Non Homologous End Joining Pathway: Mechanism and Repair
By Dr. Zubair Khalid, DVM, MS, PhD ·
Introduction to Non Homologous End Joining
DNA double-strand breaks (DSBs) are among the most cytotoxic forms of DNA damage. A single unrepaired DSB can trigger cell cycle arrest or apoptosis, while misrepaired breaks can generate chromosomal translocations and genomic instability. Cells have evolved two principal pathways to repair DSBs: non homologous end joining (NHEJ) and homologous recombination (HR). The non homologous end joining pathway is the dominant repair mechanism in mammalian cells, operating by directly ligating the two broken DNA ends without requiring a homologous template.
NHEJ is defined by its template-independent nature. The pathway recognizes broken DNA ends, brings them into physical proximity, processes them to create compatible termini, and seals the nick with a DNA ligase. Because it does not rely on sister chromatid homology, NHEJ is active throughout the cell cycle but is particularly critical in the G1 phase, when sister chromatids are unavailable for HR. Beyond genome maintenance, NHEJ is essential for V(D)J recombination, the process that generates the enormous diversity of antigen receptors in the adaptive immune system.
What is NHEJ?
NHEJ is a DSB repair pathway that directly rejoins broken DNA ends. The core reaction involves four functional steps: detection of the break, tethering of the two ends, enzymatic processing of damaged or mismatched termini, and final ligation. The pathway is considered "non homologous" because it does not require sequence homology between the two ends being joined. This distinguishes it from HR, which uses a homologous sequence—typically the sister chromatid—as a template for accurate repair.
The pathway is conserved from bacteria to humans, though the protein components become increasingly complex in higher eukaryotes. In mammalian cells, NHEJ is the predominant DSB repair pathway, handling the majority of ionizing radiation-induced breaks and essentially all programmed DSBs generated during V(D)J recombination.
NHEJ vs. Homologous Recombination
The fundamental distinction between NHEJ and HR lies in template usage. HR requires a homologous DNA sequence to direct repair synthesis, which restricts its use to the S and G2 phases of the cell cycle when sister chromatids are present. HR is generally error-free because it copies genetic information from the undamaged sister chromatid. In contrast, NHEJ operates without a template and directly ligates ends, often resulting in small insertions or deletions (indels) at the repair junction.
Pathway choice is governed by multiple factors, including cell cycle phase, chromatin context, and the nature of the break. The presence of a resected 3' single-stranded DNA overhang is a key determinant: extensive resection commits the cell to HR, while minimal resection favors NHEJ. The Homologous Recombination pathway is covered in detail elsewhere; here we focus on the mechanism, regulation, and physiological significance of NHEJ.
Key Proteins in the NHEJ Pathway
The core NHEJ machinery in mammalian cells consists of seven principal proteins: Ku70, Ku80, DNA-PKcs, Artemis, XRCC4, DNA ligase IV, and XLF. Each plays a distinct role in the repair process, from initial break recognition to final nick sealing.
Ku Heterodimer
The Ku heterodimer is composed of two subunits, Ku70 (70 kDa) and Ku80 (80 kDa), which form a basket-shaped structure that encircles DNA. Ku binds to double-stranded DNA ends with high affinity and remarkable specificity—it recognizes the transition between double-stranded and single-stranded DNA at a break, regardless of the sequence. The Ku heterodimer is abundant in the nucleus, with an estimated 400,000 molecules per human cell, ensuring rapid loading at even low-frequency DSBs.
Once bound, Ku serves as a platform for recruiting downstream NHEJ factors. It also protects DNA ends from exonucleolytic degradation and translocates inward along the DNA helix in an ATP-independent manner, allowing multiple Ku molecules to load onto a single DNA molecule. The crystal structure of Ku bound to DNA reveals that the heterodimer forms a ring that threads onto the DNA end, explaining its remarkable stability once bound.
