Recognition of cisplatin-DNA interstrand cross-links by replication protein A
Recognition of cisplatin-DNA interstrand cross-links by replication protein A
复制标题
DOI：
10.1021/bi800460d
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发表时间：
2008-09-23
期刊：
影响因子：
2.9
通讯作者：
Horn, Jeffrey M.
中科院分区：
文献类型：
--
作者：
Patrick, Steve M.;Tillison, Kristin;Horn, Jeffrey M.
复制蛋白 A (RPA) 是 DNA 复制和大多数 DNA 修复途径所需的异源三聚体蛋白。先前已证明 RPA 在核苷酸切除修复 (NER) 过程中识别和结合受损 DNA 方面发挥作用。 RPA 也被认为在补骨脂素 DNA 链间交联 (ICL) 修复中发挥作用，但在 ICL DNA 修复途径中尚未确定明确的生化活性。使用 HeLa 细胞提取物和 DNA 亲和层析，我们证明与未损坏的 DNA 相比，RPA 优先保留在顺铂链间交联 (ICL) DNA 柱上。 RPA 在顺铂链内和含有 DNA 亲和柱的 ICL 上的保留相当。使用合成 DNA 底物和纯化 RPA 进行的体外电泳迁移率变动测定 (EMSA) 表明，与未损坏的 DNA 相比，顺铂 ICL DNA 结合的亲和力更高。 RPA 与顺铂 ICL 的结合增强取决于 DNA 长度。随着顺铂 ICL 侧翼的 DNA 从 7 个碱基增加到 21 个碱基，观察到 RPA 优先结合。荧光各向异性显示，与未损坏的 DNA 相比，含有 42 聚体 DNA 的顺铂 ICL 的亲和力高出 200 倍以上，与顺铂链内损坏的 DNA 相比，亲和力高出 3-4 倍。随着 DNA 长度和结合反应严格性的增加，观察到 RPA 与顺铂 ICL DNA 的优先结合程度更高。这些数据与 RPA 在顺铂 ICL DNA 修复的初始识别和启动中的作用一致。
Replication protein A (RPA) is a heterotrimeric protein that is required for DNA replication and most DNA repair pathways. RPA has previously been shown to play a role in recognizing and binding damaged DNA during nucleotide excision repair (NER). RPA has also been suggested to play a role in psoralen DNA interstrand cross-link (ICL) repair, but a clear biochemical activity has yet to be identified in the ICL DNA repair pathways. Using HeLa cell extracts and DNA affinity chromatography, we demonstrate that RPA is preferentially retained on a cisplatin interstrand cross-link (ICL) DNA column compared with undamaged DNA. The retention of RPA on cisplatin intrastrand and ICL containing DNA affinity columns is comparable. In vitro electrophoretic mobility shift assays (EMSAs) using synthetic DNA substrates and purified RPA demonstrate higher affinity for cisplatin ICL DNA binding compared with undamaged DNA. The enhanced binding of RPA to the cisplatin ICL is dependent on the DNA length. As the DNA flanking the cisplatin ICL is increased from 7 to 21 bases, preferential RPA binding is observed. Fluorescence anisotropy reveals greater than 200-fold higher affinity to a cisplatin ICL containing 42-mer DNA compared with an undamaged DNA and a 3-4-fold higher affinity when compared with a cisplatin intrastrand damaged DNA. As the DNA length and stringency of the binding reaction increase, greater preferential binding of RPA to cisplatin ICL DNA is observed. These data are consistent with a role for RPA in the initial recognition and initiation of cisplatin ICL DNA repair.