Abstract
Regulatory mechanisms underlying γH2AX induction and the associated cell fate decision during DNA damage response (DDR) remain obscure. Here, we discover a bromodomain (BRD)-like module in DNA-PKcs (DNA-PKcs-BRD) that specifically recognizes H2AX acetyl-lysine 5 (K5ac) for sequential induction of γH2AX and concurrent cell fate decision(s). First, top-down mass spectrometry of radiation-phenotypic, full-length H2AX revealed a radiation-inducible, K5ac-dependent induction of γH2AX. Combined approaches of sequence-structure modeling/docking, site-directed mutagenesis, and biochemical experiments illustrated that through docking on H2AX K5ac, this non-canonical BRD determines not only the H2AX-targeting activity of DNA-PKcs but also the over-activation of DNA-PKcs in radioresistant tumor cells, whereas a Kac antagonist, JQ1, was able to bind to DNA-PKcs-BRD, leading to re-sensitization of tumor cells to radiation. This study elucidates the mechanism underlying the H2AX-dependent regulation of DNA-PKcs in ionizing radiation-induced, differential DDR, and derives an unconventional, non-catalytic domain target in DNA-PKs for overcoming resistance during cancer radiotherapy.