Ku70’s Role in DNA Repair and Clinical Implications

Cells are constantly exposed to factors that damage DNA, leading to mutations and genomic instability. To counteract this, cells have evolved complex repair mechanisms to maintain genetic integrity. One key player in these processes is Ku70, a protein essential for repairing double-strand breaks (DSBs), one of the most severe forms of DNA damage.

Understanding Ku70’s role in DNA repair has significant implications for disease prevention and treatment, particularly in cancer and immune system disorders. Researchers continue to investigate its molecular functions and interactions, shedding light on potential therapeutic targets.

Molecular Composition

Ku70, encoded by the XRCC6 gene, forms a heterodimer with Ku80, creating the Ku complex, a fundamental component of the non-homologous end joining (NHEJ) pathway. Structurally, Ku70 consists of three primary domains: the von Willebrand factor A (vWA) domain, the central DNA-binding domain, and the C-terminal arm. The vWA domain facilitates protein-protein interactions necessary for recruiting repair factors. The DNA-binding domain, rich in positively charged residues, enables Ku70 to recognize and associate with double-strand break sites. The C-terminal region stabilizes the Ku70-Ku80 interaction, ensuring the complex remains anchored to DNA ends throughout the repair process.

The Ku heterodimer forms a ring-like structure that encircles DNA, allowing it to slide along the helix without dissociating. This conformation prevents exonucleolytic degradation of exposed DNA ends while serving as a scaffold for downstream repair proteins. Ku70 contains lysine residues that undergo post-translational modifications, such as acetylation and ubiquitination, which modulate its DNA-binding affinity and stability. Acetylation at lysine 539 weakens Ku70’s interaction with DNA, promoting the release of the complex once repair is complete. Conversely, ubiquitination can target Ku70 for proteasomal degradation, ensuring the repair machinery does not persist longer than necessary.

Ku70 also possesses an intrinsically disordered region (IDR) that allows it to adopt multiple conformations depending on its binding partners. This flexibility is particularly advantageous in DNA repair, where Ku70 must interact with a diverse array of proteins under varying cellular conditions. Nuclear magnetic resonance (NMR) spectroscopy has shown that Ku70’s IDR undergoes conformational shifts upon DNA binding, suggesting a dynamic role in coordinating repair events. Additionally, Ku70 contains nuclear localization signals (NLS) that facilitate its transport into the nucleus, ensuring its availability at sites of DNA damage.

Role In DNA Repair Pathways

Ku70 plays a central role in repairing DNA double-strand breaks (DSBs) through the non-homologous end joining (NHEJ) pathway, a mechanism that restores genomic integrity without requiring a homologous template. When a DSB occurs, Ku70, in complex with Ku80, binds to the exposed DNA ends with high affinity, preventing degradation and facilitating the recruitment of additional repair factors. This recognition step ensures that broken DNA ends are stabilized and aligned, minimizing the risk of chromosomal rearrangements or deletions.

Once bound to DNA, Ku70 serves as a scaffold for assembling the NHEJ machinery by recruiting DNA-PKcs (DNA-dependent protein kinase catalytic subunit), forming the DNA-PK holoenzyme. This complex phosphorylates multiple repair proteins, including Artemis, a nuclease responsible for processing DNA ends to ensure compatibility before ligation. The end-processing step is particularly important when DSBs contain non-cohesive or chemically modified termini. Structural studies indicate that Ku70 helps orient Artemis at DNA ends, enhancing its ability to trim overhangs or remove obstructive lesions. Ku70 also interacts with XRCC4 and DNA ligase IV, guiding the final ligation step that restores DNA continuity.

Beyond classical NHEJ, Ku70 has been implicated in alternative end joining (alt-EJ), a backup repair pathway that operates when core NHEJ components are compromised. Although alt-EJ is generally more error-prone, Ku70 plays a role in limiting excessive resection of DNA ends, reducing the risk of large deletions. CRISPR-Cas9 knockout models have shown that loss of Ku70 increases reliance on alt-EJ, highlighting its importance in maintaining the fidelity of DSB repair. Additionally, Ku70 regulates DNA-end tethering, ensuring broken DNA fragments remain in close proximity until repair is complete, a function critical in preventing chromosomal translocations frequently observed in cancer cells with defective NHEJ components.

Interactions With Other Proteins

Ku70’s role in DNA repair is closely linked to its interactions with a network of proteins that regulate damage recognition, signaling, and resolution. Its most well-characterized partner is Ku80, with which it forms the Ku heterodimer. This interaction is essential for DNA binding, as neither protein efficiently associates with double-strand breaks alone. Crystallographic studies reveal that Ku70 and Ku80 create a ring-like structure that encircles DNA, stabilizing the break site while allowing the recruitment of additional repair factors.

