title: The dynamic alterations of H2AX complex during DNA repair detected by a proteomic approach reveal the critical roles of Ca(2+)/calmodulin in the ionizing radiation-induced cell cycle arrest.
abstract: By using DNA nuclease digestion and a quantitative "dual tagging" proteomic approach that integrated mass spectrometry, stable isotope labeling, and affinity purification, we studied the histone H2AX-associating protein complex in chromatin in mammalian cells in response to ionizing radiation (IR). In the non-irradiated control cells, calmodulin (CaM) and the transcription elongation factor facilitates chromatin transcription (FACT) were associated with H2AX. Thirty minutes after exposing cells to IR the CaM and FACT complexes dissociated, whereas two DNA repair proteins, poly(ADP-ribose) polymerase-1 and DEAH box polypeptide 30 isoform 1, interacted with H2AX. Two hours and 30 min after exposure, none of the above proteins were in the complex. H2B, nucleophosmin/B23, and calreticulin were associated with H2AX in both non-irradiated and irradiated cells. The results suggest that the H2AX complex undergoes dynamic changes upon induction of DNA damage and during DNA repair. The genuine interactions between H2AX and H2B, nucleophosmin/B23, calreticulin, poly(ADP-ribose) polymerase-1, and CaM under each condition were validated by immunoprecipitation/Western blotting and mammalian two-hybrid assays. Because multiple Ca(2+)-binding proteins were found in the H2AX complex, the roles of Ca(2+) were examined. The results indicate that Ca(2+)/CaM plays important roles in regulating IR-induced cell cycle arrest, possibly through mediating chromatin structure. The dataset presented here demonstrates that sensitive profiling of the dynamics of functional cellular protein-protein interactions can successfully lead to the dissection of important metabolic or signaling pathways.
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA.
authors.forename: Yu-Chun
authors.initials: YC
authors.lastname: Du
authors.forename: Sheng
authors.initials: S
authors.lastname: Gu
authors.forename: Jianhong
authors.initials: J
authors.lastname: Zhou
authors.forename: Tianyi
authors.initials: T
authors.lastname: Wang
authors.forename: Hong
authors.initials: H
authors.lastname: Cai
authors.forename: Mark A
authors.initials: MA
authors.lastname: Macinnes
authors.forename: E Morton
authors.initials: EM
authors.lastname: Bradbury
authors.forename: Xian
authors.initials: X
authors.lastname: Chen
mesh_terms: D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism*; D004260:DNA Repair; D003850:Deoxyribonuclease I; D016195:G2 Phase; D006657:Histones / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D040901:Proteomics / Q000379:methods*; D011839:Radiation, Ionizing
chemical_list: D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002843:Chromatin; C468783:H2AX protein, human; D006657:Histones; D003850:Deoxyribonuclease I