Fig. 5. WRN stimulates FEN-1 cleavage much more effectively than either PCNA or RPA. Reactions (20 µl) containing 10 fmol of a 1 nt 5′ flap DNA substrate, 10 fmol of FEN-1 and 80 fmol of WRN, hRPA or hPCNA were incubated under standard reaction conditions at 37°C for 15 min. % incision (mean value of three experiments) with SD.

Fig. 6. Catalytic activities of WRN are not required for stimulation of the FEN-1 cleavage reaction. Reactions (20 µl) containing 10 fmol of 1 nt 5′ flap DNA substrate and 40 fmol of FEN-1 were incubated at 37°C for 15 min under standard conditions. The reactions in the presence of either wild-type or mutant WRN contained 38 fmol of WRN. (A) WRN-K577M stimulates FEN-1 cleavage. Lane 1, no enzyme; lane 2, FEN-1; lane 3, FEN-1 + wild-type WRN; lane 4, FEN-1 + WRN-K577M; lane 5, wild-type WRN; lane 6, WRN-K577M. (B) WRN-E84A stimulates FEN-1 cleavage. Lane 1, FEN-1; lane 2, no enzyme; lane 3, FEN-1 + wild-type WRN; lane 4, FEN-1 + WRN-E84A; lane 5, wild-type WRN; lane 6, WRN-E84A.

Fig. 8. Mapping of the FEN-1 interaction domain that mediates the functional interaction between WRN and FEN-1. Reactions (20 µl) containing 10 fmol of 1 nt 5′ flap DNA substrate, 10 fmol of FEN-1 and 75 fmol of the indicated GST–WRN fragment were incubated at 37°C for 15 min under standard conditions. (A) Phosphorimage of a typical gel. Lane 1, no enzyme; lane 2, FEN-1; lane 3, FEN-1 + GST–WRN_949–1432; lane 4, FEN-1 + GST–WRN_949–1236; lane 5, FEN-1 + GST–WRN_1072–1236; lane 6, FEN-1 + GST–WRN_949–1092; lane 7, FEN-1 + GST–WRN_239–499; lane 8, GST–WRN_949–1432; lane 9, GST–WRN_949–1236; lane 10, GST–WRN_1072–1236; lane 11, GST–WRN_949–1092; lane 12, GST–WRN_239–499. (B) % incision (mean value of three experiments) with SD.

Fig. 9. WRN stimulates FEN-1 cleavage of duplex DNA substrates containing a longer 5′ flap or a nick. Reactions (20 µl) containing 10 fmol of 5 nt 5′ flap substrate (A) or a nicked duplex substrate (B) and 10 fmol of FEN-1 were incubated at 37°C for 15 min under standard conditions. The reactions in the presence of WRN contained 75 fmol of WRN. Substrate and cleavage products are as indicated. Lane 1, no enzyme; lane 2, FEN-1; lane 3, FEN-1 + wild-type WRN protein; lane 4, wild-type WRN protein. (C) % incision (mean value of three experiments) with SD for 5 nt flap and nicked duplex substrates is shown by gray and white bars, respectively.

Fig. 4. Kinetics of FEN-1 cleavage in the presence or absence of WRN. Reactions (160 µl) containing 80 fmol of 1 nt 5′ flap DNA substrate and 160 fmol of FEN-1 were incubated at 37°C under standard reaction conditions, and 20 µl aliquots were terminated at 0, 0.5, 1, 3, 6, 9, 12 and 15 min. The reactions in the presence of WRN contained 600 fmol of WRN. (A) Phosphorimage of a typical gel from a kinetic experiment. Increasing times of incubation (0–15 min) for the FEN-1 cleavage reactions conducted in the absence of WRN (lanes 3–9) or the presence of WRN (lanes 10–16) are indicated by the triangle. Fifteen minute incubations conducted in the absence of WRN + FEN-1 or FEN-1 are shown in lanes 1 and 2, respectively. (B) % incision for the reactions. Filled circles, FEN-1; open circles, FEN-1 + WRN.

Fig. 7. A C-terminal fragment of WRN retains the ability to stimulate the FEN-1 cleavage reaction. Reactions (20 µl) containing 10 fmol of 1 nt 5′ flap DNA substrate and the indicated amounts of FEN-1 were incubated at 37°C for 15 min under standard conditions. The reactions in the presence of GST–WRN fragments contained 75 fmol of WRN fragment. (A) Phosphorimage from a typical gel. Lane 1, no enzyme; lane 2, GST–WRN_949–1432; lane 3, 5 fmol of FEN-1; lane 4, GST–WRN_949–1432 + 5 fmol of FEN-1; lane 5, 5 fmol of FEN-1 + GST–WRN_1072–1236; lane 6, 10 fmol of FEN-1; lane 7, 10 fmol of FEN-1 + GST–WRN_949–1432; lane 8, 10 fmol of FEN-1 + GST–WRN_1072–1236; lane 9, 20 fmol of FEN-1; lane 10, 20 fmol of FEN-1 + GST–WRN_949–1432; lane 11, 20 fmol of FEN-1 + GST–WRN_1072–1236. (B) % incision (mean value of three experiments) with SD. Filled circles, FEN-1; open circles, FEN-1 + GST–WRN_949–1432; open squares, FEN-1 + GST–WRN_1072–1236.

