br The central role of DNA PK in

The central role of DNA-PK in non-homologous end-joining repair DNA DSBs are considered the most cytotoxic type of DNA lesion. DNA DSBs result from endogenous events such as V(D)J recombination and the production of reactive oxygen species (ROS) during cellular metabolism, as well as from exogenous sources such as ionizing radiation and radiomimetic drugs [11], [12], [13], [14]. When left unrepaired, such lesions can result in cell death. If misrepaired, DSBs have the potential to lead to chromosomal translocations and genomic instability that may result in cell death or cancer [12], [15]. NHEJ is thought to be the primary pathway for DSB repair in human somatic cell. NHEJ is initiated by binding of the ring shaped Ku70/Ku80 heterodimer (Ku) to both ends of a DSB, followed by recruitment of DNA-dependent protein kinase catalytic subunit (DNA PKcs) to the DNA-Ku complexes. Each of the Ku proteins contributes to a central DNA-binding core [16]. The N-terminus of Ku70 contains an acidic domain that is phosphorylated in vitro by DNA-PKcs [17], although this AACOCF3 may not be required for NHEJ [18]; whereas the C-terminus contains an SAP (SAF-A/B, Acinus and PIAS) domain which is a putative chromatin/DNA-binding domain [2]. The c-terminal region of Ku80 forms a long flexible arm that may be involved in protein–protein interactions [19], [20] and a conserved region at the distal C-terminus, which is required for interaction with DNA-PKcs [21], [22], [23]. Ku has strong avidity for DNA with a variety of end structures without apparent sequence specificity, including blunt, over-hanged, hair-pinned and damaged ends [24]. 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 cells [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.