The Dynamic Alterations of H2AX Complex during DNA Repair Detected by a Proteomic Approach Reveal the Critical Roles of Ca2+/Calmodulin in the Ionizing Radiation-induced Cell Cycle Arrest

• DOI: 10.1074/mcp.m500327-mcp200
• Publication Date: 2006-03-08T02:43:41Z
• Authors: Yu-Chun Du; Sheng Gu; Jianhong Zhou; Tianyi Wang; Hong Cai; Mark A. MacInnes; E. Morton Bradbury; Xian Chen
• Keywords: G2 Phase; Proteomics; 0301 basic medicine; DNA Repair; Calcium-Binding Proteins; Chromatin; Cell Line; Histones; 03 medical and health sciences; Calmodulin; Radiation, Ionizing; Deoxyribonuclease I; Humans; Protein Binding

By using DNA nuclease digestion and a quantitative "dual tagging" proteomic approach that integrated mass spectrometry, stable isotope labeling, affinity purification, we studied the histone H2AX-associating protein complex in chromatin mammalian cells response to ionizing radiation (IR). In non-irradiated control cells, calmodulin (CaM) transcription elongation factor facilitates (FACT) were associated with H2AX. Thirty minutes after exposing IR CaM FACT complexes dissociated, whereas two repair proteins, poly(ADP-ribose) polymerase-1 DEAH box polypeptide 30 isoform 1, interacted Two hours min exposure, none of above proteins complex. H2B, nucleophosmin/B23, calreticulin H2AX both irradiated cells. The results suggest undergoes dynamic changes upon induction damage during repair. genuine interactions between calreticulin, polymerase-1, under each condition validated by immunoprecipitation/Western blotting two-hybrid assays. Because multiple Ca(2+)-binding found complex, roles Ca(2+) examined. indicate Ca(2+)/CaM plays important regulating IR-induced cell cycle arrest, possibly through mediating structure. dataset presented here demonstrates sensitive profiling dynamics functional cellular protein-protein can successfully lead dissection metabolic or signaling pathways.