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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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NCL inhibits WRN helicase activity but not WRN exonuclease activity. A.WRN unwinding of a helicase substrate, 22 base pair partial duplex fork substrate (shown at left), was performed as described in Materials and Methods. Purified WRNp (5 fmol) was incubated with 40 fmol substrate and 50, 125, or 200 fmol of ΔN-NCL (lanes 12–15) or RGG fragment (lanes 5–8). Controls are GST protein (200 fmol, lane 10), RBD 3–4 fragment (200 fmol, lane 9), W, only WRNp protein (lanes 4 and 11), B- only reaction buffer (lane 3), D- heat denatured substrate (lane 2), Oligo- unreacted substrate (lane 1). In Lanes 5 and 13 the WRN protein was omitted from the reaction. Green circles point out the inhibitory effect of RGG (lane 8) or ΔN-NCL (lane 15) on WRN helicase activity. B. WRN protein (100 fmol) was incubated with the exonuclease substrate (3′-recessed DNA substrate, represented at the top of the figure) in the presence of increasing amounts of ΔN-NCL (25, 50, 100, 200, 400 fmol) under exonuclease reaction conditions for 1 h at 37°C, as described in Materials and Methods. Once the reactions were stopped, DNA products were resolved by denaturing polyacrylamide gel electrophoresis. Controls are only reaction buffer (lane 1), only 400 fmol ΔN-NCL (lane 2), only WRN protein (lane 3) and D- heat denatured substrate (lane 9). C.E. coli UvrD protein (10 fmol) was incubated with the Mix 4/3 substrate (represented to the right of the figure) in the presence of increasing amounts of ΔN-NCL (100, 250, 400 fmol) under UvrD helicase reaction conditions for 1 h at 37°C, as described in Materials and Methods. Once the reactions were stopped, DNA products were resolved by native polyacrylamide gel electrophoresis. Controls are only reaction buffer (lane 1), WRN helicase (5 fmol, lane 2), 250 or 400 fmol ΔN-NCL without helicase (lane 10–11), RGG protein without helicase (lane 12–13) and Δ- heat denatured substrate (lane 14).
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pone-0035229-g005: NCL inhibits WRN helicase activity but not WRN exonuclease activity. A.WRN unwinding of a helicase substrate, 22 base pair partial duplex fork substrate (shown at left), was performed as described in Materials and Methods. Purified WRNp (5 fmol) was incubated with 40 fmol substrate and 50, 125, or 200 fmol of ΔN-NCL (lanes 12–15) or RGG fragment (lanes 5–8). Controls are GST protein (200 fmol, lane 10), RBD 3–4 fragment (200 fmol, lane 9), W, only WRNp protein (lanes 4 and 11), B- only reaction buffer (lane 3), D- heat denatured substrate (lane 2), Oligo- unreacted substrate (lane 1). In Lanes 5 and 13 the WRN protein was omitted from the reaction. Green circles point out the inhibitory effect of RGG (lane 8) or ΔN-NCL (lane 15) on WRN helicase activity. B. WRN protein (100 fmol) was incubated with the exonuclease substrate (3′-recessed DNA substrate, represented at the top of the figure) in the presence of increasing amounts of ΔN-NCL (25, 50, 100, 200, 400 fmol) under exonuclease reaction conditions for 1 h at 37°C, as described in Materials and Methods. Once the reactions were stopped, DNA products were resolved by denaturing polyacrylamide gel electrophoresis. Controls are only reaction buffer (lane 1), only 400 fmol ΔN-NCL (lane 2), only WRN protein (lane 3) and D- heat denatured substrate (lane 9). C.E. coli UvrD protein (10 fmol) was incubated with the Mix 4/3 substrate (represented to the right of the figure) in the presence of increasing amounts of ΔN-NCL (100, 250, 400 fmol) under UvrD helicase reaction conditions for 1 h at 37°C, as described in Materials and Methods. Once the reactions were stopped, DNA products were resolved by native polyacrylamide gel electrophoresis. Controls are only reaction buffer (lane 1), WRN helicase (5 fmol, lane 2), 250 or 400 fmol ΔN-NCL without helicase (lane 10–11), RGG protein without helicase (lane 12–13) and Δ- heat denatured substrate (lane 14).

Mentions: As we have established the possibility of a physical interaction between WRNp and NCL, we next examined whether NCL affected the enzymatic activity of WRNp upon known WRN substrates. The Werner protein is both a DNA helicase [22], [23] and exonuclease [24], [29] and we examined the effect of adding NCL to WRN activity assays. The helicase activity of WRN on a 22 base pair partial duplex fork substrate was efficiently inhibited by NCL. Under the conditions used, 5 fmol WRN converted 80–90% of the duplex (40 fmol) to single-strand form within 20 minutes at 37°C (Figure 5A). This conversion was inhibited by about 50% when ΔN-NCL was present at a molar ratio of 25∶1 vs WRN. The RGG fragment, which contains the putative WRNp-NCL interacting region (Figure 2), had an even greater inhibitory effect on WRNp, with over 60% inhibition of helicase activity at a 10∶1 ratio and 90% inhibition at 25∶1 (Figure 5A). Other proteins or NCL fragments, such as GST, RBD 1–2 and RBD 3–4 (not shown), had no, or only minimal (about 20%) effect on WRN unwinding of the duplex substrate at a molar ratio of 50∶1.


