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Originally published In Press as doi:10.1074/mcp.M500327-MCP200 on March 7, 2006.
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Molecular & Cellular Proteomics 5:1033-1044, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

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*,S

Yu-Chun Du{ddagger}, Sheng Gu{ddagger}, Jianhong Zhou{ddagger}, Tianyi Wang{ddagger}, Hong Cai{ddagger}, Mark A. MacInnes{ddagger}, E. Morton Bradbury§ and Xian Chen{ddagger},¶

From the {ddagger} Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 and § Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Davis, California 95616

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 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.

Recipient of the Presidential Early Career Award for Scientists and Engineers (2000–2005). To whom correspondence should be addressed: Bioscience Division, Mail-stop M888, Los Alamos National Laboratory, Los Alamos, NM 87545. Tel.: 505-665-3197; Fax: 505-665-3024; E-mail: chen_xian{at}lanl.gov


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