Abstract
DNA alkylation damage is repaired by base excision repair (BER) initiated by alkyladenine DNA glycosylase (AAG). Despite its role in DNA repair, AAG-initiated BER promotes cytotoxicity in a process dependent on poly (ADP-ribose) polymerase-1 (PARP-1); a NAD+-consuming enzyme activated by strand break intermediates of the AAG-initiated repair process. Importantly, PARP-1 activation has been previously linked to impaired glycolysis and mitochondrial dysfunction. However, whether alkylation affects cellular metabolism in the absence of AAG-mediated BER initiation is unclear. To address this question, we temporally profiled repair and metabolism in wild-type and Aag−/− cells treated with the alkylating agent methyl methanesulfonate (MMS). We show that, although Aag−/− cells display similar levels of alkylation-induced DNA breaks as wild type, PARP-1 activation is undetectable in AAG-deficient cells. Accordingly, Aag−/− cells are protected from MMS-induced NAD+ depletion and glycolysis inhibition. MMS-induced mitochondrial dysfunction, however, is AAG-independent. Furthermore, treatment with FK866, a selective inhibitor of the NAD+ salvage pathway enzyme nicotinamide phosphoribosyltransferase (NAMPT), synergizes with MMS to induce cytotoxicity and Aag−/− cells are resistant to this combination FK866 and MMS treatment. Thus, AAG plays an important role in the metabolic response to alkylation that could be exploited in the treatment of conditions associated with NAD+ dysregulation.
Highlights
DNA alkylation damage is repaired by base excision repair (BER) initiated by alkyladenine DNA glycosylase (AAG)
The maximum AP site induction in the Aag−/− MEFs was observed 3 hours post-methyl methanesulfonate (MMS) treatment (p < 0.004), a time where AP site levels were back to baseline in wild-type cells. These results indicate that while AAG is necessary for efficient alkylation base damage processing into AP sites, it is not essential, as AP sites are still formed after alkylation treatment in AAG -deficient cells
In an effort to understand how AAG-initiated BER modulates different stages in the cellular response to base damage, we examined BER intermediate generation, poly (ADP-ribose) polymerase-1 (PARP-1) activation levels and aspects of cellular metabolism, after alkylation treatment in AAG-proficient or deficient MEFs
Summary
DNA alkylation damage is repaired by base excision repair (BER) initiated by alkyladenine DNA glycosylase (AAG). In support of a protective role for AAG against DNA base damage resulting from alkylation, oxidation and/or inflammation, Aag−/− mice were found to be more susceptible than wild-type to chronic inflammation-induced DNA damage and display increased tumor multiplicity and severity after treatment with the alkylating agent azoxymethane in combination with the inflammation-inducing compound dextran sulphate sodium[9]. Contrasting with this protective role, additional studies showed that AAG drives alkylation-induced cytotoxicity in some cell types. Unrepaired SSB intermediates of AAG activity trigger cell death via PARP-1 activation, but preventing PARP-1 activation is not sufficient to ensure survival to alkylation when DNA damage levels are high
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