DNA-bound structures and mutants reveal abasic DNA binding by APE1 DNA repair and coordination

Non-coding apurinic/apyrimidinic (AP) sites in DNA are continually created in cells both spontaneously and by damage-specific DNA glycosylases'. The b iologically critical human base excision repair enzyme APE1 cleaves the DNA sugar-phosphate backbone at a position 5' of AP sites to prime DNA repair synthesis(2-4). Here we report three co-crystal structures of human APE1 bo und to abasic DNA which show that APE1 uses a rigid, pre-formed, positively charged surface to kink the DNA helix and engulf the AP-DNA strand. APE1 i nserts loops into both the DNA major and minor grooves and binds a flipped- out AP site in a pocket that excludes DNA bases and racemized beta-anomer A P sites. Both the APE1 active-site geometry and a complex with cleaved AP-D NA and Mn2+ support a testable structure-based catalytic mechanism. Alanine substitutions of the residues that penetrate the DNA helix unexpectedly sh ow that human APE1 is structurally optimized to retain the cleaved DNA prod uct. These structural and mutational results show how APE1 probably displac es bound glycosylases and retains the nicked DNA product, suggesting that A PE1 acts in vivo to coordinate the orderly transfer of unstable DNA damage intermediates between the excision and synthesis steps of DNA repair.