Mechanistic and temporal characterisation of alkyladenine DNA glycosylase-mediated cell death.

DNA damage results from exposure to endogenous and environmental genotoxic agents such as alkylating agents. Base excision repair (BER) is a DNA repair process initiated by DNA glycosylases acting on DNA base damage. An example of such DNA glycosylases acting on alkylation base damage is the alkyladenine DNA glycosylase (AAG). AAG activity on alkylated DNA bases initiates BER and generates abasic sites (AP sites), which are further processed to form single strand breaks (SSBs). If left unrepaired, these BER intermediates are very toxic to the cell. Since BER is a multistep repair process, any imbalance in the pathway can lead to accumulation of these toxic intermediates and potentially trigger cell death via a mechanism postulated to involve hyperactivation of poly(ADP-ribose) polymerase (PARP). PARP uses energy in the form of NAD+ for the synthesis of poly(ADP-ribose) (PAR) polymers and therefore PARP hyperactivation may result in bioenergetics failure. This study characterised the BER pathway initiated by AAG and analysed cellular response to alkylation by using AAG proficient and deficient cells. The temporal changes in BER intermediate incidence were characterised. In addition, PAR synthesis and cellular levels of NAD+ and ATP as well as cell death were measured and the temporal changes were characterised. Moreover, the effects of alkylation treatment on cell cycle progression were investigated. The results show that the kinetics and magnitude of BER intermediate formation is similar in AAG proficient versus AAG deficient cells. Meanwhile, there were clear differences between these two genotypes in terms of PAR synthesis, bioenergetics, cell viability and cell cycle. This study shows that the processing of alkylated bases and formation of BER intermediates is rapid and concomitant with an increase in PAR synthesis. Importantly, this increase in PAR synthesis is only observed in AAG proficient cells suggesting that AAG activity is necessary for PAR polymer formation after SSB induction