Fig. 2. WRN and FEN-1 interact physically. (A) Beads with GST (lane 2), GST–WRN_949–1432 (lane 3) or GST–WRN_1072–1236 (lane 4) were incubated with 750 µg of HeLa NE. In control experiments, NE was omitted during binding with GST–WRN_949–1432 (lane 5) or GST–WRN_1072–1236 (lane 6). Western blotting was performed with anti-FEN-1 antibodies. The NE input (lane 1) corresponds to 10 µg. (B) Beads with GST (lane 1) or GST–WRN_949–1432 (lane 2) were incubated with 1.2 µg of recombinant FEN-1 purified from E.coli. In control experiments, FEN-1 was omitted during binding with GST–WRN_949–1432 (lane 3). Western blotting was performed with anti-FEN-1 antibodies. The FEN-1 input (lane 4) corresponds to 20 ng. (C) FEN-1–Sepharose specifically binds recombinant WRN. Purified WRN and FEN-1 were judged to be pure of DNA by analysis using SYBR Green Stain (FMC Products). Western blot of the third wash (lanes 1 and 2) or eluted fractions (lanes 3 and 4) from binding reaction of WRN to FEN-1–Sepharose (lanes 2 and 4) or BSA–Sepharose (lanes 1 and 3) is shown. (D) WRN and FEN-1 coprecipitate from HeLa cell lysate using anti-WRN antibody as demonstrated by western blotting. Top panel, blot was probed with anti-FEN-1 antibody. Bottom panel, blot was probed with anti-WRN antibody. Lane 1, control precipitate from HeLa cell lysate in which WRN antibody was omitted from immunoprecipitation; lane 2, HeLa cell lysate input; lane 3, immunoprecipitate from HeLa cell lysate using WRN antibody; lane 4, AG11395 (WS–/–) cell lysate input; lane 5, immunoprecipitate from AG11395 cell lysate using WRN antibody; lane 6, purified His-tagged FEN-1, which migrates slightly higher than NE FEN-1.

Fig. 1. Structural map of WRN protein motifs and recombinant proteins used in this study. (A) Conserved RecQ ATPase/helicase and exonuclease motifs, RecQ C-terminal region (RQC), helicase-related domain (HRDC), nuclear localization sequence (NLS) and known regions for WRN protein interactions are designated. Positions of site-directed mutations in motif I of the helicase domain (WRN-K577M) and motif I of the exonuclease domain (WRN-E84A) are indicated by asterisks. GST–WRN recombinant proteins are shown with subscripted numbers to designate the WRN amino acid sequences. (B) Purified full-length recombinant proteins (1 µg) and GST–WRN recombinant fragments (2–4 µg) used in this study were resolved on 10% polyacrylamide SDS gels run and stained with Coomassie Brilliant Blue. The 66 kDa band in the WRN protein preparations is BSA (100 µg/ml) (Orren et al., 1999). Bands migrating below GST–WRN_949–1432 (∼85 kDa) were determined to be degradation products by western blot analysis using anti-GST antibody (Santa Cruz).

Fig. 3. WRN stimulates FEN-1 cleavage activity. (A) Reactions (20 µl) containing 10 fmol of a 1 nt 5′ flap DNA substrate, 100 fmol of FEN-1 and 75 fmol of WRN were incubated at 37°C for 15 min under standard conditions as described in Materials and methods. A phosphorimage of a typical gel is shown. Substrate and cleavage products are as indicated. Lane 1, no enzyme; lane 2, FEN-1; lane 3, FEN-1 + wild-type WRN; lane 4, wild-type WRN. (B) % incision from (A) (mean value of three experiments) with standard deviation (SD) indicated by error bars. (C) Reactions (20 µl) containing 10 fmol of a 1 nt 5′ flap DNA substrate, 10 fmol of FEN-1 and the indicated amounts of WRN were incubated in the absence or presence of 40 mM KCl as indicated at 37°C for 15 min. A phosphorimage of a typical gel is shown. Lane 1, no enzyme; lane 2, 80 fmol of WRN; lane 3, FEN-1; lane 4, 80 fmol of WRN + FEN-1; lane 5, 40 fmol of WRN + FEN-1; lane 6, 20 fmol of WRN + FEN-1; lane 7, 10 fmol of WRN + FEN-1; lane 8, 5 fmol of WRN + FEN-1. (D) % incision from (C) (mean value of three experiments) with SD. Filled circles, no KCl; open circles, 40 mM KCl.