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 (OAS) that may constitute one-third of ionizing radiation damage. The antitumor drug bleomycin produces exclusively OAS in the form of C-4-keto-C-1-aldehydes in unbroken DNA strands and 3'-phosphoglycolate esters terminating strand breaks. We investigated whether two human DNA repair enzymes can mediate OAS excision in vitro: Ape1 protein (the main human abasic endonuclease (also called Hap1, Apex, or Ref1)) and DNA polymerase beta, which carries out both the abasic excision and the resynthesis steps. We used a duplex oligonucleotide substrate with one main target for bleomycin-induced damage. Ape1 catalyzed effective incision 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 location. Consistent with several previous studies, Ape1 hydrolyzed 3'-phosphoglycolates 25-fold more slowly than C-4-keto-C-1-aldehydes. DNA polymerase 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 coli Fpg (MutM) protein also excised 5'-terminal OAS, but in our hands, the RecJ protein did not. These observations help define mammalian pathways of OAS repair, point to interactions that might coordinate functional steps, and suggest that still unknown factors may contribute to removal of 3'-phosphoglycolate esters.
Xu
Y J
YJ
Department of Cancer Cell Biology, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
Kim
E Y
EY
Demple
B
B
D001483:Base Sequence; D001761:Bleomycin / Q000494:pharmacology; D019757:Carbon-Oxygen Lyases / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D019951:DNA Polymerase beta / Q000378:metabolism*; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D003855:Deoxyribose / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006801:Humans; D007700:Kinetics; D010084:Oxidation-Reduction
D029968:Escherichia coli Proteins; D001761:Bleomycin; D003855:Deoxyribose; D004247:DNA; D019951:DNA Polymerase beta; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase