Non-Homologous End Joining (NHEJ) is a pathway that repairs double-strand breaks in DNA. NHEJ is known as “non-homologous” because the break ends are directly ligated without the need for a homologous template, in contrast to homology directed repair, which requires a homologous sequence to guide repair. The term “non-homologous end joining” was coined in 1996 by Moore and Haber.
NHEJ is typically guided by short homologous DNA sequences called micro homologies. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, hallmarks of tumor cells.
Maintaining genomic integrity is imperative for the survival of an organism. Among different DNA damages, double-strand breaks (DSBs) are considered as most deleterious since they can lead to cell death if left unrepaired or chromosomal rearrangements when mis-repaired, leading to cancer. Nonhomologous DNA end joining (NHEJ) and homologous recombination (HR) are the major DSB repair pathways in higher eukaryotes. HR being a precise mechanism uses extensive homology, while NHEJ is error prone as it utilizes no or limited homology. NHEJ is a quick fix mechanism and operates throughout the cell cycle. During NHEJ, KU protein heterodimer is recruited to DNA ends followed by DNA-PKcs in association with ARTEMIS, which processes DSBs. Pol μ and/or λ fills these ends, when required, followed by ligation using XLF–XRCC4–DNA Ligase IV complex. Another repair pathway known as alternative NHEJ (A-NHEJ) or backup NHEJ repairs DSBs when the key proteins responsible for classical NHEJ are absent or defective. The choice of DSB repair pathway between NHEJ and HR depends on cell cycle, end resection, and DSB end structure, which further activate various DNA damage sensors.
NHEJ ligates two broken ends together, thereby frequently resulting in small insertions and deletions. However, post mitotic cells rely on NHEJ for repairing DSBs. Failure to faithfully repair DSBs can result in point mutations, deletions, and large genome rearrangements. However, NHEJ activity and fidelity decline with age, which may be in part due to altered expression levels, activity, and distribution of key repair enzymes. Further, mutations in NHEJ genes including Ku70 and Ku80 have been associated with shortened life spans in mice. In addition, defects in DNA-PKcs resulted in impaired telomere maintenance and shortened life span in mice.
NHEJ plays a critical role in V(D)J recombination, the process by which B-cell and T-cell receptor diversity is generated in the vertebrate immune system. In V(D)J recombination, hairpin-capped double-strand breaks are created by the RAG1/RAG2 nuclease, which cleaves the DNA at recombination signal sequences. These hairpins are then opened by the Artemis nuclease and joined by NHEJ. A specialized DNA polymerase called terminal deoxynucleotidyl transferase (TdT), which is only expressed in lymph tissue, adds non templated nucleotides to the ends before the break is joined. This process couples “variable” (V), `diversity” (D), and “joining” (J) regions, which when assembled together create the variable region of a B-cell or T-cell receptor gene. Unlike typical cellular NHEJ, in which accurate repair is the most favorable outcome, error-prone repair in V(D)J recombination is beneficial in that it maximizes diversity in the coding sequence of these genes. Patients with mutations in NHEJ genes are unable to produce functional B cells and T cells and suffer from severe combined immunodeficiency (SCID).