Alkyladenine DNA glycosylase deficiency uncouples alkylation-induced strand break generation from PARP-1 activation and glycolysis inhibition

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 AAGindependent. 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.DNA base damage is unavoidable and abundant, arising from hydrolysis, oxidation, deamination and alkylation reactions 1 . Base damage arises both as a by-product of cellular metabolism and by exposure to environmental agents and has been associated with several pathologies such as cancer, chronic inflammation and neurodegeneration 2,3 . Chiefly dealing with endogenous DNA base lesions is the base excision repair (BER) pathway. Initiation of the BER pathway occurs via the removal of a damaged base by one of many glycosylases displaying substrate specificities. Base excision leads to the formation of abasic sites and strand breaks that are subsequently processed by downstream enzymes so that DNA synthesis and ligation can take place 4 (Fig. 1A). Since many of the

A DNA repair-independent role for alkyladenine DNA glycosylase in alkylation-induced unfolded protein response

“…AAG-initiated BER leads to the generation of repair intermediates that activate PARP (poly(ADP-ribose) polymerase) (12). PARP is activated at SSBs, to synthesize a polymer of ADP-ribose (PAR) onto itself plus acceptor proteins usually associated with DNA transactions and shaping cellular outcome to a variety of stress conditions (45,46).…”

“…The nicked DNA is then ligated by DNA ligase I or the XRCC1/Ligase III complex (5,11). The flux of intermediates through this pathway must be efficiently coordinated because BER intermediates, such as abasic sites and SSB, are toxic (12)(13)(14). It has been shown that BER flux imbalance due to AAG overexpression exacerbates alkylation toxicity (15,16).…”

Excision of mutagenic replication-blocking lesions suppresses cancer but promotes cytotoxicity and lethality in nitrosamine-exposed mice

“…In addition to replication fork breakdown-associated DSBs that signal apoptosis, prior studies show that overactivity of PARP is a significant modulator of AAG-driven toxicity due to increased strand breaks and PARP-1 hyperactivation (Alhumaydhi et al, 2020; Allocca et al, 2017; Allocca et al, 2019). PARP-1 stimulates repair of SSBs by polymerizing ADP-ribose from NAD+, so an excess of DNA damage causes NAD+ depletion and a form of programmed necrotic cell death termed parthanatos (Xu et al, 2006; Yu et al, 2002; Zhao et al, 2018).…”

“…It is well established that AAG modulates responses of cells and animals to DNA methylating agents (Alhumaydhi et al, 2020; Allocca et al, 2017; Allocca et al, 2019; Calvo et al, 2016; Calvo et al, 2013; Engelward et al, 1996; Ensminger et al, 2014; Klapacz et al, 2010; Meira et al, 2009). However, prior studies have been almost exclusively focused on model methylating agents, for which human exposure is unlikely.…”

“…Interestingly, the predicted effects of Aag overexpression are very different. High levels of AAG activity cause an increase in the levels of SSBs (Alhumaydhi et al, 2020; Allocca et al, 2019; Margulies et al, 2017; Parrish et al, 2018) that cannot be bypassed by polymerases. Indeed, SSBs promote replication fork breakdown, leading to one-ended double strand breaks (DSBs) that can both signal for cell death and trigger HR.…”