hSMG-1 and ATM sequentially and independently regulate the G1 checkpoint during oxidative stress

Genotoxic stress activates the phosphatidylinositol 3-kinase-like kinases ( PIKKs) that phosphorylate proteins involved in cell cycle arrest, DNA repair and apoptosis. Previous work showed that the PIKK ataxia telangiectasia mutated ( ATM) but not ATM and Rad3 related phosphorylates p53 ( Ser15) during hyperoxia, a model of prolonged oxidative stress and DNA damage. Here, we show hSMG-1 is responsible for the rapid and early phosphorylation of p53 ( Ser15) and that ATM helps maintain phosphorylation after 24 h. Despite reduced p53 phosphorylation and abundance in cells depleted of hSMG-1 or ATM, levels of the p53 target p21 were still elevated and the G(1) checkpoint remained intact. Conditional overexpression of p21 in p53-deficient cells revealed that hyperoxia also stimulates wortmannin-sensitive degradation of p21. siRNA depletion of hSMG-1 or ATM restored p21 stability and the G(1) checkpoint during hyperoxia. These findings establish hSMG-1 as a proximal regulator of DNA damage signaling and reveal that the G1 checkpoint is tightly regulated during prolonged oxidative stress by both PIKK-dependent synthesis and proteolysis of p21.