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Nucleolin inhibits G4 oligonucleotide unwinding by Werner helicase.

Indig FE, Rybanska I, Karmakar P, Devulapalli C, Fu H, Carrier F, Bohr VA - PLoS ONE (2012)

Bottom Line: Both WRNp and NCL respond to the effects of DNA damaging agents.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).

View Article: PubMed Central - PubMed

Affiliation: 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

ABSTRACT

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.

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.

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Camptothecin induces translocation of nucleolin and Werner helicase. A.Confocal microscope images of WRNp (red) and NCL (green) distribution after U2OS cells were treated with different DNA damaging agents as detailed in Materials and Methods. Fixed cells were stained simultaneously with Rabbit anti-WRN (Novus) and Mouse anti-nucleolin (Santa Cruz). Note that WRNp re-localized from the nucleolus in all treatments while nucleolin re-localized only after CPT treatment. Merged images show co-localization (yellow) of WRNp and NCL. Images are of representative cells; At least 30 cells were analyzed for each treatment, which was repeated at least three times. B. Cells treated as above were immunoprecipitated with anti-NCL and immunoblotted as described in the legend for Figure 1. Mito C- mitomycin C, HU- hydroxyurea, CPT- camptothecin, 4NQ0-4-nitroquinoline-1-oxide, Control- untreated U2OS (WRN plus) cells; WRN cells- Ag11395 WS cells (abnormal WRN), Mo IgG- negative control mouse IgG precipitate; Control input-10% of whole cell extract used for IP. C. U2OS cells were treated with 15 µM CPT for 12 h and then washed with complete medium. Cells were fixed at times from start of treatment as indicated at the left of the images, and processed for confocal microscopy as detailed in Materials and Methods.
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pone-0035229-g003: Camptothecin induces translocation of nucleolin and Werner helicase. A.Confocal microscope images of WRNp (red) and NCL (green) distribution after U2OS cells were treated with different DNA damaging agents as detailed in Materials and Methods. Fixed cells were stained simultaneously with Rabbit anti-WRN (Novus) and Mouse anti-nucleolin (Santa Cruz). Note that WRNp re-localized from the nucleolus in all treatments while nucleolin re-localized only after CPT treatment. Merged images show co-localization (yellow) of WRNp and NCL. Images are of representative cells; At least 30 cells were analyzed for each treatment, which was repeated at least three times. B. Cells treated as above were immunoprecipitated with anti-NCL and immunoblotted as described in the legend for Figure 1. Mito C- mitomycin C, HU- hydroxyurea, CPT- camptothecin, 4NQ0-4-nitroquinoline-1-oxide, Control- untreated U2OS (WRN plus) cells; WRN cells- Ag11395 WS cells (abnormal WRN), Mo IgG- negative control mouse IgG precipitate; Control input-10% of whole cell extract used for IP. C. U2OS cells were treated with 15 µM CPT for 12 h and then washed with complete medium. Cells were fixed at times from start of treatment as indicated at the left of the images, and processed for confocal microscopy as detailed in Materials and Methods.

Mentions: Werner Syndrome cells (mutated Werner protein) are hypersensitive to the Topoisomerase I inhibitor, camptothecin [41], [42]. We have previously noted the remarkable effects of camptothecin on nucleolar protein complexes [34]. 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 (green) in the nucleoplasm (Figure 3A). Mitomycin C, bleomycin, aphidicolin (not shown) and hydrogen peroxide (not shown) did not redistribute nucleolin from the nucleolus to the nucleoplasm, although all agents caused the Werner helicase (red) to translocates from the nucleolus to the nucloplasm. Camptothecin treatment results in the formation of numerous nuclear NCL foci, some of which co-localized with WRNp foci in the nucleoplasm (Figure 3A). That only a fraction of NCL and WRN co-localize is not surprising, as both proteins interact with many other proteins and participate in several protein complexes at the same time.


