9-1-1 stimulates FEN1 activity on a variety of substrates. (A) FEN1 cleavage on a 27-nt nick flap (D₅·T₃·U_4, shown schematically at the top with * indicating ³²P) in the presence of increasing 9-1-1. Reaction mixtures with FEN1 contain 0.5 fmol of FEN1. Lane 2 contains 500 fmol of 9-1-1. Lanes 4–7 contain 50, 100, 200, and 500 fmol of 9-1-1. (B) FEN1 stimulation by 9-1-1 on a 27-nt flap with various upstream primers. Short-dashed line, 27-nt double flap (df) (D₅·T₃·U₇); solid line, 27-nt nick flap (nf) (D₅·T₃·U₄); long-dashed line, 27-nt gap flap (gf) (D₅·T₃·U₂). (C) FEN1 stimulation by 9-1-1 on a 25-nt flap with various upstream primers. Short-dashed line, 25-nt double flap (D₄·T₁·U₈); solid line, 25-nt nick flap (D₄·T₁·U₅); long-dashed line, 25-nt gap flap (D₄·T₁·U₃). (D) FEN1 stimulation by 9-1-1 on a 10-nt flap with various upstream primers. Short-dashed line, 10-nt double flap (D₅·T₄·U₇); solid line, 10-nt nick flap (D₅·T₄·U₄); long-dashed line, 10-nt gap flap (D₅·T₄·U₂). (E) FEN1 stimulation by 9-1-1 on a 1-nt flap with various upstream primers. Short-dashed line, 1-nt double flap (D₂·T₁·U₈); solid line, 1-nt nick flap (D₂·T₁·U₅); long-dashed line, 1-nt gap flap (D₂·T₁·U₃). (F) FEN1 stimulation on a nicked and gapped substrate. Solid line, nicked substrate (D₁·T₁·U₅); long-dashed line, gapped substrate (D₁·T₁·U₃).

9-1-1 uniformly stimulates FEN1 cleavage. (A) Stimulation of FEN1 cleavage was assayed in a time-course reaction (0–3.6 min) in the absence or presence of 9-1-1 on a 27-nt-nick flap substrate (D₅·T₃·U₄) as described in Materials and Methods. The starting material is 55 nt, and the 27-nt product marks the FEN1 cleavage of the flap. (B) Quantified results of stimulation time course. ▴, FEN1 alone control; ▪, FEN1 and 9-1-1 complex together. Stimulation at each time point was ≈10- to 15-fold.

Blocking 9-1-1 entry to the double strands prevents a portion of the stimulation. Stimulation of FEN1 cleavage was assayed on unblocked (lanes 1–8) and blocked (lanes 9–13) 25-nt nick-flap substrates (D₄·T₁·U₅) in the presence of increasing amounts of PCNA (3 or 6 pmol) or 9-1-1 (40 or 80 fmol) as described in Materials and Methods. The starting material is 43 nt, and FEN1 cleavage of the flap is marked by the 25- and 24-nt products. On unblocked substrate, the band densities of lanes 5–8 are 6×,9×,2×, and 8×, respectively, relative to FEN1 alone in lane 4. On blocked substrate, the band densities of lanes 10–13 are 2.5×,2×, 1.5×, and 4×, respectively, relative to FEN1 alone in lane 9.

9-1-1 does not stimulate FEN1 cleavage of a bubble substrate. Ten microliters of substrate (10 fmol) with a 37-nt flap or 24-nt bubble was mixed with 10 μl of enzymes containing 1 fmol of FEN1 and increasing amounts of PCNA (3 or 6 pmol) or 9-1-1 (0.5 or 1 pmol). The reactions took place at 37°C for 10 min. The ramps denote the different amounts of PCNA or 9-1-1. The * indicates the radiolabeled position. The sizes of substrate and cleavage product are indicated by arrows. The substrate shown in lanes 1–5 and 11–15 is derived from the annealing of D₆·T₃·U₇. The substrate shown in lanes 6–10 and 16–20 is from the annealing of D₇·T₅·U₆. The addition of PCNA to a flap substrate augments the cleavage product (lanes 4 and 5) compared with that of FEN1 only (lane 3). As with PCNA, more cleavage products are observed after addition of 9-1-1 (lanes 14 and 15) compared with those with FEN1 only (lane 13). The addition of PCNA (lanes 9 and 10) or 9-1-1 (lanes 19 and 20) to the bubble substrate does not lead to a visible cleavage product.

9-1-1 interacts with FEN1. Immunoprecipitation was performed as described in Materials and Methods. HeLa cell extract was immunoprecipitated (IP) with anti-human (h)Rad1 polyclonal antibody. Lane 1, whole cell lysate mixed only with protein G-Sepharose beads (mock immunoprecipitation containing no antibody). Lane 2, immunoprecipitation with anti-hRad1 antibody. The weak band larger than hFEN1 in Upper and the weak band smaller than hRad1 in Lower are antibody IgG heavy chain and light chain, respectively. Lane 3, supernatant fluid of third wash after immunoprecipitation. Lane 4, whole cell lysate control. Lane 5, purified recombinant human FEN1 control. Lane 6, purified recombinant human Rad1 control. Upper is blotted with anti-hFEN1 polyclonal antibody; Lower is blotted with anti-hRad1 antibody. Arrows indicate the positions of endogenous hFEN1 and hRad1. The band indicated by an asterisk is likely partially degraded recombinant hRad1. Because purified recombinant hFEN1 contains a His₆ tag, and purified recombinant hRad1 is tagged with a Flag-cAMP kinase motif, the sizes of both proteins are larger than endogenous hFEN1 and hRad1 (19).

9-1-1 fails to stimulate pol β synthesis. Synthesis by pol β was assayed in the presence of increasing amounts of PCNA (10, 50, 100, 200, and 500 fmol) or 9-1-1 (10, 50, 100, 200, and 500 fmol) on a primer–template substrate (T₆·U₁) as described in Materials and Methods. The 20-nt band marks the labeled primer, and the 110-nt product marks pol β full-length extension of the upstream primer.