title: An autonomous chromatin/DNA-PK mechanism for localized DNA damage signaling in mammalian cells.
abstract: Rapid phosphorylation of histone variant H2AX proximal to DNA breaks is an initiating event and a hallmark of eukaryotic DNA damage responses. Three mammalian kinases are known to phosphorylate H2AX in response to DNA damage. However, the mechanism(s) for damage-localized phosphorylation remains incompletely understood. The DNA-dependent protein kinase (DNA-PK) is the most abundant H2AX-modifying kinases and uniquely activated by binding DNA termini. Here, we have developed a novel approach to examine enzyme activity and substrate properties by executing biochemical assays on intact cellular structures. We apply this approach to examine the mechanisms of localized protein modification in chromatin within fixed cells. DNA-PK retains substrate specificity and independently generates break-localized γH2AX foci in chromatin. In situ DNA-PK activity recapitulates localization and intensity of in vivo H2AX phosphorylation and requires no active cellular processes. Nuclease treatments or addition of exogenous DNA resulted in genome-wide H2AX phosphorylation, showing that DNA termini dictated the locality of H2AX phosphorylation in situ. DNA-PK also reconstituted focal phosphorylation of structural maintenance of chromatin protein 1, but not activating transcription factor 2. Allosteric regulation of DNA-PK by DNA termini protruding from chromatin constitutes an autonomous mechanism for break-localized protein phosphorylation that generates sub-nuclear foci. We discuss generalized implications of this mechanism in localizing mammalian DNA damage responses.
authors: Life Sciences Division, Lawrence Berkeley National Laboratory, Mail Stop 84-171, 1 Cyclotron Road, Berkeley, CA 94720, USA.
authors: Denise P
authors: DP
authors: Muñoz
authors: Misako
authors: M
authors: Kawahara
authors: Steven M
authors: SM
authors: Yannone
mesh_terms: D051698:Activating Transcription Factor 2 / Q000378:metabolism; D000494:Allosteric Regulation; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D002843:Chromatin / Q000235:genetics / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D004249:DNA Damage; D051747:DNA-Activated Protein Kinase / Q000378:metabolism* / Q000502:physiology; D004789:Enzyme Activation; D006657:Histones / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity; D000071857:Tumor Suppressor p53-Binding Protein 1; D000098550:Structural Maintenance of Chromosome Protein 1
chemical_list: C497105:ATF2 protein, human; D051698:Activating Transcription Factor 2; D018797:Cell Cycle Proteins; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; C468783:H2AX protein, human; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; C498301:TP53BP1 protein, human; D000071857:Tumor Suppressor p53-Binding Protein 1; D000098550:Structural Maintenance of Chromosome Protein 1; C494918:AKT1 protein, human; D051747:DNA-Activated Protein Kinase; D051057:Proto-Oncogene Proteins c-akt