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)

NCL inhibits WRN helicase activity but not WRN exonuclease activity. A.WRN unwinding of a helicase substrate, 22 base pair partial duplex fork substrate (shown at left), was performed as described in Materials and Methods. Purified WRNp (5 fmol) was incubated with 40 fmol substrate and 50, 125, or 200 fmol of ΔN-NCL (lanes 12–15) or RGG fragment (lanes 5–8). Controls are GST protein (200 fmol, lane 10), RBD 3–4 fragment (200 fmol, lane 9), W, only WRNp protein (lanes 4 and 11), B- only reaction buffer (lane 3), D- heat denatured substrate (lane 2), Oligo- unreacted substrate (lane 1). In Lanes 5 and 13 the WRN protein was omitted from the reaction. Green circles point out the inhibitory effect of RGG (lane 8) or ΔN-NCL (lane 15) on WRN helicase activity. B. WRN protein (100 fmol) was incubated with the exonuclease substrate (3′-recessed DNA substrate, represented at the top of the figure) in the presence of increasing amounts of ΔN-NCL (25, 50, 100, 200, 400 fmol) under exonuclease reaction conditions for 1 h at 37°C, as described in Materials and Methods. Once the reactions were stopped, DNA products were resolved by denaturing polyacrylamide gel electrophoresis. Controls are only reaction buffer (lane 1), only 400 fmol ΔN-NCL (lane 2), only WRN protein (lane 3) and D- heat denatured substrate (lane 9). C.E. coli UvrD protein (10 fmol) was incubated with the Mix 4/3 substrate (represented to the right of the figure) in the presence of increasing amounts of ΔN-NCL (100, 250, 400 fmol) under UvrD helicase reaction conditions for 1 h at 37°C, as described in Materials and Methods. Once the reactions were stopped, DNA products were resolved by native polyacrylamide gel electrophoresis. Controls are only reaction buffer (lane 1), WRN helicase (5 fmol, lane 2), 250 or 400 fmol ΔN-NCL without helicase (lane 10–11), RGG protein without helicase (lane 12–13) and Δ- heat denatured substrate (lane 14).
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Related In: Results  -  Collection

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pone-0035229-g005: NCL inhibits WRN helicase activity but not WRN exonuclease activity. A.WRN unwinding of a helicase substrate, 22 base pair partial duplex fork substrate (shown at left), was performed as described in Materials and Methods. Purified WRNp (5 fmol) was incubated with 40 fmol substrate and 50, 125, or 200 fmol of ΔN-NCL (lanes 12–15) or RGG fragment (lanes 5–8). Controls are GST protein (200 fmol, lane 10), RBD 3–4 fragment (200 fmol, lane 9), W, only WRNp protein (lanes 4 and 11), B- only reaction buffer (lane 3), D- heat denatured substrate (lane 2), Oligo- unreacted substrate (lane 1). In Lanes 5 and 13 the WRN protein was omitted from the reaction. Green circles point out the inhibitory effect of RGG (lane 8) or ΔN-NCL (lane 15) on WRN helicase activity. B. WRN protein (100 fmol) was incubated with the exonuclease substrate (3′-recessed DNA substrate, represented at the top of the figure) in the presence of increasing amounts of ΔN-NCL (25, 50, 100, 200, 400 fmol) under exonuclease reaction conditions for 1 h at 37°C, as described in Materials and Methods. Once the reactions were stopped, DNA products were resolved by denaturing polyacrylamide gel electrophoresis. Controls are only reaction buffer (lane 1), only 400 fmol ΔN-NCL (lane 2), only WRN protein (lane 3) and D- heat denatured substrate (lane 9). C.E. coli UvrD protein (10 fmol) was incubated with the Mix 4/3 substrate (represented to the right of the figure) in the presence of increasing amounts of ΔN-NCL (100, 250, 400 fmol) under UvrD helicase reaction conditions for 1 h at 37°C, as described in Materials and Methods. Once the reactions were stopped, DNA products were resolved by native polyacrylamide gel electrophoresis. Controls are only reaction buffer (lane 1), WRN helicase (5 fmol, lane 2), 250 or 400 fmol ΔN-NCL without helicase (lane 10–11), RGG protein without helicase (lane 12–13) and Δ- heat denatured substrate (lane 14).
Mentions: As we have established the possibility of a physical interaction between WRNp and NCL, we next examined whether NCL affected the enzymatic activity of WRNp upon known WRN substrates. The Werner protein is both a DNA helicase [22], [23] and exonuclease [24], [29] and we examined the effect of adding NCL to WRN activity assays. The helicase activity of WRN on a 22 base pair partial duplex fork substrate was efficiently inhibited by NCL. Under the conditions used, 5 fmol WRN converted 80–90% of the duplex (40 fmol) to single-strand form within 20 minutes at 37°C (Figure 5A). This conversion was inhibited by about 50% when ΔN-NCL was present at a molar ratio of 25∶1 vs WRN. The RGG fragment, which contains the putative WRNp-NCL interacting region (Figure 2), had an even greater inhibitory effect on WRNp, with over 60% inhibition of helicase activity at a 10∶1 ratio and 90% inhibition at 25∶1 (Figure 5A). Other proteins or NCL fragments, such as GST, RBD 1–2 and RBD 3–4 (not shown), had no, or only minimal (about 20%) effect on WRN unwinding of the duplex substrate at a molar ratio of 50∶1.

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