gssg synthesis NHEJ does not require sequence homology betwe

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 gssg synthesis 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 As mentioned above, there are two major pathways for the DSB repair in mammalian cells, NHEJ and HR/HDR (homology-directed repair) [13], [51]. NHEJ is active throughout the cell cycle, predominately during the G0 and G1 phases and is considered the major pathway for the DSB repair in human cells [52], [53]. HR/HDR is widely regarded as an accurate form of repair, which requires an undamaged sister chromatid with sequence homology of approximately 100 base pairs and more [54], to act as a DNA template and functions only after DNA replication [13], [53]. As a critical enzyme in the NHEJ repair pathway, DNA-PK regulates HR process too. However, how a cell chooses between repairing a double strand break (DSB) by non-homologous end joining (NHEJ) or by homologous recombination (HR) is a central and largely unanswered question. Direct comparisons revealed that DNA-PKcs binds to synthetic 4-way junctions with an affinity similar to open DNA ends. Protein kinase assays, using p53 as a phosphorylation target, showed that DNA-PKcs adopts an active conformation in the complex with 4-way junctions. The recognition of Holliday junctions by DNA-PKcs may simply reflect a general affinity for profoundly kinked or bent DNA structures [55]. A structural similarity between 4-way junctions and the DNA at the entry and exit point of nucleosomes might be indicative of a constitutive interaction of DNA-PKcs with chromatin filaments.