br General mechanism of NHEJ
General mechanism of NHEJ NHEJ is an amazingly versatile pathway that can select specific enzymes which bind to, process, and finally mediate the direct re-ligation of a wide range of DSBs including those that are complex, have incompatible ends, and contain lose your own voice damages , , , . After induction of the DSB (A), the general mechanism of NHEJ include the following steps: (B) recognition of the DNA double strand break by the Ku70/80 heterodimer (Ku); (C) assembly and stabilization of the NHEJ complex at the DNA damage site; (D) bridging of the DNA ends and promotion of end stability; (E) activation of the DNA dependent protein kinase catalytic subunit (DNA-PKcs) kinase activity; (F) DNA end processing, if required; (G) ligation of the broken ends by the DNA ligase IV-X-ray cross complementing protein 4 (XRCC4) complex with the assistance of the XRCC4-like factor (XLF, also named Cernunnos); and (H) dissolution of the NHEJ complex and completion of the repair process (Fig. 1). It should be noted that although these steps are listed sequentially, the order in which a number of these steps occur in particular steps C–F are unknown. In this review, we will highlight the important NHEJ proteins and mechanisms modulating NHEJ with emphasis given to the DNA-PK complex and the versatility of this DSB repair pathway.
Ku70/80 NHEJ is initiated by the sensing and binding of the DSB by the Ku heterodimer, which consists of the Ku70 and Ku80 subunits , . Ku is an abundant protein that has an extremely high affinity for dsDNA ends that forms a ring-shaped protein that slides onto the ends of the broken DNA molecule in a sequence independent manner , , , . Ku70/80 binds to the sugar backbone of DNA and not to the bases, which explains its ability to bind to DNA in a sequence independent manner . Depending on the length of the DNA substrate, multiple Ku molecules can slide onto naked DNA in vitro , . However, recent data shows that typically only one Ku heterodimer is bound to each end of the DSB in vivo . Following induction a DSB, Ku70/80 binds to the DNA damage site within seconds of its creation and does so in all cell cycle phases , , . Once bound to the DSB, Ku then performs its primary function in NHEJ, which is to serve as a scaffold to recruit the NHEJ machinery to the DNA lesion. The Ku heterodimer directly interacts with each canonical NHEJ factor, DNA-PKcs , XRCC4 , , , DNA ligase IV  and XLF , as well with the majority of the DNA end processing factors . The recruitment of the NHEJ machinery to the site of the DSB does not occur in a step-wise sequential manner but via a dynamic assembly , . For example, the NHEJ factors required for the terminal ligation step, XRCC4, DNA ligase IV, and XLF, localize to DSBs independently of DNA-PKcs, which had long thought to be required for their recruitment to DSBs , . The canonical NHEJ factors appear to collectively stabilize the entire NHEJ machinery at the DNA damage site and do so by a number of protein-protein interactions between themselves , . A secondary function that the Ku heterodimer performs at DSBs is a general role in binding to and maintaining the stability of the ends of the broken DNA molecule in all cell cycle phases . Ku maintains the two ends of the broken DNA molecule together via forming a synaptic complex in vitro and is also required for the positional stability of DSB ends in vivo , , , . The role of Ku in maintaining and stabilizing DSB ends is likely to protect them from non-specific processing. Specifically, Ku has been shown to block DNA end processing enzymes including exonuclease 1 and the Mre11/Rad50/Nbs1 complex in vitro . Blocking non-specific processing of a DSB is of importance because it protects against chromosomal aberrations and genomic instability. This is supported by the fact that Ku deficient cells have severe chromosomal instability following DSB induction in S phase cells, suggesting that Ku has a general function in protecting the genome even when HR is the likely preferred DSB repair pathway . Although Ku plays a role in maintaining DSB ends in all cell cycle phases, it should be noted that Ku-mediated end-joining may also be detrimental to cell survival, in particular end-joining of DSBs at replication forks. For example, Ku is required for the cell killing of cells following combined treatment with camptothecin and inhibition of the ataxia–telangiectasia mutated (ATM) protein , treatment of HR-deficient cells with poly(ADP-ribose) polymerase (PARP) inhibitors , and in Fanconi Anemia (FA)-deficient cells .