Nucleolin inhibits G4 oligonucleotide unwinding by Werner helicase.

Indig FE, Rybanska I, Karmakar P, Devulapalli C, Fu H, Carrier F, Bohr VA - PLoS ONE (2012)

Camptothecin induces translocation of nucleolin and Werner helicase. A.Confocal microscope images of WRNp (red) and NCL (green) distribution after U2OS cells were treated with different DNA damaging agents as detailed in Materials and Methods. Fixed cells were stained simultaneously with Rabbit anti-WRN (Novus) and Mouse anti-nucleolin (Santa Cruz). Note that WRNp re-localized from the nucleolus in all treatments while nucleolin re-localized only after CPT treatment. Merged images show co-localization (yellow) of WRNp and NCL. Images are of representative cells; At least 30 cells were analyzed for each treatment, which was repeated at least three times. B. Cells treated as above were immunoprecipitated with anti-NCL and immunoblotted as described in the legend for Figure 1. Mito C- mitomycin C, HU- hydroxyurea, CPT- camptothecin, 4NQ0-4-nitroquinoline-1-oxide, Control- untreated U2OS (WRN plus) cells; WRN cells- Ag11395 WS cells (abnormal WRN), Mo IgG- negative control mouse IgG precipitate; Control input-10% of whole cell extract used for IP. C. U2OS cells were treated with 15 µM CPT for 12 h and then washed with complete medium. Cells were fixed at times from start of treatment as indicated at the left of the images, and processed for confocal microscopy as detailed in Materials and Methods.
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pone-0035229-g003: Camptothecin induces translocation of nucleolin and Werner helicase. A.Confocal microscope images of WRNp (red) and NCL (green) distribution after U2OS cells were treated with different DNA damaging agents as detailed in Materials and Methods. Fixed cells were stained simultaneously with Rabbit anti-WRN (Novus) and Mouse anti-nucleolin (Santa Cruz). Note that WRNp re-localized from the nucleolus in all treatments while nucleolin re-localized only after CPT treatment. Merged images show co-localization (yellow) of WRNp and NCL. Images are of representative cells; At least 30 cells were analyzed for each treatment, which was repeated at least three times. B. Cells treated as above were immunoprecipitated with anti-NCL and immunoblotted as described in the legend for Figure 1. Mito C- mitomycin C, HU- hydroxyurea, CPT- camptothecin, 4NQ0-4-nitroquinoline-1-oxide, Control- untreated U2OS (WRN plus) cells; WRN cells- Ag11395 WS cells (abnormal WRN), Mo IgG- negative control mouse IgG precipitate; Control input-10% of whole cell extract used for IP. C. U2OS cells were treated with 15 µM CPT for 12 h and then washed with complete medium. Cells were fixed at times from start of treatment as indicated at the left of the images, and processed for confocal microscopy as detailed in Materials and Methods.
Mentions: Werner Syndrome cells (mutated Werner protein) are hypersensitive to the Topoisomerase I inhibitor, camptothecin [41], [42]. We have previously noted the remarkable effects of camptothecin on nucleolar protein complexes [34]. 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 (green) in the nucleoplasm (Figure 3A). Mitomycin C, bleomycin, aphidicolin (not shown) and hydrogen peroxide (not shown) did not redistribute nucleolin from the nucleolus to the nucleoplasm, although all agents caused the Werner helicase (red) to translocates from the nucleolus to the nucloplasm. Camptothecin treatment results in the formation of numerous nuclear NCL foci, some of which co-localized with WRNp foci in the nucleoplasm (Figure 3A). That only a fraction of NCL and WRN co-localize is not surprising, as both proteins interact with many other proteins and participate in several protein complexes at the same time.

Bottom Line: Both WRNp and NCL respond to the effects of DNA damaging agents.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).

View Article: PubMed Central - PubMed

Affiliation: 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

ABSTRACT

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.

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.

Show MeSH
Related in: MedlinePlus