DNA-PK Complex
The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a 460 kDa serine/threonine kinase that belongs to the phosphatidylinositol 3-kinase-like kinase (PIKK) family. DNA-PKcs is recruited to DNA ends by Ku, forming the active DNA-PK holoenzyme. The kinase activity of DNA-PKcs is essential for NHEJ; it autophosphorylates and phosphorylates downstream targets, including Artemis and XRCC4.
DNA-PKcs autophosphorylation induces a conformational change that opens the DNA-PK complex, allowing access of processing enzymes to the DNA ends. This autophosphorylation is also required for the release of DNA-PKcs from DNA, a necessary step before ligation can occur. The kinase activity is stimulated by DNA binding and requires ATP; in vitro assays typically use 1–2 mM ATP in kinase buffer to measure DNA-PK activity.
Processing Enzymes
Artemis is a nuclease that possesses both 5'→3' exonuclease and endonuclease activities. It is recruited to DNA-PK complexes and becomes activated upon phosphorylation by DNA-PKcs. Artemis is essential for opening hairpin structures generated during V(D)J recombination and for processing overhangs at radiation-induced DSBs. Its endonuclease activity can cleave 5' and 3' overhangs, as well as hairpin loops, generating blunt or partially complementary ends that can be ligated.
Additional processing enzymes include the polymerases Pol μ and Pol λ, which fill in gaps at DNA ends, and the nuclease Aprataxin (APTX), which removes abortive ligation intermediates. The MRN complex (Mre11-Rad50-Nbs1) also participates in NHEJ, particularly in the processing of complex breaks, though its role is more prominent in HR.
Ligation Complex
The ligation complex consists of XRCC4, DNA ligase IV, and XLF (also known as Cernunnos). DNA ligase IV is the only ligase that functions in NHEJ; it catalyzes the formation of a phosphodiester bond between the 3' hydroxyl and 5' phosphate at the DNA ends. XRCC4 stabilizes ligase IV and enhances its activity, while XLF interacts with XRCC4 to promote the ligation of non-complementary ends.
The XRCC4-ligase IV complex has a distinctive filamentous structure that can bridge two DNA molecules. XLF forms homodimers that interact with XRCC4, creating a filament that promotes the synapsis of DNA ends. This filamentous architecture explains how NHEJ can ligate ends that are not perfectly complementary, as the protein scaffold holds the ends in proximity while processing enzymes modify them.
Step-by-Step Mechanism of NHEJ
The NHEJ reaction proceeds through a series of ordered steps, each mediated by specific protein complexes. The entire process typically completes within 30 minutes to several hours in mammalian cells, depending on the complexity of the break.
Recognition and Binding
The first step in NHEJ is the recognition and binding of the broken DNA ends by the Ku heterodimer. Ku loads onto DNA ends within seconds of break formation, with an association rate constant of approximately 10⁷ M⁻¹s⁻¹. The Ku heterodimer binds in a sequence-independent manner, recognizing the structural feature of a DNA end rather than specific nucleotides.
Once bound, Ku undergoes a conformational change that creates a high-affinity binding site for DNA-PKcs. The recruitment of DNA-PKcs to Ku-bound DNA ends forms the active DNA-PK holoenzyme. This complex protects the DNA ends from degradation and serves as a scaffold for the assembly of the complete NHEJ machinery. The binding of DNA-PKcs to Ku-DNA complexes is ATP-independent but requires the C-terminal region of Ku80, which interacts with DNA-PKcs.
Synapsis and Bridging
Synapsis refers to the physical tethering of the two broken DNA ends. This step is mediated by the DNA-PK complex, which can bridge two DNA molecules. The kinase activity of DNA-PKcs is required for synapsis; autophosphorylation induces conformational changes that allow the two DNA-PK complexes at each end to interact.
The XRCC4-ligase IV-XLF complex also contributes to synapsis. XLF and XRCC4 form alternating filaments that can bridge DNA ends, stabilizing the synaptic complex. This filamentous structure is particularly important for ligating ends that have limited complementarity, as it holds the ends in close proximity while processing enzymes act.
Synapsis is a dynamic process; the ends can undergo multiple rounds of processing and realignment before ligation. This flexibility contributes to the error-prone nature of NHEJ, as the ends may be aligned in different registers, creating deletions or insertions at the junction.
End Processing
Most DSBs generated by ionizing radiation or chemotherapeutic agents do not have clean, ligatable ends. They may contain damaged nucleotides, abasic sites, or overhangs that prevent direct ligation. End processing enzymes modify these termini to create ligatable ends.
The processing step involves several enzymes working in concert:
- Artemis cleaves hairpin structures and overhangs. Its endonuclease activity is activated by DNA-PKcs phosphorylation.
- Pol μ and Pol λ fill in gaps by template-dependent or template-independent synthesis.
- Aprataxin removes adenylate groups from abortive ligation intermediates.
- PNKP (polynucleotide kinase 3'-phosphatase) removes 3' phosphates and adds 5' phosphates.
- Mre11 (as part of the MRN complex) can resect ends in a limited fashion.
The extent of processing determines the fidelity of repair. Minimal processing results in accurate repair, while extensive processing creates deletions. The balance between these outcomes is regulated by the availability of microhomology—short regions of complementarity (2–6 nucleotides) between the two ends that can guide alignment.
Ligation
The final step is ligation, catalyzed by DNA ligase IV in complex with XRCC4 and XLF. DNA ligase IV seals the nick by forming a phosphodiester bond between the 3' hydroxyl and 5' phosphate at the junction. The reaction requires ATP and proceeds through an adenylated enzyme intermediate.
Ligation is the rate-limiting step of NHEJ. The XRCC4-ligase IV complex must undergo a conformational change to access the DNA ends, and this change is promoted by XLF. Once ligation is complete, the NHEJ factors dissociate from the repaired DNA, and the DNA is restored to its native state.
End Processing and DNA Polymerases
The processing of incompatible DNA ends is a defining feature of NHEJ. Unlike HR, which can accurately repair complex breaks using a template, NHEJ must make the ends ligatable through enzymatic modification. This processing is inherently mutagenic, as it can remove or add nucleotides at the junction.
Nuclease Activity
Artemis is the primary nuclease in NHEJ. It belongs to the metallo-β-lactamase family and possesses both exonuclease and endonuclease activities. The endonuclease activity of Artemis is cryptic—it requires phosphorylation by DNA-PKcs to become active. Once activated, Artemis can cleave:
- 5' overhangs
- 3' overhangs
- Hairpin structures
- Single-stranded DNA at the junction between single-stranded and double-stranded regions
The nuclease activity of Artemis is particularly important for V(D)J recombination, where it opens the hairpin intermediates generated by RAG proteins. In the context of radiation-induced DSBs, Artemis processes complex ends containing damaged nucleotides.
Other nucleases contribute to end processing in specific contexts. The MRN complex, particularly Mre11, possesses both endonuclease and 3'→5' exonuclease activities. While MRN is more critical for HR, it can participate in NHEJ when breaks contain secondary structures or protein adducts. The nuclease activity of Mre11 is stimulated by its interaction with Rad50 and Nbs1, and it requires manganese as a cofactor.
Polymerase Activity
DNA polymerases μ and λ are specialized polymerases that function in NHEJ. Unlike replicative polymerases, these enzymes can synthesize DNA in a template-independent manner, meaning they can add nucleotides even without a complementary template strand.
Pol μ is unique among DNA polymerases in its ability to perform template-independent synthesis. It can add nucleotides to a 3' overhang, creating a complementary sequence that can anneal to the other end. This activity is particularly important during V(D)J recombination, where it generates junctional diversity by adding random nucleotides (N-nucleotides) at the coding joints.
Pol λ is more template-dependent than Pol μ but can still incorporate nucleotides opposite damaged bases or abasic sites. It also possesses a 5'-deoxyribose-5-phosphate lyase activity, allowing it to process abasic sites at DNA ends.
