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  • As mentioned above there are two major pathways for the


    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 ORY-1001 sale 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. It has been suggested, that NHEJ is the initial default pathway for DSB repair and, when NHEJ fails, HR may take over [56], [57]. Thus, the affinity of DNA-PKcs for the crossover region of Holliday junctions could anticipate an unexpected backup mechanism for the down regulation of improper recombination events. An active regulatory role of DNA-PKcs appears to be important for the resetting of stalled replication forks, as indicated by the observation that DNA replication-dependent spontaneous HR rates are inhibited by DNA-PKcs more efficiently than strand exchanges after exogenously induced DSBs [58]. A possible 4-way junction DNA arising during DNA replication includes the chicken-foot-like intermediate formed by backward migration of stalled replication forks. This transient 4-way intermediate is supposed to facilitate the replicative bypass of damaged templates before the correct replication fork is regenerated [59]. The recognition of chicken foot intermediates by DNA-PKcs could prevent branch migration in the wrong direction or avoid the endonucleolytic resolution of this particular kind of Holliday junction. Moreover, data showing that blocking phosphorylation at PQR in the ABCDE mutant (i.e., the combined mutant) substantially enhances HR and abrogates the severe radiosensitivity observed by blocking ABCDE alone, suggests that phosphorylation of PQR is critical to DNA-PK\'s ability to inhibit HR. Since ABCDE and PQR are phosphorylated in trans, DNA-PK bound to a single DNA end (e.g., at a collapsed replication fork) should not be phosphorylated at either ABCDE or PQR. Although the DNA-PK mutant that cannot phosphorylate either cluster cannot function normally to promote NHEJ, it strongly promotes HR [60]. This hypothesis, although speculative, is supported by the rapid recruitment of DNA-PKcs to sites where replication forks run across a damaged template [61]. Meek group recently have demonstrated that certain DNA-PKcs phosphorylations clearly promote HR while inhibiting NHEJ [62]. Although DNA-PK has been shown to phosphorylate many substrates both in vitro and in vivo, the only phosphorylation substrate of DNA-PK that has been demonstrated to result in a physiological alteration in NHEJ efficiency is autophosphorylation of DNA-PKcs itself [63]. The requirement for DNA-PK\'s enzymatic activity to inhibit HR might be explained by the requirement for a particular autophosphorylation event. The emerging data from Jeggo and colleagues demonstrate slowed kinetics of 3′ end resection (as measured by Rad51 foci induced after damage) in cells expressing DNA-PKcs that cannot phosphorylate ABCDE [64]. These observations have suggested that the phosphorylation of ABCDE and PQR clusters may reciprocally regulate end access at early points after damage, but since these sites are not required for autophosphorylation induced kinase dissociation, these impaired complexes that cannot regulate end access still eventually dissociate allowing other pathways (such as MRN signaling) access to damaged ends [38].