Nucleolin Inhibits G4 Oligonucleotide Unwinding by Werner Helicase

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.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).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

NCL inhibits WRN unwinding of G4 tetraplex DNA.

<p>The preparation of G4 tetraplex substrate was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035229#s2" target="_blank">Materials and Methods</a>. Purified WRNp (5 fmol) was incubated with 40 fmol G4 DNA substrate and 125 or 200 fmol ΔN-NCL (lanes 9–10) or 40, 125 or 200 fmol RGG fragment (lanes 15–17). Other lanes contain controls-ΔN-only ΔN-NCL (40 fmol, lane 3), 1-1-RBD 1–2 fragment (200 fmol, lanes 4, 11and 12), 3-4-RBD 3–4 fragment (200 fmol, lanes 5, 13 and 14), GST- GST protein (200 fmol, lane 19), Only WRN protein on lanes 8 and 18, B-only reaction buffer (lane 2), Δ- heat denatured substrate (lane 1). Reactions were terminated after 20 min at 37°C and run out on 8% polyacrylamide gels. A representative intact gel is shown.</p

Camptothecin induces translocation of nucleolin and Werner helicase. A.

<p>Confocal microscope images of WRNp (red) and NCL (green) distribution after U2OS cells were treated with different DNA damaging agents as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035229#s2" target="_blank">Materials and Methods</a>. 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>B.</b> Cells treated as above were immunoprecipitated with anti-NCL and immunoblotted as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035229#pone-0035229-g001" target="_blank">Figure 1</a>. 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. <b>C.</b> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035229#s2" target="_blank">Materials and Methods</a>.</p

<i>In vitro</i> binding of WRNp and NCL. A-B.

<p>The WRNp binding domain of nucleolin is in the C-terminus. Indirect ELISA was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035229#s2" target="_blank">Materials and Methods</a>. Purified GST-nucleolin fragments (A) or purified His₆-Werner proteins (B) were coated onto 96-well microtiter plate wells. Coated protein was incubated with His₆-Werner protein (A) or GST-nucleolin (B). Results shown were derived from a single plate, with samples analyzed in quadruplicate and error bars showing the standard deviation from the mean. The co-efficient of variation was usually less than 10%. Experiments were replicated at least three times with similar results. See section D for nucleolin fragment names and mapping. BSA, plate coated with only BSA; Ab, plate coated with only anti-WRN antibody. <b>C.</b> Different GST-WRN fragments were used to pull down nucleolin from nuclear extract as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035229#s2" target="_blank">Materials and Methods</a> section. Upper panel shows the detection of nucleolin only in the WRN fragments *HRDC+(amino acid (a.a.) residues 1072–1432) and **C-terminal (a.a. residues 949–1432), but not in other fragments. NBR is the likely Nucleolin Binding Region of the Werner protein. Membrane was stripped and immunoblotted with anti-GST antibody (lower panel). MW in kDa are indicated at right for each panel. <b>D.</b> Different GST-nucleolin fragments were used to pull down full length purified His₆-WRN. In the upper panel WRNp is present only in the nucleolin fragments containing the RGG domain- RGG and ΔN-NCL. Same membrane was stripped and immunoblotted with GST antibody (lower panel). MW in kDa are indicated at left for each panel. <b>E</b>. WRNp does not bind to NCL N-terminal domain. Constructs were transfected into HeLa cells, which were extracted and immunoblotted as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035229#s2" target="_blank">Materials and Methods</a>. (A) Western Blot analysis of Nucleolin fragments from HeLa cells transiently transfected with either pEGFP (lane 1), GFP-NCL 1–283 (N-terminal domain, lane 2) and GFP-NCL 284–707 (ΔN-NCL, lane 3). (B) Immunoprecipitation with anti-WRN antibody of the above HeLa cell extracts, and detection with anti-GFP. Lanes as above. Only in lane 3 (GFP-ΔN-NCL) is a GFP signal detected.</p

NCL inhibits WRN helicase activity but not WRN exonuclease activity. A.

<p>WRN unwinding of a helicase substrate, 22 base pair partial duplex fork substrate (shown at left), was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035229#s2" target="_blank">Materials and Methods</a>. 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>B.</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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035229#s2" target="_blank">Materials and Methods</a>. 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). <b>C. </b><i>E. coli</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035229#s2" target="_blank">Materials and Methods</a>. 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).</p

GFP-NCL and RFP-WRN co-localize in the nucleoplasm after CPT treatment.

<p>U2OS cells were transfected with GFP-NCL (green) and RFP-WRN (red) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035229#s2" target="_blank">Materials and Methods</a>. Cells were treated with 15 µM CPT and immediately imaged in a time series obtained with a Zeiss 710 confocal. <b>A.</b> Still images from a 120 minute time series at 0, 30 and 120 minutes after the addition of CPT. <b>B.</b> Still images from a 14 hour time series at 0, 44 and 360 and 600 minutes after the addition of CPT. <b>C.</b> A 3x zoom on two cells from the 120 minute time series, comparing the distribution of GFP-NCL and RFP-WRN at 0 and 30 minutes. Arrows point to co-localizing WRN-NCL foci (orange-yellow) in the nucleoplasm. <b>D.</b> A 4x zoom on a cell from the overnight time series, comparing the distribution of GFP-NCL and RFP-WRN at 0, 44 and 360 minutes. Arrows point to co-localizing WRN-NCL foci (orange-yellow) in the nucleoplasm. Note the intense co-localization of NCL and WRNp in the nucleoli at 0 min, and that most of the WRNp and some of the NCL have translocated to the nucleoplasm within 30 (A) and 44 (B) minutes, where co-localizing NCL-WRN foci can be already detected (C and D).</p