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Submitted on October 4, 2005
Biosciences, Los Alamos Natl Lab, Los Alamos, NM 87544
Corresponding Author: chen_xian{at}lanl.gov
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 FACT were associated with H2AX. Thirty min after exposing cells to IR the CaM and FACT complexes dissociated, while two DNA repair proteins, poly (ADP-ribose) polymerase-1 (PARP-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, PARP-1, and CaM under each condition were validated by immunoprecipitation/Western blotting, and mammalian two hybrid assays. Because multiple Ca2+ binding proteins were found in the H2AX complex, the roles of Ca2+ were examined. The results indicate that Ca2+/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
Revised on January 18, 2006
Accepted on March 7, 2006
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
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