Ultimately, DNA ligase IV (LIG4), which forms a constitutive complex with XRCC4, catalyzes ligation. Notably, LIG4 can tolerate certain terminal mismatches and damaged bases, a unique feature among vertebrate ligases. In addition to its catalytic role in ligation, accumulating evidence implicates LIG4 in DNA end synapsis. LIG4 is required for stable synapsis in both human cell and Xenopus egg extracts and in reconstitutions of human NHEJ factors.

The authors state: "Ligation is the only essential step in NHEJ, and is performed by one of the three mammalian ligases, DNA ligase IV (LIG4; Lieber et al., 1997)." They further note that "LIG4 is recruited to broken ends through participation in a complex of core NHEJ factors including XRCC4, the Ku 70/80 heterodimer (Ku), and XLF." This work examines how LIG4 structure regulates the paired-end complex in cellular NHEJ.

The review defines canonical NHEJ as the pathway that uses Ku, DNA ligase IV (Lig4), and associated factors to repair certain double-strand breaks in DNA. It explicitly states: "Lig4 is the DNA ligase required for, and specific to, c-NHEJ." The authors further note that complete loss of this obligatory and specific c-NHEJ factor appears incompatible with mammalian life, based on mouse models and human ligase IV syndrome.

This review of mammalian NHEJ states that classical NHEJ is mediated by a core set of factors including Ku70–Ku80, DNA-PKcs, XRCC4, XLF and DNA ligase IV. It explains that "XRCC4–DNA ligase IV is the dedicated ligase complex for classical NHEJ" and contrasts this with alternative end-joining mechanisms that can utilize DNA ligase III. The article also notes that LIG4 deficiency severely compromises classical NHEJ and V(D)J recombination.

DNA ligase IV and X-ray repair cross-complementing protein 4 (XRCC4) are the most central components of NHEJ in eukaryotes. XRCC4 stimulates DNA ligase IV enzyme activity in biochemical assays. Ku has a high affinity for DNA ends and can promote the binding of XRCC4–DNA ligase IV to the DNA ends. This Ku–XRCC4–DNA ligase IV complex is required for the efficient reconstitution of the NHEJ pathway using human proteins. For the joining of two blunt DNA ends, Ku and XRCC4–DNA ligase IV are sufficient, and the addition of other NHEJ proteins does not substantially improve the joining.

Non-homologous DNA end joining (NHEJ) is the predominant repair mechanism of any type of DNA double-strand break (DSB) during most of the cell cycle. Direct ligation of the DNA ends is performed by the XRCC4–DNA ligase 4 (LIG4) complex, an activity that is enhanced by XRCC4-like factor (XLF) and/or by paralogue of XRCC4 and XLF (PAXX). DNA ends that are incompatible for direct ligation by LIG4 are processed by the nuclease Artemis or by polymerases to become compatible for ligation.

Non-homologous end-joining (NHEJ) is a major pathway for the repair of DNA double strand breaks in mammalian cells. The final ligation step of classical NHEJ is carried out by the XRCC4–DNA ligase IV complex, frequently with the accessory factor XLF. DNA ligase IV is specialized for this pathway and functions in complex with XRCC4 to catalyze phosphodiester bond formation between the aligned DNA ends.

Classical non-homologous end joining (c-NHEJ) repairs the majority of DNA double-strand breaks in mammalian cells. In the canonical pathway, Ku70–Ku80 and DNA-PKcs assemble at the break, followed by recruitment of XRCC4–DNA ligase IV (LIG4), which executes the ligation step. Genetic studies have established that LIG4 is essential for V(D)J recombination and for efficient c-NHEJ, and its loss results in severe combined immunodeficiency in humans and mice.

The curated human pathway entry describes that during NHEJ, "XRCC4:LIG4 ligates DNA DSB ends" and that the accessory protein NHEJ1 (XLF) facilitates this reaction. The reaction notes that the "DNA ligase activity of ... XRCC4:LIG4:NHEJ1" acts on extended ligatable DNA DSB ends in the nucleoplasm, reflecting the model that XRCC4-bound DNA ligase IV carries out the ligation step of classical NHEJ in human cells.

The article explains: "Among the three human DNA ligases, DNA LIG4 is the one that repairs DNA-DSBs through the non-homologous end joining (NHEJ) pathway." It describes the repair process as beginning with Ku binding and recruitment of "other NHEJ proteins such as DNA-PKcs and the DNA LIG4/XRCC4 complex," highlighting that LIG4 functions in a ligation complex at the final step. The paper emphasizes that hypomorphic LIG4 mutations impair DNA damage repair mechanisms via NHEJ, causing a severe combined immunodeficiency phenotype.

While it has been known that Ku-dependent NHEJ requires DNA ligase IV, it is unclear which DNA ligase(s) is required for Ku-independent MHEJ. In this study, we show that human DNA ligases I and III, but not ligase IV, are required for efficient microhomology-mediated end joining (MHEJ). These data indicate that the core NHEJ pathway that depends on Ku requires ligase IV, whereas an alternative, microhomology-mediated pathway uses ligases I and III instead.

Non-homologous end joining (NHEJ) is one of two eukaryotic pathways carrying out DNA double-strand break (DSB) repair. The final step in NHEJ repair involves ligation of the DNA ends by DNA ligase IV in a complex that also includes XRCC4 and Ku. The proteins DNA ligase IV and XRCC4 are essential in the final step of the NHEJ pathway: DNA ligase IV is the enzyme responsible for joining the two ends of the DNA breaks.

Although DNA ligase IV is the canonical ligase for classical non-homologous end joining, cells can also repair double-strand breaks via alternative end-joining mechanisms. These backup pathways are typically active when core c-NHEJ components such as LIG4 are absent or compromised and can employ DNA ligase III or I together with microhomology. However, alternative end joining is genetically and mechanistically distinct from classical NHEJ and is generally more error-prone.

The clinical overview notes: "DNA Ligase IV is a component of the protein complex that mediates repair of DNA double-strand breaks by nonhomologous end-joining (NHEJ)." It specifies that DNA ligase IV forms a complex with XRCC4 and Cernunnos and "is involved in the ligation of blunt DNA ends, compatible DNA overhangs, and incompatible short DNA overhangs." The article also explains that mutations in LIG4 result in radiosensitive severe combined immunodeficiency due to defective V(D)J recombination, which depends on NHEJ.

In mammalian cells, non-homologous end-joining (NHEJ) is the major pathway for repairing DNA double-strand breaks (DSBs) caused by ionizing radiation and other factors. 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. DNA ligase IV functions in a complex with XRCC4 and other factors to seal the DNA break.

For the majority of the cell cycle DSBs are repaired by NHEJ, a process that robustly ligates even damaged or incompatible DNA ends. Our work uses single-molecule and biochemical approaches to understand how the NHEJ machinery, including Ku, DNA-PKcs, XRCC4 and Ligase IV, recognizes, synapses and ligates DNA ends. A central question is how the Ligase IV complex carries out ligation of diverse DNA end structures characteristic of classical NHEJ.

Knockout mouse models lacking DNA ligase IV show embryonic lethality or severe combined immunodeficiency, with a near-complete block in V(D)J recombination, indicating that classical NHEJ in developing lymphocytes relies on LIG4 for efficient sealing of RAG-induced DNA double-strand breaks. Human LIG4 syndrome patients similarly exhibit impaired c-NHEJ and radiosensitivity, although other ligases can support low levels of alternative end joining.