The activities of these polymerases are coordinated with the nucleases. The processing of DNA ends is not a one-step process; rather, it involves iterative cycles of nucleolytic digestion and polymerization until the ends become ligatable. This iterative processing contributes to the variability of NHEJ junctions.
Regulation of NHEJ During the Cell Cycle
The choice between NHEJ and HR is tightly regulated during the cell cycle. This regulation ensures that the appropriate repair pathway is used based on the availability of homologous templates and the requirements of the cell.
Cell Cycle Phase Preference
NHEJ is active throughout the cell cycle but is the dominant DSB repair pathway in G1 phase. In G1, cells have a 2N DNA content, and sister chromatids are not available. HR cannot function effectively without a homologous template, so NHEJ is the only option for DSB repair.
In S and G2 phases, both NHEJ and HR are active. HR becomes available because sister chromatids are present following DNA replication. However, NHEJ still contributes significantly to DSB repair in these phases, particularly for breaks that are not suitable for HR.
The cell cycle regulation of NHEJ is less stringent than that of HR. While HR is essentially restricted to S/G2, NHEJ can operate at any time. This makes NHEJ the default pathway for DSB repair, with HR serving as an alternative when conditions permit.
Competition with Homologous Recombination
The competition between NHEJ and HR is governed by the process of DNA end resection. Resection—the nucleolytic degradation of the 5' ends to create 3' single-stranded overhangs—is the critical commitment step for HR. If resection proceeds beyond a threshold length (typically 100–200 nucleotides), the break is committed to HR and NHEJ cannot occur.
The decision to resect is regulated by cyclin-dependent kinases (CDKs). In G1, CDK activity is low, and resection is inhibited. The resection inhibitor 53BP1 is recruited to DSBs and blocks the access of resection factors. In S/G2, CDK activity is high, and 53BP1 is phosphorylated and inactivated, allowing resection to proceed.
This regulatory mechanism ensures that NHEJ is favored in G1, while HR is favored in S/G2. The balance between these pathways is critical for genome stability; inappropriate pathway choice can lead to genomic rearrangements or loss of heterozygosity.
NHEJ in V(D)J Recombination and Immune Diversity
The adaptive immune system relies on the generation of diverse antigen receptors through V(D)J recombination. This process creates DSBs as intermediates, and NHEJ is essential for their resolution.
RAG Proteins and NHEJ
V(D)J recombination is initiated by the RAG1 and RAG2 proteins, which introduce DSBs at recombination signal sequences (RSSs) flanking the variable (V), diversity (D), and joining (J) gene segments. RAG1/RAG2 introduce a nick at the RSS, followed by a transesterification reaction that generates a hairpin at the coding end and a blunt signal end.
The hairpin coding ends are opened by Artemis, which is activated by DNA-PKcs. This generates short overhangs that are processed by the NHEJ machinery. The processing includes:
- Opening of hairpins by Artemis
- Addition of N-nucleotides by terminal deoxynucleotidyl transferase (TdT) or Pol μ
- Fill-in synthesis by Pol μ or Pol λ
- Ligation by XRCC4-ligase IV-XLF
The joining of coding ends is imprecise, generating junctional diversity that contributes to the enormous repertoire of antigen receptors. In contrast, the joining of signal ends is precise, as these ends are blunt and can be ligated directly.
Defects Leading to Immunodeficiency
Mutations in NHEJ genes cause severe combined immunodeficiency (SCID) in humans and mice. The immunodeficiency arises because V(D)J recombination cannot be completed, leading to a failure to generate mature B and T lymphocytes.
The severity of the immunodeficiency depends on the specific gene affected. Mutations in Artemis cause a radiosensitive form of SCID (RS-SCID), characterized by the absence of both B and T cells. Mutations in DNA-PKcs, XRCC4, and ligase IV also cause immunodeficiency, though the phenotypes can be more variable.
In addition to immunodeficiency, NHEJ defects cause growth retardation and neurological abnormalities. These phenotypes are thought to result from the accumulation of unrepaired DSBs during development, particularly in the developing brain where extensive neural proliferation occurs.
