Deciphering the antioxidative role of p53 against H2O2-induced DNA damage using ion mobility mass spectrometry

The transcription factor p53 is exquisitely sensitive and selective to a broad variety of cellular environments. Several studies have reported that oxidative stress weakens the p53-DNA binding affinity for certain promoters depending on the oxidation mechanism. Despite this body of work, the precise mechanisms by which the physiologically relevant DNA-p53 tetramer complex senses cellular stresses caused by H2O2 are still unknown. Here, we employed native mass spectrometry (MS) and ion mobility (IM)-MS coupled to chemical labelling and chemical oxidation to examine the mechanism of redox regulation of the p53-p21 complex. Our approach has found that two reactive cysteines in p53 protect against H2O2-induced oxidative DNA damage by forming sulfenates. A harsh H2O2 insult that exceeds the antioxidant capability of p53 causes DNA oxidation followed by p53 dissociation. The DNA-free p53 is further oxidized to form disulfides leading to function and structure impairment.