The role of GAPDH in maintaining the functional state of the DNA repair enzyme APE1
Apurinic/apyrimidinic (AP) sites are highly mutagenic DNA lesions occurring either spontaneously or by the action of DNA damaging agents. In human cells, AP sites are processed by the major DNA repair enzyme APE1 through the base excision repair (BER) pathway. APE1 is a multifunctional protein that has AP endonuclease/3’-diesterase activities in addition to its role as a redox factor in activating many transcription factor. Recently, it has been shown that APE1 interacts with the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an interaction that results in the activation of APE1 by reduction. Interestingly, depletion of GAPDH sensitized the cells to DNA damaging agents and induced an increase in spontaneous AP sites frequency. Moreover, cells knocked-down for GAPDH showed defects in proliferation. Here we set up to investigate the effects of GAPDH knockdown on cell cycle progression, APE1 subcellular localization and to identify the cysteine residue(s) of APE1, target(s) of GAPDH reduction. Our studies showed that GAPDH deficient cells arrested in G1 phase of the cell cycle. The defect in cell cycle progression is most probably due to accumulation of DNA damage which activates checkpoints leading to a delay in the cell cycle to allow DNA repair. Furthermore, in GAPDH deficient cells, APE1 formed nuclear foci-like structures that could represent aggregates of the oxidized form of APE1 or inactive APE1 foci on DNA lesions. Using site-directed mutagenesis, we created seven APE1 cysteine to alanine mutants which were cloned into a mammalian expression vector. We expect that at least one of these mutants is likely to resist the inactivation by oxidation as it cannot engage in disulfide bridge formation. Therefore, the expression of this mutant(s) in GAPDH knockdown cells is expected to restore a normal APE1 cellular distribution, rescue the cell cycle defects, and render the cells less sensitive to DNA damaging agents. In conclusion, our results show a new role of GAPDH in maintaining genomic stability under oxidative stress by maintaining APE1 in its functional state.
APE1 GAPDH Cysteine Site AP APE1 GAPDH GAPDH knockdown DNA damage cysteine AP sites