title: Evidence for a remodelling of DNA-PK upon autophosphorylation from electron microscopy studies.
abstract: The multi-subunit DNA-dependent protein kinase (DNA-PK), a crucial player in DNA repair by non-homologous end-joining in higher eukaryotes, consists of a catalytic subunit (DNA-PKcs) and the Ku heterodimer. Ku recruits DNA-PKcs to double-strand breaks, where DNA-PK assembles prior to DNA repair. The interaction of DNA-PK with DNA is regulated via autophosphorylation. Recent SAXS data addressed the conformational changes occurring in the purified catalytic subunit upon autophosphorylation. Here, we present the first structural analysis of the effects of autophosphorylation on the trimeric DNA-PK enzyme, performed by electron microscopy and single particle analysis. We observe a considerable degree of heterogeneity in the autophosphorylated material, which we resolved into subpopulations of intact complex, and separate DNA-PKcs and Ku, by using multivariate statistical analysis and multi-reference alignment on a partitioned particle image data set. The proportion of dimeric oligomers was reduced compared to non-phosphorylated complex, and those dimers remaining showed a substantial variation in mutual monomer orientation. Together, our data indicate a substantial remodelling of DNA-PK holo-enzyme upon autophosphorylation, which is crucial to the release of protein factors from a repaired DNA double-strand break.
authors: Structural Electron Microscopy Group, Section of Structural Biology, The Institute of Cancer Research, London SW3 6JB, UK.
authors: Edward P
authors: EP
authors: Morris
authors: Angel
authors: A
authors: Rivera-Calzada
authors: Paula C A
authors: PC
authors: da Fonseca
authors: Oscar
authors: O
authors: Llorca
authors: Laurence H
authors: LH
authors: Pearl
authors: Laura
authors: L
authors: Spagnolo
mesh_terms: D004247:DNA / Q000378:metabolism; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase / Q000378:metabolism / Q000648:ultrastructure*; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron / Q000379:methods; D010766:Phosphorylation
chemical_list: D004247:DNA; D051747:DNA-Activated Protein Kinase