The major human AP endonuclease (Ape1) is involved in the nucleotide incision repair pathway.
In nucleotide incision repair (NIR), an endonuclease nicks oxidatively damaged DNA in a DNA glycosylase-independent manner, providing the correct ends for DNA synthesis coupled to the repair of the remaining 5'-dangling modified nucleotide. This mechanistic feature is distinct from DNA glycosylase-mediated base excision repair. Here we report that Ape1, the major apurinic/apyrimidinic endonuclease in human cells, is the damage- specific endonuclease involved in NIR. We show that Ape1 incises DNA containing 5,6-dihydro-2'-deoxyuridine, 5,6-dihydrothymidine, 5-hydroxy-2'-deoxyuridine, alpha-2'-deoxyadenosine and alpha-thymidine adducts, generating 3'-hydroxyl and 5'-phosphate termini. The kinetic constants indicate that Ape1-catalysed NIR activity is highly efficient. The substrate specificity and protein conformation of Ape1 is modulated by MgCl2 concentrations, thus providing conditions under which NIR becomes a major activity in cell-free extracts. While the N-terminal region of Ape1 is not required for AP endonuclease function, we show that it regulates the NIR activity. The physiological relevance of the mammalian NIR pathway is discussed.
Gros
Laurent
L
Groupe Réparation de l'ADN', UMR 8113 CNRS, LBPA-ENS Cachan, Institut Gustave Roussy, 39, rue Camille Desmoulins, 94805 Villejuif Cedex, France.
Ishchenko
Alexander A
AA
Ide
Hiroshi
H
Elder
Rhoderick H
RH
Saparbaev
Murat K
MK
D002457:Cell Extracts; D004249:DNA Damage; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009711:Nucleotides / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation / Q000187:drug effects; D013379:Substrate Specificity
D002457:Cell Extracts; D009711:Nucleotides; D009838:Oligodeoxyribonucleotides; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D008274:Magnesium