Long Patch Base Excision Repair proceeds via coordinated stimulation of the multienzyme repair complex

Abstract

Base excision repair, a major repair pathway in mammalian cells, is responsible for correcting DNA damage and maintaining genomic integrity. Recent reports show that the Rad 9‐Rad1‐Hus1 complex (9‐1‐1) stimulates enzymes proposed to perform long patch base excision repair (LP‐BER) including DNA glycosylases, apurinic/apyrimidinic endonuclease 1 (APE1), DNA Polymerase β(Polβ), flap endonuclease 1 (FEN‐1) and DNA Ligase 1 (Lig 1). Since these stimulatory effects were studied in incomplete enzyme systems, we reconstituted the entire LP‐BER pathway in vitro using purified human proteins to characterize the functional interactions in the multi‐enzyme repair complex. We found that Pol βstimulated the flap cleavage activity of FEN‐1, the highest value, 12‐fold, in connection with strand displacement synthesis. Even in the absence of dNTPs or using a Pol βsynthesis‐deficient mutant, Pol βstimulated FEN‐1 nearly five‐fold. We also observed a two‐fold stimulation of Lig1 activity by Polβ. Stimulation of FEN‐1 and Lig 1 activities by Pol βare likely responsible for the diminished responsiveness of these proteins to the 9‐1‐1 complex in reconstituted LP‐BER. Observed stimulations suggest a significant role for Pol βactivity in base excision repair and provide further evidence for the existence of LP‐BER in vivo.This work was supported by National Institutes of Health Grant GM024441