Abstract
BackgroundThe 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 FindingsHere 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/SignificanceThese 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.
Highlights
The nucleolus is a nuclear domain long known to play a central role in ribosome biogenesis and RNA metabolism [1,2,3]
This is unsurprising, as both NCL and WRN are multi-functional proteins that participate in several different protein complexes at the same time, and only a fraction of each protein is present in each complex
When U2OS cells were treated with camptothecin and several other DNA damaging agents, we found that only in the presence of camptothecin did we observe NCL in the nucleoplasm (Figure 3A)
Summary
The nucleolus is a nuclear domain long known to play a central role in ribosome biogenesis and RNA metabolism [1,2,3]. A key nucleolar protein is nucleolin, a RNA binding phosphoprotein [4], that plays a major role in nucleolar organization and function, especially ribosome genesis [5]. 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. We have investigated if these nuclear proteins interact and if this interaction has a possible functional significance in DNA damage repair
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