EXCISION OF C-4'-OXIDIZED DEOXYRIBOSE LESIONS FROM DOUBLE-STRANDED DNA BY HUMAN APURINIC APYRIMIDINIC ENDONUCLEASE (APE1 PROTEIN) AND DNA-POLYMERASE-BETA/

Oxidative damage to DNA deoxyribose generates oxidized abasic sites (O AS) that may constitute one-third of ionizing radiation damage. The an titumor drug bleomycin produces exclusively OAS in the farm of C-4-ket o-C-1-aldehydes in unbroken DNA strands and 3'-phosphoglycolate esters terminating strand breaks. We investigated whether two human DNA repa ir enzymes can mediate OAS excision in vitro: Ape1 protein (the main h uman abasic endonuclease (also called Hap1, Apex, or Ref1)) and DNA po lymerase beta, which carries out both the abasic excision and the resy nthesis steps. We used a duplex oligonucleotide substrate with one mai n target for bleomycin-induced damage. Ape1 catalyzed effective incisi on at the C-4-keto-C-1-aldehyde sites at a rate that may be only a few -fold lower than incision of hydrolytic abasic sites at the same locat ion. Consistent with several previous studies, Ape1 hydrolyzed 3'-phos phoglycolates 25-fold more slowly than C-4-keto-C-1-aldehydes. DNA pol ymerase beta excised the 5'-terminal OAS formed by Ape1 incision at a rate similar to its removal of unmodified abasic residues. Polymerase beta-mediated excision of 5'-terminal OAS was stimulated by Ape1 as it is for unmodified abasic sites. Escherichia coil Fpg (MutM) protein a lso excised 5'-terminal GAS, but in our hands, the RecJ protein did no t. These observations help define mammalian pathways of OAS repair, po int to interactions that might coordinate functional steps, and sugges t that still unknown factors may contribute to removal of 3'-phosphogl ycolate esters.