The DNA-dependent Protein Kinase Is Inactivated by Autophosphorylation of the Catalytic Subunit

Department of Biological Sciences, University of Calgary, 2500 University Drive, N.W., Calgary, Alberta, T2N 1N4, Canada.
Journal of Biological Chemistry (Impact Factor: 4.57). 05/1996; 271(15):8936-41. DOI: 10.1074/jbc.271.15.8936
Source: PubMed


The DNA-dependent protein kinase (DNA-PK) requires for activity free ends or other discontinuities in the structure of double strand DNA. In vitro, DNA-PK phosphorylates several transcription factors and other DNA-binding proteins and is thought to function in DNA damage recognition or repair and/or transcription. Here we show that in vitro DNA-PK undergoes autophosphorylation of all three protein subunits (DNA-PKcs, Ku p70 and Ku p80) and that phosphorylation correlates with inactivation of the serine/threonine kinase activity of DNA-PK. Significantly, activity is restored by the addition of purified native DNA-PKcs but not Ku, suggesting that inactivation is due to autophosphorylation of DNA-PKcs. Our data also suggest that autophosphorylation results in dissociation of DNA-PKcs from the Ku-DNA complex. We suggest that autophosphorylation is an important mechanism for the regulation of DNA-PK activity.

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    • "Following DNA damage, DNA-PKcs becomes auto-phosphorylated on S2056 and is additionally phosphorylated on a cluster of threonine residues by the related PIKK family kinases ATM and ATR [15], [16]. DNA-PK activity is required for re-joining of the DNA ends but not the initial recruitment to the break whereas auto-phosphorylation reduces kinase activity and destabilizes the interaction with DNA ends [15], [17]–[20]. "
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    ABSTRACT: A growing body of evidence suggests that Polycomb group (PcG) proteins, key regulators of lineage specific gene expression, also participate in the repair of DNA double-strand breaks (DSBs) but evidence for direct recruitment of PcG proteins at specific breaks remains limited. Here we explore the association of Polycomb repressive complex 1 (PRC1) components with DSBs generated by inducible expression of the AsiSI restriction enzyme in normal human fibroblasts. Based on immunofluorescent staining, the co-localization of PRC1 proteins with components of the DNA damage response (DDR) in these primary cells is unconvincing. Moreover, using chromatin immunoprecipitation and deep sequencing (ChIP-seq), which detects PRC1 proteins at common sites throughout the genome, we did not find evidence for recruitment of PRC1 components to AsiSI-induced DSBs. In contrast, the S2056 phosphorylated form of DNA-PKcs and other DDR proteins were detected at a subset of AsiSI sites that are predominantly at the 5' ends of transcriptionally active genes. Our data question the idea that PcG protein recruitment provides a link between DSB repairs and transcriptional repression.
    PLoS ONE 07/2014; 9(7):e102968. DOI:10.1371/journal.pone.0102968 · 3.23 Impact Factor
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    • "Upon Ku binding by DNA-PKcs, Ku heterodimer may translocate away from the DNA end, allowing DNA-PKcs to bind the DNA termini [56] (Fig. 3ii and iii). DNA-PK promotes NHEJ in several ways; by promoting the synapsis of DSB termini [57] [58] (Fig. 3iii), by a coordinated series of autophosphorylation events that auto-regulate the retention/stability of DNA-PKcs [59] [60] [61] (Fig. 3iv), and by facilitating DNA end processing (see below; Fig. 3v) [62] [63] [64], and by promoting recruitment/retention of XRCC4–Lig4 [65] [66] [67] (Fig. 3vi). In the latter case, trans-phosphorylation of Ku, XRCC4 and DNA ligase IV (Lig4) enhances the stability of NHEJ complexes promotes DNA ligation. "
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    DNA repair 03/2014; 17. DOI:10.1016/j.dnarep.2014.02.019 · 3.11 Impact Factor
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    • "In contrast, for many DNA repair proteins (e.g. Ku70/80), only one or two molecules [45–47] are recruited to sites of DNA damage, such as DSBs, during their repair and therefore do not form readily visible RIF, even following immuno-staining. With low LET IR, the homogeneous distribution of induced damage within the nucleus makes it difficult to visualize one to two molecules of fluorescently tagged repair proteins as foci above the background fluorescence levels. "
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