Ku70 also engages with DNA-PKcs to form the DNA-PK holoenzyme, a key mediator of DSB repair. Upon DNA binding, Ku70 facilitates DNA-PKcs recruitment, triggering its autophosphorylation and activation. This event initiates a cascade of phosphorylation events that regulate end processing and ligation. Ku70’s interaction with DNA-PKcs enhances the kinase’s affinity for DNA ends, ensuring efficient signaling and coordination of repair factors. Additionally, Ku70 modulates Artemis, the nuclease that trims incompatible DNA termini, ensuring proper processing before ligation.

Ku70 further interacts with XRCC4 and DNA ligase IV, proteins responsible for the final ligation step in NHEJ. Structural analyses show that Ku70 helps position these enzymes at DNA ends, ensuring precise alignment before ligation occurs. This function is particularly important in complex DSBs, where Ku70’s ability to tether DNA fragments maintains end proximity. Ku70 also associates with PAXX, a relatively recent addition to the NHEJ protein network. PAXX stabilizes Ku70’s interaction with XRCC4-ligase IV, reinforcing the repair complex under conditions of high genomic stress. Loss-of-function studies have demonstrated that cells deficient in both Ku70 and PAXX exhibit severe repair defects, underscoring their cooperative role in maintaining genome integrity.

V(D)J Recombination

Ku70 plays an instrumental role in V(D)J recombination, a process that generates diverse antigen receptor repertoires by rearranging variable (V), diversity (D), and joining (J) gene segments. The recombination-activating gene (RAG) complex introduces site-specific double-strand breaks (DSBs) at recombination signal sequences adjacent to V, D, and J segments. Ku70, along with Ku80, rapidly associates with the DNA ends, preventing degradation and facilitating the recruitment of repair machinery.

V(D)J recombination requires precise end processing to ensure that coding and signal ends are appropriately joined. Ku70’s ability to tether DNA ends is critical, as this process involves the formation of hairpin structures at coding ends that must be carefully resolved. Ku70 interacts with Artemis, the nuclease responsible for opening DNA hairpins, ensuring that coding ends are processed correctly before ligation. Ku70-deficient models exhibit aberrant joining events, resulting in deletions or insertions that compromise antigen receptor integrity.

Post-Translational Modifications

Ku70 undergoes post-translational modifications that regulate its stability, DNA-binding affinity, and interactions with repair factors. Acetylation at lysine 539 weakens its association with DNA, facilitating the release of the Ku complex once repair is complete. Histone deacetylases (HDACs), particularly SIRT6, counteract this modification, reinforcing Ku70’s DNA-binding affinity and prolonging its presence at damage sites. Hyperacetylation of Ku70 impairs non-homologous end joining (NHEJ) efficiency, leading to increased genomic instability.

Ubiquitination plays a role in modulating Ku70 function. Polyubiquitination targets Ku70 for proteasomal degradation, ensuring repair proteins do not accumulate excessively after DSB repair. The E3 ubiquitin ligase CHIP mediates this degradation under prolonged DNA damage conditions. Monoubiquitination, in contrast, enhances Ku70’s recruitment to chromatin in response to radiation-induced DNA damage. SUMOylation has been linked to Ku70’s nuclear retention and interaction with chromatin remodeling factors, reinforcing its role in repair complex assembly.

Potential Clinical Implications

Ku70’s role in maintaining genomic integrity has implications for cancer and neurodegenerative diseases. In tumor biology, Ku70 expression influences the sensitivity of cancer cells to DNA-damaging therapies such as ionizing radiation and chemotherapy. Overexpression of Ku70 has been observed in radioresistant tumors, where enhanced repair capacity allows malignant cells to survive genotoxic treatments. Conversely, reduced Ku70 expression increases mutation rates and chromosomal instability, predisposing cells to oncogenic transformation.

In neurodegeneration, defective DNA repair contributes to the accumulation of genomic lesions in neurons. Ku70 mutations have been linked to ataxia-telangiectasia-like disorder (ATLD), characterized by progressive neurodegeneration and impaired DNA damage response. Ku70-deficient neurons exhibit increased susceptibility to oxidative stress-induced DNA breaks, leading to apoptosis. Studies in model organisms suggest that reduced Ku70 expression correlates with shorter lifespan and increased susceptibility to age-associated diseases. Modulating Ku70 activity presents a potential avenue for addressing DNA repair deficiencies in aging-related disorders.