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    • br DNA PK After sensing and binding

    2019-07-27


    DNA-PK After sensing and binding to the DSB, Ku quickly recruits DNA-PKcs to the site of the DNA break. Similar to Ku70/80, recruitment of DNA-PKcs to DSBs occurs within seconds of their creation [12]. The interaction between Ku70/80 and DNA-PKcs requires the presence of dsDNA and the complex formed at the DSB consisting of DNA, Ku70/80, and DNA-PKcs is referred to as “DNA-PK” [35]. DNA-PKcs is a member of phosphatidylinositol-3 (PI-3) kinase-like kinase family (PIKK), which also includes the two DNA damage responsive proteins, ATM and ATM and Rad3-related protein (ATR) [36], [37]. The N-terminal region of DNA-PKcs is composed of HEAT (Huntington-elongation-A-subunit-TOR) repeats that likely serve as a protein-protein interaction interface and the C-terminal region of the protein contains the PI3 kinase domain, which is flanked N-terminally by the FAT (FRAP, ATM, TRRAP) domain and C-terminally by the FATC (FAT C-terminal) domain [38], [39]. Structural studies of DNA-PKcs show that the N-terminal portion of the protein produces a pincer-shaped structure that forms a central channel that likely binds to dsDNA and the C-terminal domains form a crown structure that sits on top of the pincer-shaped structure [40], [41]. Binding of DNA-PKcs to the DNA-Ku complex results in translocation of the Ku heterodimer inward on the dsDNA strand and ultimately results in activation of the DNA-PKcs kinase activity [42], [43]. Once Ku recruits DNA-PKcs to the DSB ends, it has been shown that the large DNA-PKcs molecule also forms a distinct structure at the DNA termini that forms a synaptic complex responsible for holding the two ends of the broken DNA molecule together [27], [28], [29]. This synaptic complex consisting of DNA-PKcs and Ku is stable at DNA termini and blocks processing by nucleases and ligases and ultimately is required for DNA-PKcs kinase activation [29], [44]. It is likely that Ku70/80 recruits DNA-PKcs to the DSB via multiple contacts between the two proteins, which is supported by predictions from low RO4929097 is structure of the DNA-Ku70/80-DNA-PKcs complex [45], [46]. Small angle X-ray scattering analysis shows the Ku80 C-terminal region may play a role in retaining DNA-PKcs at DSB ends and keeping the DNA-PK complex in a synaptic complex at the DSB site [47]. Although the C-terminal region of Ku80 helps retain DNA-PKcs at DSB termini, it is not required for the ability of DNA-PKcs to localize to DSBs in vivo as previously believed [48], [49], [50], [51]. The central cavity formed by the N-terminal region of DNA-PKcs results in DNA threading through the channel and ultimately stabilization of the DNA-PKcs-Ku-DNA complex and it is this portion of the protein that is required for the ability of DNA-PKcs to interact with the Ku-DNA complex [40], [41], [52].
    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 resolution 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.