X4L4 does not stimulate WRN helicase. Helicase assays. WRN (lanes 2–5) or BLM (lanes 8–11) and increasing amount of X4L4 (lanes 3–6 and 9–12) were preincubated on ice. DNA substrate (37A/37B) was added to the proteins and incubated. Products were analyzed by nondenaturing PAGE.

WRN generates ligatable DNA ends. (A) Schematic representation of the reaction is indicated. (B) Ku (100 fmol in lanes 2–5 and 8), X4L4 (400 fmol in lanes 2–4, 6 and 9), and WRN (200 fmol in lane 3, and 400 fmol in lanes 4–7) were preincubated. The DNA substrate (66A/66B) was added to the reaction and incubated. Reaction products were analyzed by denaturing PAGE. (C) Ku (200 fmol in lanes 1 and 3), X4L4 (400 fmol in lanes 1 and 3), and WRN (200 fmol in lane 1) were preincubated. The DNA substrates (66A/66B in lanes 1 and 2, and 64A/64B in lane 3) were added to the reaction and incubated. S indicates substrates, and P indicates ligated products in B and C.

WRN interacts with X4L4. (A, B, and C) Co-IP assay. Protein complexes were immunoprecipitated from nuclear extracts using control mouse IgG (lane 2 in A) or mouse antibody against XRCC4 (lane 3 in A), using control rabbit IgG (lane 3 in B) or antibody against WRN (lane 2 in B), or using control rabbit IgG (lane 2 in C), rabbit antibody against XRCC4 (lane 3 in C), or rabbit antibody against WRN (lane 4 in C). Proteins immunoprecipitated from HeLa cell nuclear extracts were analyzed by Western blot analysis with rabbit antibody against WRN (upper panel in A) and BLM (lower panel in A), with mouse antibody against XRCC4 (B), or with rabbit antibody against DNA ligase IV (C). 10% of IP input is shown in lane 1 (A, B, and C). (D) ELISA. Wells were coated with 9 nM WRN (●) or BLM (○) and incubated with increasing concentrations of X4L4 (0.36, 0.72, 1.4, 2.9, 5.8, 12, and 23 nM). Bound X4L4 was detected using mouse anti-XRCC4 antibody. (E) ELISA. Wells were coated with 14.4 nM X4L4 (●) or dimeric XRCC4 (○). Increasing amounts of WRN (3.0, 6.1, 9.2, 12, 24, 49, and 73 nM) was added to the wells. Bound WRN was detected using rabbit polyclonal anti-WRN antibody. Absorbance values were corrected for BSA background and plotted against X4L4 (D) or WRN (E). Values and error bars are the mean of at least two independent experiments.

X4L4 stimulates WRN exonuclease activity. Exonuclease assays. (A) 100 fmol of WRN (lanes 2–5) and increasing amounts of X4L4 (25 fmol in lane 3, 50 fmol in lane 4, and 100 fmol in lanes 5 and 6) were preincubated on ice for 10 min, followed by the addition of 10 fmol of DNA substrate (49A/49B). Reactions were analyzed by denaturing PAGE. (B) Increasing amounts of XRCC4 or X4L4 (36, 72, 144, and 288 fmol) and WRN (36 fmol) were preincubated on ice for 10 min, followed by the addition of 10 fmol of the radiolabeled substrate (49C/49D) in lanes 2–5 and 7–10. XRCC4 (288 fmol) alone, lane 6; X4L4 (288 fmol) alone, lane 11; WRN (36 fmol) alone, lane 12. (C) The amount of the labeled probe excised by WRN was calculated as a fraction of the total DNA for each lane in B.