Non-Homologous End Joining (NHEJ) in DNA Double-Strand Break Repair

What is non-homologous end joining?

Non-homologous end joining (NHEJ) is a primary pathway for repairing double-strand breaks (DSBs) in cells. It is crucial because it operates throughout the cell cycle, unlike other repair mechanisms limited to specific phases. NHEJ is particularly important in repairing DSBs caused by ionizing radiation, reactive oxygen species and other random DNA breaks. It works by directly joining the broken DNA ends, a process that is essential for maintaining genomic integrity and preventing severe consequences like carcinogenesis. In mammalian cells, NHEJ often acts as the default repair pathway. Its speed is advantageous for cell survival, but it is less precise than other repair pathways.

Steps in the non-homologous end joining process

The NHEJ DNA repair mechanism is generally broken down into three steps: recognition of the free DNA ends, processing of the DNA ends and finally ligation and final repair.

Recognition of DNA ends

The first challenge in the non-homologous end joining DNA repair process is recognizing that a double-strand break has occurred, identifying the free ends of DNA. The Ku70/Ku80 heterodimer plays a critical role in this step, binding to the DNA ends where it recruits other key proteins that help hold the DNA ends near to each other and help prevent degradation and loss of DNA from nearby nucleases that may opportunistically or specifically recognize free DNA ends as a threat to genome stability.

Processing of DNA ends

A second challenge in the NHEJ process is the direct ligation of DNA ends. Often, the ends of a DNA DSB are not immediately compatible for ligation due to damage or irregularities. For example, complex DNA structures like overhangs, hairpins or fragmented nucleotides prevent direct ligation of the strands. NHEJ employs Artemis nuclease and DNA polymerases mu (Pol μ) and lambda (Pol λ) to process the DNA ends so they are suitable for ligation. This process of trimming or filling in nucleotides in preparation for ligation is often responsible for introducing mutations.

Ligation and final repair

The final step of the non-homologous end joining mechanism is the ligation, or joining together, of the free DNA ends. This is accomplished by the XRCC4-DNA ligase IV complex which enzymatically seals the break by creating phosphodiester bonds to join the free DNA ends together. Accessory proteins such as XLF (Cernunnos) and PAXX help bridge DNA ends and stabilize the ligase complex, especially when ends are not perfectly aligned.

Key proteins involved in non-homologous end joining DNA repair

Multiple proteins and complexes are involved in the NHEJ process. Key components include the Ku70/Ku80 heterodimer, DNA-dependent protein kinase (DNA-PKcs), artemis nuclease, DNA polymerase mu (Pol μ) and DNA polymerase lambda (Pol λ), XRCC4-DNA ligase IV complex and additional factors.

Ku70/Ku80 heterodimer

In the NHEJ pathway, the DSB repair process begins with the action of the Ku70/Ku80 heterodimer. This protein complex acts as the first responder to DSBs and binds to the DNA ends with high affinity and specificity. Its role is crucial because it stabilizes broken DNA ends while providing a platform for assembling other NHEJ factors.

DNA-dependent protein kinase (DNA-PKcs)

Another key player in NHEJ is the DNA-dependent protein kinase (DNA-PK). This enzyme becomes activated upon its interaction with the DNA-Ku complex. The activated DNA-PK is central in coordinating the repair process by phosphorylating a series of substrates. These include the Artemis nuclease, crucial for processing DNA ends, the XRCC4-DNA Ligase IV complex, essential for the ligation step, and the DNA-PK itself, as part of a regulatory feedback mechanism.

Artemis nuclease

Artemis nuclease is involved in processing the DNA ends to make them suitable for ligation. It plays a key role in opening hairpin structures and trimming damaged nucleotides, ensuring the DNA ends are properly prepared for the final ligation step.

DNA polymerase mu (Pol μ) and DNA polymerase lambda (Pol λ)

Both Pol μ and Pol λ share the ability to perform template-independent DNA synthesis, which is crucial in non-homologous end joining where a complementary DNA strand may not be available. These polymerases are particularly important for gap-filling during the repair process, ensuring that any gaps created during DNA end-processing are accurately filled. Their activity contributes significantly to the efficiency and fidelity of the NHEJ repair mechanism.

