FIGURE 7.

hChlR1 interacts with Fen1 and stimulates its endonuclease activity. A, 1 mg of lysates from 293 cells transiently expressing FLAG-Fen1 alone (lane 2) or together with constitutively expressed ChlR1-HA (lane 4) were incubated with HA antibody as indicated at the top of the immunoblots; specific interactions were detected by Western blotting using antibodies to hChlR1 (top) and Fen1 (bottom). B, reciprocal immunoprecipitations in 293 cells expressing ChlR1-HA and FLAG-tagged Fen1 performed with protein A beads in combination with Fen1 antibodies (lane 2) or preimmune serum (lane 3), as noted at the top of the immunoblot; specific interactions were detected by Western blotting using antibodies to hChlR1 (top), and Fen1 (bottom). C, hChlR1 directly interacts with Fen1. Recombinant His-tagged-Fen1 (5.6 pmol) and recombinant FLAG-tagged ChlR1 (5 pmol) were incubated with FLAG-M2 antibody beads (lanes 3 and 4); proteins were eluted in the absence (lane 3) or presence (lane 4) of 1 mg/ml FLAG peptide, and specific interactions were detected by Western blotting using antibodies specific to hChlR1 (top) and Fen1 (bottom). Input represents 10% of total amount of lysate/recombinant protein used for immunoprecipitation. D, hChlR1 stimulates Fen1 endonuclease activity. Fen1 (5 fmol) was incubated in the absence (lanes 2–5) or presence (lanes 6–9) of the indicated amounts of RPA with or without hChlR1 (lanes 7–9 and lanes 3–5, respectively) in standard flap endonuclease reaction mixtures (described in Ref. 36) in the presence of the 729/5TBG substrate (described in Table 1), whose structure is shown on the left. IP, immunoprecipitation.