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
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.
Available from: Harshil Patel
- "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 , . 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 , –. "
[Show abstract] [Hide abstract]
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
Available from: Keith W Caldecott
- "Upon Ku binding by DNA-PKcs, Ku heterodimer may translocate away from the DNA end, allowing DNA-PKcs to bind the DNA termini  (Fig. 3ii and iii). DNA-PK promotes NHEJ in several ways; by promoting the synapsis of DSB termini   (Fig. 3iii), by a coordinated series of autophosphorylation events that auto-regulate the retention/stability of DNA-PKcs    (Fig. 3iv), and by facilitating DNA end processing (see below; Fig. 3v)   , and by promoting recruitment/retention of XRCC4–Lig4    (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. "
[Show abstract] [Hide abstract]
ABSTRACT: The repair of DNA double strand breaks is essential for cell survival and several conserved pathways have evolved to ensure their rapid and efficient repair. The non-homologous end joining pathway is initiated when Ku binds to the DNA break site. Ku is an abundant nuclear heterodimer of Ku70 and Ku80 with a toroidal structure that allows the protein to slide over the broken DNA end and bind with high affinity. Once locked into placed, Ku acts as a tool-belt to recruit multiple interacting proteins, forming one or more non-homologous end joining complexes that act in a regulated manner to ensure efficient repair of DNA ends. Here we review the structure and functions of Ku and the proteins with which it interacts during non-homologous end joining.
DNA repair 03/2014; 17. DOI:10.1016/j.dnarep.2014.02.019 · 3.11 Impact Factor
Available from: Pamela Reynolds
- "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. "
[Show abstract] [Hide abstract]
ABSTRACT: The formation of DNA lesions poses a constant threat to cellular stability. Repair of endogenously and exogenously produced lesions has therefore been extensively studied, although the spatiotemporal dynamics of the repair processes has yet to be fully understood. One of the most recent advances to study the kinetics of DNA repair has been the development of laser microbeams to induce and visualize recruitment and loss of repair proteins to base damage in live mammalian cells. However, a number of studies have produced contradictory results that are likely caused by the different laser systems used reflecting in part the wavelength dependence of the damage induced. Additionally, the repair kinetics of laser microbeam induced DNA lesions have generally lacked consideration of the structural and chemical complexity of the DNA damage sites, which are known to greatly influence their reparability. In this review, we highlight the key considerations when embarking on laser microbeam experiments and interpreting the real time data from laser microbeam irradiations. We compare the repair kinetics from live cell imaging with biochemical and direct quantitative cellular measurements for DNA repair.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 05/2013; 756(1-2). DOI:10.1016/j.mrgentox.2013.05.006 · 3.68 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.