Methods to Study NHEJ
Several experimental approaches are used to study NHEJ, each providing different types of information about the pathway.
Reporter Assays
Reporter assays are the most common method for measuring NHEJ efficiency in cells. These assays use a reporter construct containing a DSB that must be repaired by NHEJ to restore expression of a selectable marker.
The most widely used reporter is the EJ (end joining) assay, which contains a GFP gene interrupted by an I-SceI recognition site. Expression of I-SceI creates a DSB; NHEJ repair that results in a +1 frameshift restores GFP expression. The frequency of GFP-positive cells provides a quantitative measure of NHEJ efficiency.
A variation of this assay uses a reporter that distinguishes between NHEJ and HR. The DR-GFP reporter contains two GFP genes, one of which is mutated. HR between the two genes restores GFP expression, while NHEJ does not. This allows the relative activities of NHEJ and HR to be measured in the same cell population.
Live-Cell Imaging
Live-cell imaging allows the visualization of NHEJ protein recruitment to DSBs in real time. This approach typically uses laser microirradiation to create DSBs in a defined region of the nucleus, followed by time-lapse microscopy to track the recruitment of fluorescently tagged NHEJ proteins.
These studies have revealed the kinetics of NHEJ protein recruitment. Ku is recruited within seconds of break formation, followed by DNA-PKcs within minutes. The XRCC4-ligase IV complex is recruited later, consistent with its role in the final ligation step.
Live-cell imaging has also revealed the dynamic behavior of NHEJ complexes. The DNA-PK complex undergoes repeated rounds of association and dissociation at DNA ends, suggesting that the processing of complex breaks requires multiple attempts.
Biochemical Reconstitution
Biochemical reconstitution involves the assembly of the NHEJ reaction from purified components. This approach has been used to define the minimal requirements for NHEJ and to study the mechanisms of individual steps.
A typical reconstitution reaction contains:
- 100 nM Ku
- 100 nM DNA-PKcs
- 50 nM XRCC4-ligase IV
- 50 nM XLF
- 1 mM ATP
- 10 mM MgCl₂
- 25 mM Tris-HCl (pH 7.5)
- 50 mM KCl
- 1 mM DTT
The reaction is incubated at 37°C for 30–60 minutes, and the products are analyzed by gel electrophoresis. This approach has been used to demonstrate that Ku, DNA-PKcs, XRCC4-ligase IV, and XLF are sufficient for the ligation of compatible ends, while Artemis and polymerases are required for the processing of incompatible ends.
Pathological Consequences of NHEJ Defects
Mutations in NHEJ genes cause a spectrum of human diseases, ranging from severe immunodeficiency to cancer predisposition.
SCID and Radiosensitivity
Severe combined immunodeficiency is the most direct consequence of NHEJ defects. Patients with mutations in Artemis, DNA-PKcs, or ligase IV present with a complete absence of T and B lymphocytes, leading to life-threatening infections in infancy.
The radiosensitivity of NHEJ-deficient cells is a defining feature. Cells with mutations in NHEJ genes are hypersensitive to ionizing radiation and radiomimetic drugs, reflecting their inability to repair radiation-induced DSBs. This radiosensitivity is used diagnostically; fibroblasts from patients with suspected NHEJ defects are tested for their sensitivity to ionizing radiation.
Cancer and Genomic Instability
NHEJ defects are associated with genomic instability and cancer predisposition. The genomic instability arises from the misrepair of DSBs, which can generate chromosomal translocations, deletions, and amplifications.
Mice with hypomorphic mutations in NHEJ genes develop thymic lymphomas with high frequency. These lymphomas are characterized by chromosomal translocations involving the T-cell receptor loci, reflecting the role of NHEJ in V(D)J recombination.
In humans, polymorphisms in NHEJ genes are associated with increased cancer risk, though the effect sizes are modest. Somatic mutations in NHEJ genes are also found in some tumors, suggesting that NHEJ dysfunction can contribute to tumor progression.