XRCC4-DNA ligase IV complex

The proteins DNA ligase IV and XRCC4 (X-ray repair cross-complementing protein 4) 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. XRCC4 binds to and stabilizes DNA ligase IV, enhancing its enzymatic activity. XRCC4 also helps align the DNA ends, which is essential for their accurate and efficient ligation. XRCC4 also recruits other necessary factors to the break site.

Additional factors (PAXX, XLF, TdT)

Additional components such as PAXX, XLF, and TdT are also involved in NHEJ. PAXX (Paralog of XRCC4 and XLF) is a protein with structural similarities to XRCC4 and XLF and is believed to help stabilize DNA ends. XLF (also known as Cernunnos or NHEJ1) works in tandem with XRCC4 and helps bridge DNA ends, facilitating the ligation step. Terminal Deoxynucleotidyl Transferase (TdT) is a DNA polymerase that adds nucleotides in a template-independent manner. While not a part of the classical NHEJ pathway, TdT is vital in the immune system for V(D)J recombination, a specialized form of NHEJ.

Interplay of non-homologous end joining with other signaling pathways

The NHEJ pathway does not operate in isolation within the cell but interacts with various other signaling pathways. This crosstalk helps maintain cellular health by preventing the propagation of damaged DNA, which could lead to genomic instability and potentially to oncogenesis.

One key interaction is with cell cycle regulation machinery. Upon the detection of DNA damage like DSBs, the DNA-PK complex signals to key cell cycle regulators, including cyclins D, E, A, and B, cyclin-dependent kinases (CDKs), and the p53 tumor suppressor protein. This signaling activates cell cycle checkpoints, including G1/S the checkpoint, and triggers a pause in cell cycle progression, giving the cell time to address the damage. Once the DNA has been repaired, these checkpoint signals are deactivated, and the cell cycle can resume.

If damage is too severe to be repaired via NHEJ, the cell can initiate programmed cell death (apoptosis) as a protective measure. p53, in coordination with ATM, evaluates whether the damage can be repaired or if the cell should undergo apoptosis. The latter case results in the activation of pro-apoptotic genes such as BAX, PUMA and NOXA, which facilitate the cell death process.

Non-homologous end joining (NHEJ) vs homologous recombination (HR)

NHEJ is most active during the G0 and G1 phases of the cell cycle, and in mammalian cells, it is the most commonly used pathway for repairing DSBs. The NHEJ process involves directly rejoining the broken DNA ends without requiring a homologous sequence. While this is efficient, small deletions or insertions can occur at the repair site.

In contrast, homologous recombination (HR) is a highly accurate repair process that typically occurs during the late S and G2 phases of the cell cycle. The accuracy of this process comes from its reliance on a sister chromatid, which provides a homologous sequence as a template for repair. By using this template, HR ensures that the genetic information at the break site is accurately restored.

Classical NHEJ vs Alternative NHEJ (alt-NHEJ/MMEJ)

When classical NHEJ factors (e.g., Ku or Ligase IV) are missing or inhibited, cells may activate alternative end joining, also called microhomology-mediated end joining (MMEJ). This pathway aligns DNA ends via short (2–25 bp) microhomologies before ligation, often leading to larger deletions and increased chromosomal rearrangements. While alt-NHEJ is normally suppressed, it can become prominent in cancer cells or after targeted DNA-PK inhibition. It is also exploited in certain CRISPR gene-editing strategies.

Health consequences of non-homologous end joining dysfunction

Malfunctions in NHEJ can result in improper repair of DNA DSBs, leading to genomic instability such as chromosomal aberrations, translocations and mutations. These kinds of genomic alterations are not just errors at the molecular level. They disrupt vital genetic information and can cause other cellular processes to malfunction. In particular, the misrepair of DSBs can directly contribute to the development and progression of cancers by activating oncogenes or deactivating tumor suppressor genes.

