title: The complexity of phosphorylated H2AX foci formation and DNA repair assembly at DNA double-strand breaks.
abstract: The maintenance of genome stability requires efficient DNA double-stranded break (DSB) repair mediated by the phosphorylation of multiple histone H2AX molecules near the break sites. The phosphorylated H2AX (gammaH2AX) molecules form foci covering many megabases of chromatin. The formation of gamma-H2AX foci is critical for efficient DNA damage response (DDR) and for the maintenance of genome stability, however, the mechanisms of protein organization in foci is largely unknown. To investigate the nature of gammaH2AX foci formation, we analyzed the distribution of gammaH2AX and other DDR proteins at DSB sites using a variety of techniques to visualize, expand and partially disrupt chromatin. We report here that gammaH2AX foci change composition during the cell cycle, with proteins 53BP1, NBS1 and MRE11 dissociating from foci in G(2) and mitosis to return at the beginning of the following G(1). In contrast, MDC1 remained colocalized with gamma-H2AX during mitosis. In addition, while gammaH2AX was found to span large domains flanking DSB sites, 53BP1 and NBS1 were more localized and MDC1 colocalized in doublets in foci. H2AX and MDC1 were found to be involved in chromatin relaxation after DSB formation. Our data demonstrates that the DSB repair focus is a heterogeneous and dynamic structure containing internal complexity.
authors.affiliation: Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA. nakamuraa@mail.nih.gov
authors.forename: Asako J
authors.initials: AJ
authors.lastname: Nakamura
authors.forename: V Ashutosh
authors.initials: VA
authors.lastname: Rao
authors.forename: Yves
authors.initials: Y
authors.lastname: Pommier
authors.forename: William M
authors.initials: WM
authors.lastname: Bonner
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D016193:G1 Phase; D016195:G2 Phase; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D000076228:MRE11 Homologue Protein; D008938:Mitosis; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D015534:Trans-Activators / Q000378:metabolism; D000071857:Tumor Suppressor p53-Binding Protein 1
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; C413163:MDC1 protein, human; C121766:MRE11 protein, human; C112156:NBN protein, human; D009687:Nuclear Proteins; C498301:TP53BP1 protein, human; D015534:Trans-Activators; D000071857:Tumor Suppressor p53-Binding Protein 1; D000076228:MRE11 Homologue Protein