Common Pitfalls and Exam Tips for NHEJ
Misconceptions
Several misconceptions about NHEJ are common among students:
"NHEJ is always error-prone." While NHEJ is often mutagenic, it can also be accurate. If the DNA ends are compatible and require no processing, NHEJ can ligate them with perfect fidelity. The error-prone nature of NHEJ arises from the processing of incompatible ends, not from the ligation step itself.
"NHEJ only occurs in G1." NHEJ is active throughout the cell cycle. It is the dominant pathway in G1, but it also contributes to DSB repair in S and G2. The key point is that HR is restricted to S/G2, not that NHEJ is restricted to G1.
"Ku binds to single-stranded DNA." Ku binds specifically to double-stranded DNA ends. It does not bind to single-stranded DNA or to internal double-stranded regions. This specificity is essential for its role in recognizing DSBs.
"DNA-PKcs is a helicase." DNA-PKcs is a kinase, not a helicase. It does not unwind DNA; rather, it phosphorylates proteins to regulate their activity. The confusion may arise because DNA-PKcs is a large protein with ATPase activity, but this activity is used for autophosphorylation, not for DNA unwinding.
"Artemis is a restriction enzyme." Artemis is a structure-specific nuclease, not a restriction enzyme. It does not recognize specific DNA sequences; rather, it cleaves specific DNA structures, such as hairpins and overhangs.
Key Takeaways
For exam preparation, focus on the following points:
- The four steps of NHEJ: recognition (Ku), synapsis (DNA-PK), processing (Artemis, polymerases), and ligation (XRCC4-ligase IV-XLF).
- The distinction from HR: NHEJ is template-independent and error-prone; HR is template-dependent and accurate.
- The cell cycle regulation: NHEJ is active throughout the cell cycle but dominant in G1; HR is restricted to S/G2.
- The role in V(D)J recombination: NHEJ is essential for generating immune diversity.
- The disease associations: NHEJ defects cause SCID, radiosensitivity, and cancer predisposition.
- The key proteins: Ku70/Ku80, DNA-PKcs, Artemis, XRCC4, ligase IV, and XLF.
Frequently Asked Questions
What is the non homologous end joining pathway?
The non homologous end joining pathway is a DNA double-strand break repair mechanism that directly ligates broken DNA ends without requiring a homologous template. It is the dominant DSB repair pathway in mammalian cells and is active throughout the cell cycle. The pathway involves the recognition of DNA ends by the Ku heterodimer, the recruitment of DNA-PKcs, the processing of incompatible ends by nucleases and polymerases, and the final ligation by the XRCC4-ligase IV-XLF complex. For a comprehensive overview, see the Non Homologous End Joining Repair article.
How does NHEJ differ from homologous recombination?
NHEJ differs from homologous recombination in three fundamental ways. First, NHEJ does not require a homologous template, while HR uses the sister chromatid as a template. Second, NHEJ is error-prone, often introducing small insertions or deletions at the repair junction, while HR is generally error-free. Third, NHEJ is active throughout the cell cycle, while HR is restricted to the S and G2 phases when sister chromatids are available. The choice between the two pathways is governed by DNA end resection, which commits the break to HR.
What are the main proteins involved in NHEJ?
The main proteins involved in NHEJ are Ku70 and Ku80 (which form the Ku heterodimer that recognizes DNA ends), DNA-PKcs (a kinase that regulates the pathway), Artemis (a nuclease that processes DNA ends), XRCC4 and XLF (scaffold proteins), and DNA ligase IV (the enzyme that seals the break). Additional factors include Pol μ and Pol λ (polymerases that fill in gaps) and Aprataxin (which removes abortive ligation intermediates). The Non Homologous End Joining Proteins article provides a detailed description of each component.
Why is NHEJ important for the immune system?
