br DNA PK kinase activity As

DNA-PK kinase activity As previously stated, DNA-PKcs recruitment to the DSB results in translocation of the Ku heterodimer inward on the dsDNA allowing DNA-PKcs to interact directly access DSB end, which results in activation of the catalytic activity of the enzyme [42], [43]. DNA-PKcs has no to limited kinase activity in the absence of Ku70/80 and DNA, thus making it truly a DNA-dependent protein kinase [53], [54]. The mechanism by which binding to the Ku–DNA complex stimulates the catalytic activity of DNA-PKcs is not clearly understood. It is likely that multiple regions/motifs of the protein play a role in this process. Low SKF 81297 hydrobromide australia structures showed that binding to the Ku–DNA complex induces a conformational change in the FAT and FATC domains surrounding the PIK3 kinase domain and this conformation change is predicted to result in the alteration of the catalytic groups and/or the ATP binding pocket of DNA-PKcs and ultimately full activation of its kinase activity [45], [46], [55]. Surprisingly, the N-terminus also plays a role in modulating the enzymatic activity of DNA-PKcs [52], [56]. Deletion of the N-terminal region of DNA-PKcs and N-terminally restraining DNA-PKcs results in spontaneous activation of its kinase activity suggesting that the N-terminus keeps DNA-PKcs basal activity low and that a perturbation of the N-terminus results in a conformational change that results in an increase in basal kinase activity. The kinase activity of DNA-PKcs is essential for NHEJ, but the exact role of the enzymatic activity of DNA-PKcs plays in NHEJ is not fully understood [57]. In vitro, DNA-PKcs can phosphorylate each of the canonical NHEJ factors including Ku70/80 [58], [59], XRCC4 [60], [61], DNA ligase IV [62], and XLF [63], but surprisingly none of these phosphorylations are required for NHEJ. DNA-PKcs has also been shown to phosphorylate a number of other factors implicated in NHEJ and the DNA damage response, including Artemis, polynucleotide kinase/phosphatase (PNKP), the histone H2AX and p53 [9], [64], [65]. But, considering these factors are also phosphorylated by ATM, the role of DNA-PKcs-mediated phosphorylation of these proteins is not exactly clear. However, it was recently found that phosphorylation of the implicated NHEJ factor Werner (WRN) by DNA-PKcs is required for efficient DSB repair, possibly identifying a DNA-PKcs mediated phosphorylation of a substrate that is important for NHEJ [66]. Furthermore, a number of new in vivo substrates of DNA-PKcs have emerged [65]. Proteins that are phosphorylated by DNA-PKcs following DNA damage include Akt/PKB [67], [68], the nuclear receptor 4A [69], heat shock protein HSP90α [70], [71], and the scaffold attachment factor A [72], but it is not known if phosphorylation of any of these proteins is important for NHEJ or the cellular response to DSBs. In respect to the NHEJ pathway, the best characterized DNA-PKcs substrate is DNA-PKcs itself as it autophosphorylates itself on a number of residues in different regions of the polypeptide [73], [74], [75], [76]. Autophosphorylation of DNA-PKcs results in kinase inactivation and dissociation from the DNA-Ku70/80 complex in vitro, but even the specific role that DNA-PKcs autophosphorylation plays in NHEJ is not completely understood [73], [77].