Laboratory of Clinical Investigation, Intramural Research Program, National Institute on Aging, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland, United States of America.
PLoS One. 2012;7(6):e35229. doi: 10.1371/journal.pone.0035229. Epub 2012 Jun 4.
The Werner protein (WRNp), a member of the RecQ helicase family, is strongly associated with the nucleolus, as is nucleolin (NCL), an important nucleolar constituent protein. Both WRNp and NCL respond to the effects of DNA damaging agents. Therefore, we have investigated if these nuclear proteins interact and if this interaction has a possible functional significance in DNA damage repair.
METHODOLOGY/PRINCIPAL FINDINGS: Here we report that WRNp interacts with the RNA-binding protein, NCL, based on immunoprecipitation, immunofluorescent co-localization in live and fixed cells, and direct binding of purified WRNp to nucleolin. We also map the binding region to the C-terminal domains of both proteins. Furthermore, treatment of U2OS cells with 15 µM of the Topoisomerase I inhibitor, camptothecin, causes the dissociation of the nucleolin-Werner complex in the nucleolus, followed by partial re-association in the nucleoplasm. Other DNA damaging agents, such as hydroxyurea, Mitomycin C, and aphidicolin do not have these effects. Nucleolin or its C-terminal fragment affected the helicase, but not the exonuclease activity of WRNp, by inhibiting WRN unwinding of G4 tetraplex DNA structures, as seen in activity assays and electrophoretic mobility shift assays (EMSA).
CONCLUSIONS/SIGNIFICANCE: These data suggest that nucleolin may regulate G4 DNA unwinding by WRNp, possibly in response to certain DNA damaging agents. We postulate that the NCL-WRNp complex may contain an inactive form of WRNp, which is released from the nucleolus upon DNA damage. Then, when required, WRNp is released from inhibition and can participate in the DNA repair processes.
Werner 蛋白（WRNp）是 RecQ 解旋酶家族的成员，强烈与核仁相关，核仁组成蛋白（NCL）也是核仁的重要组成部分。WRNp 和 NCL 均对 DNA 损伤剂的作用有响应。因此，我们研究了这些核蛋白是否相互作用，以及这种相互作用是否在 DNA 损伤修复中有潜在的功能意义。
方法/主要发现： 在这里，我们报道了 WRNp 与 RNA 结合蛋白 NCL 相互作用，这是基于免疫沉淀、活细胞和固定细胞中的免疫荧光共定位以及纯化的 WRNp 与核仁蛋白的直接结合。我们还将结合区域映射到两个蛋白质的 C 端结构域。此外，用 15µM 的拓扑异构酶 I 抑制剂喜树碱处理 U2OS 细胞会导致核仁中核仁蛋白- Werner 复合物的解离，随后在核质中部分重新结合。其他 DNA 损伤剂，如羟基脲、丝裂霉素 C 和阿霉素，没有这些作用。核仁蛋白或其 C 端片段通过抑制 WRNp 对 G4 四链体 DNA 结构的解旋，影响 WRNp 的解旋酶，但不影响外切核酸酶活性，如在活性测定和电泳迁移率变动分析（EMSA）中所见。
结论/意义： 这些数据表明，核仁蛋白可能通过 Werner 蛋白调节 G4 DNA 的解旋，可能是对某些 DNA 损伤剂的响应。我们推测 NCL-WRNp 复合物可能包含一种无活性的 Werner 蛋白形式，该形式在 DNA 损伤时从核仁中释放出来。然后，当需要时，WRNp 从抑制中释放出来并能参与 DNA 修复过程。
