三链结构的抑制，以“真的”和无效的修复合成的效率衡量，在ICL位点停止；(Ii)在HeLa细胞中，通过核苷酸切除修复(NER)途径的ICL移除在存在邻近的三链的情况下延迟；以及(Iii)重组的着色性干皮病A蛋白与ICL的结合受到第三条DNA链的存在的损害，该蛋白参与了切开前NER因子的招募。这些数据表征了在特定步骤中三链诱导的ICL修复途径的调节，这可能对靶向基因组修饰的受控诱导和相关的细胞反应具有意义。
Targeting DNA-damaging agents to specific DNA sites by using sequence-specific DNA ligands has been successful in directing genomic modifications. The understanding of repair processing of such targeted damage and the influence of the adjacent complex is largely unknown. In this way, directed interstrand cross-links (ICLs) have already been generated by psoralen targeting. The mechanisms responsible for ICL removal are far from being understood in mammalian cells, with the proposed involvement of both mutagenic and recombinogenic pathways. Here, a unique ICL was introduced at a selected site by photoactivation of a psoralen moiety with the use of psoralen conjugates of triplex-forming oligonucleotides. The processing of psoralen ICL was evaluated in vitro and in cells for two types of cross-linked substrates, either containing a psoralen ICL alone or with an adjacent triple-stranded structure. We show that the presence of a neighbouring triplex structure interferes with different stages of psoralen ICL processing: (i) the ICL-induced DNA repair synthesis in HeLa cell extracts is inhibited by the triplex structure, as measured by the efficiency of 'true' and futile repair synthesis, stopping at the ICL site; (ii) in HeLa cells, the ICL removal via a nucleotide excision repair (NER) pathway is delayed in the presence of a neighbouring triplex; and (iii) the binding to ICL of recombinant xeroderma pigmentosum A protein, which is involved in pre-incision recruitment of NER factors is impaired by the presence of the third DNA strand. These data characterize triplex-induced modulation of ICL repair pathways at specific steps, which might have implications for the controlled induction of targeted genomic modifications and for the associated cellular responses.