NHEJ is a majormechanism of DSBR (68).
The Ku complex, a heterodimer of twoprotein subunits Ku70 and Ku80, acts as a sensor for DSBs. The presence of Kucomplex at the DNA ends of DSB is essential for subsequent DNA end processingand ligation (69, 70). DNA-dependent protein kinase catalyticsubunit (DNA-PKcs) is recruited to Ku at DNA ends of DSB, becomes activated andcatalyzes synapsis of the DNA ends by dimerization (69, 70). Another core complex, XRCC4-lig4-XRCC4 like factor (XLF), is recruitedto sites of damage. XRCC4-lig4-XLF induces DNA-PKcs autophosphorylation,dissociation, translocation of the Ku heterodimer along the DNA away from theends, and recruitment of end processing factors (70).
A number of end processing enzymes andpolymerases are recruited to the site of damage by interacting with the corecomplex (62,70, 71). PNKP is one of the key enzymes required for the processing ofDSBs. PNKP is recruited to the site ofdamage by interacting with phosphorylated XRCC4 via the PNKP FHA domain (72). Ligation of DNA ends is catalyzed by lig4. Finally, the Ku complex isremoved from DNA ends by proteasomal degradation mediated by ubiquitination byCullin and RNF8 (70).
Figure 1.3 shows a model depicting thesteps involved in the conventional NHEJ pathway.A substituteof classical NHEJ, alt-NHEJ, is available when the cell is deficient in NHEJcomponents and relies on microhomology (MH), the tendency of the two DNA endsto align themselves at short regions of homology within the two ends (73-75). The main attributes of alt-NHEJ are largedeletions, insertions and a high frequency of chromosomal translocations (76). The damage is detected in this situation byPARP-1, which facilitates the recruitment of the MRN complex to bridge the DNA ends,following the loading of CtlP to facilitate MH searches (77-79).
The ligation process is mainly driven bythe activity of the XRCC1/lig3 complex and on some occasions lig1, where MH isnot required (80). In recent studies, it was shown thatpolymerase ? (Pol ?) performs terminal transferase activity at the 3?-termini of resected DSBs to mediate MH (81,82). PNKP is also involved in this process and is likely partiallyregulated by IR-induced phosphorylation of PNKPby Ataxia telangiectasia mutated (ATM) and DNA-PKcs (83,84). An in vivo study showedthat the inactivation of DNA-PKcs and/or ATM led to reduced PNKP levels at DNAdamage sites (84).
Based on the observation that depletion ofPNKP does not alter the level of sister chromatid exchanges, it was surmisedthat PNKP does not participate in HR, the other major DSB repair pathway (85).