Action of human apurinic endonuclease (Ape1) on C1'-oxidized deoxyribose damage in DNA.
Oxidative damage to DNA includes diverse lesions in the sugar-phosphate backbone. The chemical "nuclease" bis(1,10-phenanthroline)copper complex [(OP)(2)Cu] is believed to generate a mixture of direct oxidative strand breaks and C1'-oxidized abasic sites (2-deoxyribonolactone; dL). We found that, under our conditions, the lesions produced by (OP)(2)Cu (50 microM) in synthetic duplex DNA were predominantly dL, accompanied by approximately 30% direct strand breaks with 3'-phosphates. For enzymatic studies, (OP)(2)Cu was used to introduce damage with limited sequence-selectivity, while photolysis of a site-specific 2'-deoxyuridine-1'-t-butyl ketone generated dL at a defined position. The results showed that Ape1, the major human abasic endonuclease, catalyzed 5'-incision of dL sites, but acted at least 10-fold less effectively to remove the 3'-phosphates at direct strand breaks. Kinetic analysis of Ape1 incision using the site-specific dL substrate revealed the same k(cat) for dL and regular (glycosylase-generated) abasic sites, but with K(m) approximately five-fold higher for dL substrate. The efficiency of Ape1 acting on dL, and the abundance of this enzyme in vivo, indicate that dL sites in vivo would be rapidly processed by the endonuclease. The recent observation that Ape1-cleaved dL sites can covalently trap DNA polymerase beta during the abasic excision process suggests that efficient incision of dL by Ape1 may potentiate further problems in DNA repair.
Xu
Yong-jie
YJ
Department of Cancer Cell Biology, Harvard School of Public Health, Boston, MA 02115, USA.
DeMott
Michael S
MS
Hwang
Jae Taeg
JT
Greenberg
Marc M
MM
Demple
Bruce
B
D019757:Carbon-Oxygen Lyases / Q000378:metabolism*; D004249:DNA Damage / Q000502:physiology*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D003855:Deoxyribose / Q000378:metabolism*; D006801:Humans; D009841:Oligonucleotides / Q000378:metabolism; D010618:Phenanthrolines / Q000378:metabolism; D013379:Substrate Specificity / Q000502:physiology
D009841:Oligonucleotides; D010618:Phenanthrolines; D003855:Deoxyribose; D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; C025205:1,10-phenanthroline