The human Rad9-Rad1-Hus1 checkpoint complex stimulates flap endonuclease 1

The toroidal damage checkpoint complex Rad9-Rad1-Hus1 (9-1-1) has been characterized as a sensor of DNA damage. Flap Endonuclease 1 (FEN1) is a structure-specific Nuclease involved both in removing initiator RNA from Okazaki fragments and in DNA repair pathways. FEN1 activity is stimulated by proliferating cell nuclear antigen (PCNA), a toroidal sliding clamp that acts as a platform for DNA replication and repair complexes. We show that 9-1-1 also binds and stimulates FEN1. Stimulation is observed on a variety of FLAP, nick, and gapped substrates simulating repair intermediates. Blocking 9-1-1 entry to the double strands prevents a portion of the stimulation. Like PCNA stimulation, 9-1-1 stimulation cannot circumvent the tracking mechanism by which FEN1 enters the substrate; however, 9-1-1 does not substitute for PCNA in the stimulation of DNA Polymerase beta. This suggests that 9-1-1 is a damage-specific activator of FEN1.