Uncoupling of the base excision and nucleotide incision repair pathways reveals their respective biological roles

Abstract

The multifunctional DNA repair enzymes apurinic͞apyrimidinic (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 ␣-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. apurinic͞apyrimidinic endonuclease ͉ oxidative DNA damage ͉ tert-butyl hydroperoxide ͉ 3Ј-blocking groups ͉ ␣-anomeric 2Ј-deoxyadenosine

Alternative Excision Repair Pathways

“…Several questions were addressed as to the new functions of APendonuclease: whether endonuclease activity for base damage is different from AP endonuclease activity, how active NIR is within human cells as compared with BER, and how AP endonucleases are able to recognize various types of base damage. For the first question, mutagenesis of the E. coli nfo gene showed that three mutants-H69A, H109A, and G149D-were proficient in the AP endonuclease and 3 0 phosphodiesterase activities but deficient in NIR activity, indicating that AP endonuclease and base damage nicking activities are separate mechanisms (Ishchenko et al 2006). These nfo mutants complemented the hypersensitivity of an E. coli nfo-deletion mutant to alkylation but not to oxidative DNA damage, suggesting that NIR is responsible for repair of oxidative base damage in E. coli cells.…”

A unified view of base excision repair: Lesion-dependent protein complexes regulated by post-translational modification

“…For example, APE1 has been found to initiate DNA glycosylase-independent repair of oxidized cytosines [20][21][22]. This alternate repair pathway has been defined as Nucleotide Incision Repair (NIR), yet can also be considered a minor sub-pathway of BER with a unique APE1-mediated repair initiation event.…”

Uncoupling of the apyrimidinic/apurinic endonucleolytic and 3′→5′ exonucleolytic activities of the Nfo protein of Mycoplasma pneumoniae through mutation of specific amino acid residues

“…This mutant, which represents the counterpart of Nfo Mpn mutant H72N from this study, was found to lack one of the three active-site Zn 2+ ions (Zn 1 , as predicted) and also showed other rearrangements within the active site, such as an increased conformational flexibility of residue H109, which is, like H69, involved in the interaction with Zn 1 in the wild-type protein (Golan et al, 2010). Like Nfo Eco mutant H69A, Nfo Mpn mutant H72N showed a significantly reduced AP cleavage activity in comparison with the wild-type protein; however, both proteins showed considerably higher activities in the presence of divalent cations in the reaction (this study; Ishchenko et al, 2006 This notion is based on the finding that mutation of the aforementioned active site residues not only affects the AP endonucleolytic activity (in particular in the presence of EDTA), but also affects the exonucleolytic activity of Nfo Mpn . Nevertheless, it is clear that the latter activity has more stringent amino acid sequence and/or structural requirements than the AP cleavage activity.…”