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Uncoupling of the Base Excision and Nucleotide Incision Repair Pathways Reveals Their Respective Biological Roles
Alexander A. Ishchenko, Eric Deprez, Andrei Maksimenko, Jean-Claude Brochon, Patrick Tauc and Murat K. Saparbaev
Proceedings of the National Academy of Sciences of the United States of America
Vol. 103, No. 8 (Feb. 21, 2006), pp. 2564-2569
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/30049453
Page Count: 6
You can always find the topics here!Topics: HeLa cells, DNA, 3T3 cells, Enzymes, DNA repair, DNA damage, Atoms, Mutant proteins, Fluorescence, Oligonucleotides
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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, Apel, incises DNA containing α-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.
Proceedings of the National Academy of Sciences of the United States of America © 2006 National Academy of Sciences