Nucleolin inhibits G4 oligonucleotide unwinding by Werner helicase.
BACKGROUND
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.


RESULTS
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
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.
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. indigf@mail.nih.gov
Fred E
FE
Indig
Ivana
I
Rybanska
Parimal
P
Karmakar
Chakravarty
C
Devulapalli
Haiqing
H
Fu
France
F
Carrier
Vilhelm A
VA
Bohr
D002166:Camptothecin / Q000494:pharmacology; D002460:Cell Line; D002466:Cell Nucleolus / Q000187:drug effects / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D054856:G-Quadruplexes / Q000187:drug effects; D006801:Humans; D047468:Immunoprecipitation; D009841:Oligonucleotides / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D053484:RecQ Helicases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity / Q000187:drug effects; D000071657:Werner Syndrome Helicase; D000097796:Nucleolin
D002166:Camptothecin; D005090:Exodeoxyribonucleases; D009841:Oligonucleotides; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D053484:RecQ Helicases; D011993:Recombinant Fusion Proteins; D000071657:Werner Syndrome Helicase; D000097796:Nucleolin; C098785:WRN protein, human