Uncoupling of the base excision and nucleotide incision repair pathways reveals their respective biological roles.
The multifunctional DNA repair enzymes apurinic/apyrimidinic (AP) endonucleases cleave DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases in the base excision repair pathway. Alternatively, in the nucleotide incision repair (NIR) pathway, the same AP endonucleases incise DNA 5' of a number of oxidatively damaged bases. At present, the physiological relevance of latter function remains unclear. Here, we report genetic dissection of AP endonuclease functions in base excision repair and NIR pathways. Three mutants of Escherichia coli endonuclease IV (Nfo), carrying amino acid substitutions H69A, H109A, and G149D have been isolated. All mutants were proficient in the AP endonuclease and 3'-repair diesterase activities but deficient in the NIR. Analysis of metal content reveals that all three mutant proteins have lost one of their intrinsic zinc atoms. Expression of the nfo mutants in a repair-deficient strain of E. coli complemented its hypersensitivity to alkylation but not to oxidative DNA damage. The differential drug sensitivity of the mutants suggests that the NIR pathway removes lethal DNA lesions generated by oxidizing agents. To address the physiological relevance of the NIR pathway in human cells, we used the fluorescence quenching mechanism of molecular beacons. We show that in living cells a major human AP endonuclease, Ape1, incises DNA containing alpha-anomeric 2'-deoxyadenosine, indicating that the intracellular environment supports NIR activity. Our data establish that NIR is a distinct and separable function of AP endonucleases essential for handling lethal oxidative DNA lesions.
Groupe Réparation de l'ADN, Unité Mixte de Recherche 8126, Centre National de la Recherche Scientifique, Institut Gustave Roussy, 94805 Villejuif Cedex, France.
Alexander A
AA
Ishchenko
Eric
E
Deprez
Andrei
A
Maksimenko
Jean-Claude
JC
Brochon
Patrick
P
Tauc
Murat K
MK
Saparbaev
D000818:Animals; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D003839:Deoxyadenosines / Q000737:chemistry / Q000378:metabolism; D043223:Deoxyribonuclease IV (Phage T4-Induced) / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D006801:Humans; D051379:Mice; D009154:Mutation; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D013379:Substrate Specificity; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
D002614:Chelating Agents; D003839:Deoxyadenosines; D029968:Escherichia coli Proteins; D016877:Oxidants; D004247:DNA; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D015032:Zinc