NHEJ is essential for V(D)J recombination, the process that generates the diversity of antigen receptors on B and T lymphocytes. During V(D)J recombination, the RAG1/RAG2 proteins introduce double-strand breaks at recombination signal sequences. The coding ends form hairpins that must be opened and processed by the NHEJ machinery before ligation. This processing is imprecise, generating junctional diversity that contributes to the enormous repertoire of antigen receptors. Defects in NHEJ proteins cause severe combined immunodeficiency because V(D)J recombination cannot be completed.
What happens if NHEJ is defective?
Defects in NHEJ cause severe combined immunodeficiency, characterized by the absence of mature B and T lymphocytes. Patients with NHEJ defects also exhibit radiosensitivity, growth retardation, and neurological abnormalities. At the cellular level, NHEJ defects lead to the accumulation of unrepaired DNA double-strand breaks, genomic instability, and increased cancer predisposition. The severity of the phenotype depends on which NHEJ gene is mutated and the nature of the mutation.
Is NHEJ error-prone?
NHEJ is often error-prone, but it can also be accurate. The error-prone nature of NHEJ arises from the processing of incompatible DNA ends. When the ends are compatible and require no processing, NHEJ ligates them with perfect fidelity. However, most DSBs induced by ionizing radiation or chemotherapeutic agents have damaged or incompatible ends that require processing. This processing can remove nucleotides (creating deletions) or add nucleotides (creating insertions), resulting in mutations at the repair junction.
When does NHEJ occur in the cell cycle?
NHEJ occurs throughout the cell cycle but is the dominant DSB repair pathway in the G1 phase. In G1, sister chromatids are not available, so homologous recombination cannot function. NHEJ is also active in the S and G2 phases, where it competes with HR for DSB repair. The balance between NHEJ and HR in S/G2 is regulated by CDK activity, which controls the initiation of DNA end resection that commits breaks to HR.
Key Takeaways
- NHEJ is the dominant DNA double-strand break repair pathway in mammalian cells, operating without a homologous template and functioning throughout the cell cycle.
- The core NHEJ machinery includes Ku70/Ku80, DNA-PKcs, Artemis, XRCC4, DNA ligase IV, and XLF, which work together to recognize, process, and ligate broken DNA ends.
- NHEJ is error-prone because end processing often introduces small insertions or deletions at repair junctions, distinguishing it from the accurate homologous recombination pathway.
- The pathway is essential for V(D)J recombination, and defects in NHEJ genes cause severe combined immunodeficiency, radiosensitivity, and cancer predisposition.
- NHEJ is regulated during the cell cycle, with CDK-dependent control of DNA end resection determining whether breaks are repaired by NHEJ or homologous recombination.
- NHEJ can be studied using reporter assays, live-cell imaging, and biochemical reconstitution, each providing complementary information about the pathway.
- The Non Homologous End Joining CRISPR application exploits NHEJ to generate targeted gene knockouts, highlighting the pathway's utility in genome engineering.
Further Reading
- Caracciolo D et al. Alternative Non-Homologous End-Joining: Error-Prone DNA Repair as Cancer's Achilles' Heel. Cancers. 2021. PubMed 33808562
- van Gent DC, van der Burg M. Non-homologous end-joining, a sticky affair. Oncogene. 2007. PubMed 18066085
- Saika H, Nishizawa-Yokoi A, Toki S. The non-homologous end-joining pathway is involved in stable transformation in rice. Frontiers in plant science. 2014. PubMed 25368624
- Menon V, Povirk LF. End-processing nucleases and phosphodiesterases: An elite supporting cast for the non-homologous end joining pathway of DNA double-strand break repair. DNA repair. 2016. PubMed 27262532
- Revy P, Malivert L, de Villartay JP. Cernunnos-XLF, a recently identified non-homologous end-joining factor required for the development of the immune system. Current opinion in allergy and clinical immunology. 2006. PubMed 17088645
- Rooney S, Chaudhuri J, Alt FW. The role of the non-homologous end-joining pathway in lymphocyte development. Immunological reviews. 2004. PubMed 15242400