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  • DNA bound Ku proteins recruit

    2020-10-29

    DNA-bound Ku proteins recruit DNA-PKcs; and thereby translocate into the duplex by one helical turn, leaving DNA-PKcs near the DNA terminus to assist in tethering the broken ends together [25]. In addition to keeping the DSBs in close proximity, DNA-PKcs may prevent exonucleolytic degradation of the ends [26], mediate the alignment of the DNA strands in search for sequence microhomologies [27], [28], and serve as a landing platform for DNA polymerases and ligation factors. The final rejoining of DNA ends is driven by a dimeric factor that consists of DNA ligase IV and XRCC4 [29]. Participation of DNA polymerase X family members to fill in short gaps prior to ligation is suggested by their colocalization with DNA-PKcs [30] and direct interactions with Ku and the DNA ligase IV-XRCC4 complex [31] (Fig. 1B). In the absence of DNA-PKcs, Ku interacts with the distal termini of DNA ends. In the presence of DNA-PKcs, Ku translocates inward (by about one helical turn) and DNA-PKcs has direct contacts with 10bp at the terminus of a DNA end. Although DNA-PKcs has innate affinity (itself) for DNA ends [5], Ku is required for targeting DNA-PKcs to damaged DNA in physiologic conditions and in living KH CB19 australia [32]. Extensive evidence supports that the access to DNA ends by serial enzymes, such as DNA ligase IV, XRCC 4, Artemis, DNA polymerase mu etc., is mediated by autophosphorylation of DNA-PKcs in trans over the synaptic cleft between two tethered DNA molecules [31], [33], [34], [35], [36], [37]. These studies clearly show that DNA-PKcs plays a central role during the NHEJ process. This enzyme not only captures and tethers the two ends of a broken DNA molecule but also regulates the access of modifying enzymes and ligases to the DNA termini. NHEJ does not require sequence homology between the DNA ends as a prerequisite for ligation. As a result, this process can be error prone while HR is generally considered to be an error-free process. However, how a cell chooses whether to repair a DSB through NHEJ or HR, and how DNA-PK is activated or deactivated in response to DNA damage, is still relatively unknown [38], although the downstream signaling of DNA-PK has been extensively studied [39], [40]. In the downstream signaling, DNA-PK directly induces the Ser139 phosphorylation of H2AX (γ-H2AX) or indirectly phosphorylates the H2AX via the Akt/GSK3b signaling [41]. γ-H2AX is not only a measuring indicator for DNA damage [41], [42], but also a signaling to recruits the DNA ligase IV-XRCC4 complex, Artemis and pol μ/λ etc. to the DNA damage sites. In turn, both serine/threonine protein phosphatase PP2A and PP6 have been reported to directly or indirectly dephosphorylated γ-H2AX foci [43], [44]. SiRNA knock-down of either PP6 or PP2A leads to sustained phosphorylation of histone H2AX on serine 139 after IR, inefficient DNA repair, and hypersensitive to DNA damage [43], [44]. Although H2A.X does not affect nucleosome conformation, it has a de-stabilizing effect that is enhanced by the DNA-PK-mediated phosphorylation and results in an impaired histone H1 binding, which may facilitate DNA repair [45]. The telomere is a nucleoprotein complex located at the ends of each eukaryotic chromosome. It is very important to distinguish normal telomere ends from pathologic DSBs for maintaining genome integrity. Curiously, DNA-PKcs and Ku, the very core NHEJ subunits that trigger the rejoining of DSBs, exert a seemingly opposite action at telomeres by inhibiting the fusion of chromosomal ends. DNA-PKcs, Ku70, and Ku80 have all been located at mammalian telomeres [46], [47], and knockout mutations in any of these three DNA-PK subunits cause spontaneous end-to-end fusions of chromosomes [46], [48]. It appears that DN-PKcs and Ku operate at telomeres by distinct mechanisms, as Ku80 has been reported to act as a negative regulator of the telomerase complex [49] whereas DNA-PKcs cooperates with this specialized enzyme in telomere elongation [50].
    The essential role of DNA-PK in homologous recombination repair