Genetic disorders and cancer risks

Deficiencies in NHEJ components are linked to several genetic disorders characterized by increased sensitivity to DNA-damaging agents and a predisposition to cancer. For instance, mutations in the genes encoding DNA ligase IV, XRCC4, and Artemis are associated with severe combined immunodeficiency (SCID) and other syndromes. These conditions often present with developmental abnormalities, immunodeficiency, and a heightened risk of malignancies. The direct link between NHEJ dysfunction and cancer highlights the pathway's role in safeguarding against uncontrolled cell growth and tumor development.

Impact on aging and cellular senescence

Beyond cancer, NHEJ dysfunction is also implicated in the aging process and cellular senescence. As cells age, they lose their ability to efficiently repair DNA, partly due to the declining functionality of repair pathways like NHEJ. This decline contributes to the accumulation of DNA damage and can trigger cellular senescence, a state where cells cease to divide. While senescence is a protective mechanism against cancer, it also contributes to aging by diminishing tissue regeneration and function.

Influence on immune system function

Additionally, NHEJ has implications for immune system functioning, particularly in the development of lymphocytes. The V(D)J recombination process, which is essential for generating diverse antibody and T-cell receptor repertoires, relies on mechanisms similar to those used in NHEJ. Errors or dysfunctions in NHEJ can lead to immune deficiencies or, conversely, to inappropriate activation of immune responses.

Therapeutic potential of non-homologous end joining components

NHEJ pathway components offer significant therapeutic potential, particularly in the fields of cancer therapy and gene editing. This potential stems from the ability to manipulate the NHEJ pathway to either enhance or inhibit DNA repair processes, depending on the therapeutic goal.

Targeting NHEJ in cancer therapy

Cancer cells have high rates of DNA damage, so they rely heavily on NHEJ for survival. Inhibiting key proteins in the NHEJ pathway, such as DNA-PK, Ku70/Ku80, or DNA ligase IV, can make cancer cells more sensitive to DNA damage and thus more vulnerable to conventional treatments like chemotherapy and radiation therapy, which induce DNA damage. Specific inhibitors of NHEJ components are also being explored for use in combination therapies to make existing cancer treatments more efficient and reduce the likelihood of resistance development.

Gene editing and NHEJ

Another area of therapeutic interest involves the role of NHEJ in gene editing, particularly with technologies like CRISPR-Cas9. Gene editing often involves creating targeted DNA double-strand breaks, which are then repaired by the cell's own repair mechanisms. Researchers would like to harness NHEJ to introduce specific mutations or deletions at the site of the break. This capability is important for studying gene function and developing gene therapies. However, the error-prone nature of NHEJ can also be a limitation in this context, as it may lead to off-target effects. Understanding and controlling the balance between NHEJ and more accurate repair mechanisms like HR is a key focus in refining gene editing techniques for therapeutic applications.

The exploration of NHEJ components in therapeutic contexts highlights the pathway's significance beyond its basic biological function. By targeting these components, researchers and clinicians can develop more effective cancer treatments and advance the field of gene editing, offering new avenues for treating a wide range of genetic diseases and disorders.

References

1. Gillyard T, Davis J. DNA double-strand break repair in cancer: a path to achieving precision medicine. Int Rev Cell Mol Biol. 2021;364:111–137.
2. Scully R, Panday A, Elango R, Willis NA. DNA double-strand break repair-pathway choice in somatic mammalian cells. Nat Rev Mol Cell Biol. 2019;20(11):698–714.
3. Sławińska N, Krupa R. Molecular aspects of senescence and organismal ageing–DNA damage response, telomeres, inflammation and chromatin. Int J Mol Sci. 2021;22(2):590.
4. Vogt A, He Y. Structure and mechanism in non-homologous end joining. DNA Repair (Amst). 2023;130:103547.
5. Vogt A, He Y, Lees-Miller SP. How to fix DNA breaks: new insights into the mechanism of non-homologous end joining. Biochem Soc Trans. 2023;51(5):1789–1800.
6. Xue C, Greene EC. DNA repair pathway choices in CRISPR-Cas9-mediated genome editing. Trends Genet. 2021;37(7):639–656.
7. Zhao B, Rothenberg E, Ramsden DA, Lieber MR. The molecular basis and disease relevance of non-homologous DNA end joining. Nat Rev Mol Cell Biol. 2020;21(12):765–781.