Figure1. Non-homologous end-joining pathway.
Overview of non-homologous end-joining pathway
DNA double-strand break (DSB) is one of the most dangerous forms of DNA damage. Unrepaired DSBs will lead cells to undergo apoptosis or senescence. On the other side, mis-processing of DSBs will also result in genomic instability and carcinogenesis. Non-homologous end-joining (NHEJ) is one important pathway in eukaryotic cells responsible for the repair of DSBs. A lot of highly efficient DSB repair pathways are involved in eukaryotic cells, and among them, non-homologous end-joining (NHEJ) likely plays the largest role in DSB repair in humans. The NHEJ pathway is also important for V(D)J recombination during T- and B-cell lymphocyte development. NHEJ pathway has the potential to relegate any type of DNA ends. Unlike the other classically studied DSB repair mechanisms, such as homologous recombination (HR), NHEJ does not require a homologous template for repair of DSB. Therefore, NHEJ is not restricted to a certain phase of the cell cycle. However, it is believed that HR is only active during S and G2 phases of the cell cycle when a homologous template via the sister chromatid is available.
Non-homologous end-joining pathway mechanisms
There are several steps of NHEJ pathway. The initial step in NHEJ is the recognition and binding of the Ku heterodimer to the DSB. The Ku heterodimer consists of Ku70 and Ku80. Ku70/80 has been shown to localize to laser-generated DSBs within seconds of their creation. There are some crystallographic studies of Ku70/80 showing that the heterodimer produces a ring-shaped structure which can accommodate a double strand DNA helix and allows the Ku heterodimer to slide onto the DNA step. Once the heterodimer is bound to the DSB ends, it then serves as a scaffold to recruit the other NHEJ factors to the damage site. Ku physically interacts with the XRCC4-DNA Ligase IV complex and recruits it to the DNA ends in vitro and in vivo. XRCC4 may be a second NHEJ scaffold which is responsible for the recruitment of a number of NHEJ factors to the DSB factors to the DSB ends. In particular, it may play a role in securing the ability of the processing enzymes to interact with the DSB region. Finally, a lot of factors have been said to stabilize the NHEJ complex at DSBs including Ku, DNA-PKcs, the kinase activity of DNA-PKcs, and XRCC4.
Ku itself can hold the two termini of a linearized plasmid in a synaptic complex further suggesting that Ku can bind to and protect DNA ends. Using a live cell imaging approach, it was said that Ku80, but not the Mre11-Rad50-Nbs1 complex or the structural maintenance of chromosomes protein 1, is required for the positional stability of DSB ends in asynchronized mouse cellsd. Ku’s function of protecting the DSB ends may happen in all cell cycle phases as some research shows that accumulation and dissociation kinetics of Ku80 at micro-laser induced DSBs are independent of cell cycle. Ku80 functions at DSB ends even when HR is the preferred repair pathway. Furthermore, XRCC4 via Arg54, Leu65, and Leu15 in XRCC4 directly interact with the globular head domain of XLF, which creates a head to head interface between the two proteins.
The next step, if necessary, is processing of the DNA ends to create ligatable ends. Different DNA end processing enzymes are required, including those that resect DNA ends, fill in gaps, remove blocking end groups, and make the ends ligatable. Most of those DNA end processing enzymes are recruited to the DSBs likely by a Ku-XRCC4 scaffold.
And a lot of factors have been shown to have the function of removing blocking end groups in order to make the ends of DSBs ligatable. There is a surprising new discovery showing that Ku has enzymatic activity, the 5’ deoxyribose-5-phosphate (5’-dRP)/AP lyase activity. Ku excises abasic sites near DSBs in vitro and this activity is highest when the abasic site is within a short 5’ overhang at the DSB end. And the proteins implicate in resecting DNA ends for NHEJ consist of Artemis, WRN, and APLF.
The last step in the repair of a DSB is ligation of the broken ends by DNA ligase IV, which has activity on its own. XRCC4 stabilizes ligase IV which stimulates the ligation activity of ligase IV. In addition, XRCC4 stimulates the activity of Ligase IV by promoting the adenylation of DNA Lagese IV. Little is known about the mechanisms which regulate the dissolution of the NHEJ complex from the DSB ends. Recent research with human cells showed that E3 ubiquitin ligase RNF8 may mediate the dissociation of Ku from DNA ends. In addition, a solution structure of DNA-PKcs obtained by small-angle X-ray scattering (SAXS) revealed that autophosphorylation of DNA-PKcs induces a conformational change which likely releases DNA-PKcs from DNA ends.
As for DNA-PKcs, it has no to limited kinase activity making it truly a DNA dependent kinase. Its kinase domain of DNA-PKcs is surrounded by the FAT and FATC domains. Some studies have shown that the N-terminus keeps DNA-PKcs basal activity low and that perturbation of the N-terminus is likely to result in a conformational change that opens the ATP binding pocket of the protein resulting in an increase in basal kinase activity. It is also believed that kinase activity of DNA-PKcs is essential for NHEJ as inactivation of DNA-PKcs activity via point mutations or small molecular chemical inhibition results in radiosensitivity and a defect in DSB repair.
Relations with diseases
Cancer’s fundamental feature is genome activity. An increase in cancer frequency is observed in mice and humans with germline mutations in genes which encode for proteins that are responsible for the response and repair of DSBs, underlying the importance of DSB repair proteins for protecting the genome. NHEJ and its factors have long been shown to play a role in maintaining genome activity. A lot of works showing the role of NHEJ factors in promoting genome stability have been worked out using mice deficient in one of the NHEJ factors. In addition, translocations between DSBs on different chromosomes increase in the absence of Ku70 in mammalian cells. XRCC4 and DNA ligase IV null mice die during the embryogenesis phase via massive neuronal apoptosis. Furthermore, studies with human clinical samples have implicated a connection between the DNA-PKcs activity and genomic instability and cancer incidence. As DNA-PKcs activity decreases, dicentric chromosomes and an excess in chromosomal fragmentation increase, and this increase in DNA-PKcs activity is related with a risk in breast and cervical cancer. In addition, a point mutation at threonine 2609 is identified in a breast cancer tumor sample, which suggests that phosphorylation at the T2609 cluster may be important for suppressing breast cancer. All in all, it is clear that the NHEJ pathway plays an important role in maintaining genome stability and protecting the cell from becoming cancerous.