DNA Double-Strand Break Repair by Non-Homologous End Joining

Eukaryotic DNA is perpetually under siege from various threats, including ultraviolet radiation and chemical damage. Among the forms of DNA damage that can result, double-stranded breaks (DSBs) are particularly perilous because they can cause chromosomal rearrangements and disrupt vital genetic information. If these breaks are not promptly and accurately repaired, severe effects such as carcinogenesis can occur. Cells have evolved sophisticated DNA repair mechanisms to counteract these threats. Non-homologous end joining (NHEJ) is one such defense mechanism, playing a crucial role in maintaining genomic stability by efficiently repairing DSBs.

Causes of DNA Double-Strand Breaks

Environmental and Biological Sources

DSBs can arise from various sources, both external and internal to the cell. One of the most common external causes is ionizing radiation, such as X-rays and gamma rays. This type of radiation, commonly encountered in medical imaging and radiation therapy and from natural environmental sources like cosmic rays, carries enough energy to directly break the DNA strands.

Another significant cause of DSBs is exposure to chemical mutagens, including chemotherapeutic drugs, environmental pollutants, and industrial chemicals. These substances can interact with DNA directly or induce breaks indirectly by generating reactive intermediates. In addition, lifestyle factors like smoking and prolonged exposure to ultraviolet (UV) radiation can contribute to DSB formation.

Internal cellular processes can also result in DSBs. One key source is replication stress, during which DNA secondary structures, tightly bound proteins, or unrepaired ingle-strand breaks can cause replication fork stalling and collapse, resulting in DSBs. This is particularly prevalent under conditions of high replication stress. Reactive oxygen species (ROS), which are byproducts of normal cellular metabolism, can also attack the DNA backbone and lead to strand breaks.

Mechanical stress during cell division, especially in mitosis and meiosis, can also cause DSBs. The chromosomes experience significant stress during these phases, and any excessive manipulation or movement can strain the DNA structure, leading to breaks. Furthermore, in developing lymphocytes, enzymes involved in processes like V(D)J recombination intentionally create DSBs to generate antibody diversity.

Cells’ Response to DSBs: DNA Repair Mechanisms

Cells recognize DSBs as severe damage and respond by activating various mechanisms. These include arresting the cell cycle to prevent the propagation of damaged DNA, activating DNA repair mechanisms to address the breaks, and – if the damage cannot be repaired – triggering  programmed cell death (apoptosis) to eliminate the damaged cell. This multifaceted response allows cells to maintain genomic integrity and prevent deleterious effects from unrepaired DSBs.

Cells have evolved two primary mechanisms for repairing DSBs: Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ). Both of these pathways are crucial for maintaining genomic stability by repairing DSBs, but they operate under different contexts and with distinct mechanisms.

Homologous Recombination vs. Non-Homologous End Joining

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.

In contrast, NHEJ operates under a different set of rules. Unlike HR, it does not require a homologous sequence for repair, making it a more versatile but error-prone process. 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, and while this is efficient, small deletions or insertions can occur at the repair site.

If these primary repair pathways are compromised, cells may resort to an alternative repair mechanism known as Alternative End Joining (alt-EJ) or Microhomology-Mediated End Joining (MMEJ). This method relies on microhomologous sequences, which are short, identical or nearly identical DNA sequences found at or near the break sites, to facilitate the repair process. These microhomologous sequences are usually a few to several dozen base pairs long and act as brief templates for aligning and joining the broken DNA strands. However, MMEJ is more error-prone than other repair mechanisms and often results in deletions or insertions.

The decision regarding which DNA repair mechanism to employ involves balancing accuracy against efficiency. HR, known for its high accuracy, operates more slowly and is restricted to specific phases of the cell cycle. In contrast, NHEJ provides a quicker response, which is essential for preventing the severe consequences of unrepaired double-strand breaks (DSBs), but this speed comes with an increased likelihood of introducing mutations.

Key Components in the Non-Homologous End Joining Pathway

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 the broken DNA ends while providing a platform for assembling other NHEJ factors.

DNA-Dependent Protein Kinase (DNA-PK)

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 adequately prepared for the final ligation step.

DNA Polymerases Mu (Pol μ) and Lambda (Pol λ)

Both Pol μ and Pol λ share the ability to perform template-independent DNA synthesis, which is crucial in NHEJ 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.

XRCC4 and DNA Ligase IV

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 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.

Processing of DNA Ends in NHEJ

One 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 the direct ligation of the strands. NHEJ employs a range of nucleases and polymerases to process the DNA ends so they are suitable for ligation.

Nucleases like the MRE11-Rad50-NBS1 complex, with both exonuclease and endonuclease activities, can resect damaged DNA ends, while Artemis is able to open hairpin structures and process overhangs. Polymerases such as pol µ, pol λ, and TdT add nucleotides to the processed ends, ensuring that any gaps created during the trimming process are filled. This end processing is crucial for restoring the length and sequence of the DNA strand and preparing it for a successful ligation.

Interplay 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 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 reversed, 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.

Health Consequences of NHEJ 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 the inappropriate activation of immune responses.

Therapeutic Potential of NHEJ 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.

Further Reading

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. doi:10.1016/BS.IRCMB.2021.06.003
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. doi:10.1038/S41580-019-0152-0
3. Sławińska N, Krupa R. Molecular aspects of senescence and organismal ageing–DNA damage response, telomeres, inflammation and chromatin. International Journal of Molecular Sciences 2021, Vol 22, Page 590. 2021;22(2):590. doi:10.3390/IJMS22020590
4. Vogt A, He Y. Structure and mechanism in non-homologous end joining. DNA Repair (Amst). 2023;130:103547. doi:10.1016/J.DNAREP.2023.103547
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. doi:10.1042/BST20220741
6. Xue C, Greene EC. DNA repair pathway choices in CRISPR-Cas9-mediated genome editing. Trends in Genetics. 2021;37(7):639-656. doi:10.1016/J.TIG.2021.02.008
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. doi:10.1038/s41580-020-00297-8