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.
Wang
Wensheng
W
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Brandt
Patrick
P
Rossi
Marie L
ML
Lindsey-Boltz
Laura
L
Podust
Vladimir
V
Fanning
Ellen
E
Sancar
Aziz
A
Bambara
Robert A
RA
D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000502:physiology*; D004249:DNA Damage; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000502:physiology*; D004720:Endonucleases / Q000502:physiology*; D004789:Enzyme Activation; D045585:Flap Endonucleases / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D029702:Schizosaccharomyces pombe Proteins
D018797:Cell Cycle Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D029702:Schizosaccharomyces pombe Proteins; C106332:hus1 protein, S pombe; C069930:rad9 protein; D004720:Endonucleases; D045585:Flap Endonucleases