TMEJ in DNA Repair: Insights into a Vital Pathway

Cells rely on multiple pathways to repair DNA damage, ensuring stability and preventing mutations that could lead to disease. One such pathway, polymerase theta-mediated end joining (TMEJ), plays a crucial role in repairing double-strand breaks, particularly when homologous recombination is unavailable. While highly error-prone, TMEJ is essential for cellular survival under certain conditions.

Beyond genome maintenance, TMEJ has been implicated in cancer progression and resistance to therapy. Understanding its mechanisms provides insights into both normal cellular processes and potential therapeutic targets.

Molecular Steps In TMEJ

TMEJ is a specialized repair mechanism that operates when cells face double-strand breaks (DSBs), particularly when homologous recombination (HR) and classical non-homologous end joining (c-NHEJ) are compromised. This pathway relies on polymerase theta (Polθ), an enzyme that facilitates repair through microhomology-mediated annealing. Unlike HR, which requires extensive sequence homology, TMEJ utilizes short complementary base sequences—typically 2 to 6 nucleotides—flanking the break site to guide repair. This process introduces insertions and deletions (indels), making it inherently mutagenic but indispensable in certain contexts.

The repair process begins with the recognition of DSBs, where Polθ is recruited to the damaged site. Single-stranded DNA overhangs, generated by nucleolytic processing, expose microhomologous sequences that serve as repair templates. Polθ, which possesses both polymerase and helicase activity, stabilizes these regions and initiates DNA synthesis. The enzyme extends one strand while displacing the complementary strand, creating a structure that facilitates annealing.

Once the microhomologies align, Polθ continues extending the DNA strand, filling gaps and stabilizing the repair intermediate. Template slippage often occurs, leading to characteristic indels. The final step involves sealing the repaired DNA ends, a process thought to involve DNA ligase III in conjunction with XRCC1. Unlike c-NHEJ, which relies on Ku70/Ku80 and DNA-PKcs for end bridging and ligation, TMEJ operates independently of these factors, making it a distinct repair pathway.

Key Proteins And Enzymes

TMEJ relies on a distinct set of proteins to repair double-strand breaks through microhomology-mediated mechanisms. Central to this process is polymerase theta (Polθ), encoded by the POLQ gene, which possesses both helicase and polymerase domains. This dual functionality enables it to unwind DNA while synthesizing new strands, a capability not found in other repair polymerases. Studies in Nature Structural & Molecular Biology have shown that Polθ preferentially binds to single-stranded DNA overhangs at break sites, stabilizing these regions to facilitate microhomology annealing. Its polymerase activity extends the annealed strand, incorporating nucleotides in a manner prone to indels.

Beyond Polθ, additional proteins enhance the efficiency and specificity of this repair pathway. DNA ligase III, in complex with XRCC1, plays a fundamental role in sealing the repaired DNA ends. Unlike c-NHEJ, which relies on DNA ligase IV and the Ku70/Ku80 heterodimer, TMEJ depends on ligase III. Research in Molecular Cell has shown that XRCC1 stabilizes ligase III at repair sites, enhancing its ability to catalyze the final ligation step. The absence of XRCC1 significantly reduces TMEJ efficiency, leading to increased genomic instability.

Replication protein A (RPA) also regulates DNA end processing. While traditionally associated with homologous recombination, recent findings in Cell Reports suggest that RPA transiently binds to single-stranded DNA at DSBs before Polθ engagement, preventing excessive degradation and preserving microhomology regions. Additionally, structural studies indicate that Polθ interacts with RAD51, a recombinase typically involved in HR, though in TMEJ, this interaction inhibits RAD51 filament formation, favoring the error-prone repair pathway.

Coordination With Poly(ADP) Ribose Polymerases

Poly(ADP) ribose polymerases (PARPs) facilitate TMEJ by modulating the early stages of DNA damage recognition and repair. Among the PARP family, PARP1 and PARP2 are particularly relevant due to their ability to detect single-strand breaks and convert them into signals that recruit repair factors. These enzymes catalyze the addition of poly(ADP-ribose) (PAR) chains onto target proteins, altering chromatin structure and enhancing repair site accessibility. Experimental evidence from Molecular Cell has shown that PARP inhibition reduces TMEJ efficiency, suggesting a functional interaction between these enzymes and Polθ.

PARP1 also regulates DNA end processing, ensuring that single-stranded DNA overhangs form in a manner conducive to microhomology-mediated repair. By modifying chromatin-associated proteins such as histones and DNA-binding factors, PARP1 creates a repair-permissive environment that facilitates Polθ engagement. This coordination is especially relevant in tumor cells with HR deficiencies, where TMEJ serves as a compensatory pathway. Studies demonstrate that cancer cells with BRCA1 or BRCA2 mutations rely on both PARP1 and Polθ, making them particularly sensitive to PARP inhibitors.

Role In Genome Integrity

Cells must constantly defend their genetic material from damage, as even a single double-strand break can threaten stability. TMEJ plays a compensatory role in preserving genome integrity, particularly in cells with homologous recombination deficiencies, such as those with BRCA1 or BRCA2 mutations. While this mechanism enables continued survival, it introduces small insertions and deletions, increasing mutational burden over time.

The error-prone nature of TMEJ can lead to genomic instability, yet its function remains indispensable under certain conditions. In rapidly dividing cells, such as those in embryonic development or highly proliferative tissues, the ability to repair DSBs quickly may outweigh the risks of imperfect repair. Additionally, in stress conditions like replication fork collapse, where stalled replication machinery results in DNA breaks, TMEJ provides a rapid, albeit imprecise, means of restoring continuity. While preventing immediate genomic catastrophe, its long-term consequences contribute to structural variations that can drive evolutionary changes and disease progression.

Associations With Disease Mechanisms

The mutagenic nature of TMEJ has significant implications for disease development, particularly in cancer. While this pathway serves as a backup repair mechanism, its propensity to introduce indels contributes to genomic instability. Tumors with homologous recombination deficiencies, such as BRCA1/2-mutant breast and ovarian cancers, often rely heavily on TMEJ for survival. Studies have shown that POLQ inhibition sensitizes HR-deficient cancer cells to DNA damage, highlighting its potential as a therapeutic target. Beyond BRCA-associated malignancies, an overactive TMEJ pathway has been observed in glioblastomas and lung cancers, where it enables rapid adaptation to genotoxic stress.

Beyond cancer, TMEJ has been implicated in neurodegenerative disorders linked to defective DNA repair. Conditions such as ataxia-telangiectasia and some forms of amyotrophic lateral sclerosis (ALS) involve mutations in genes that regulate DNA damage responses. When classical repair pathways are impaired, TMEJ may take over, leading to an accumulation of mutations that accelerate disease progression. Additionally, its error-prone nature has been suggested as a factor in age-related genomic deterioration, where increased reliance on mutagenic repair mechanisms correlates with cellular senescence and decline. These findings underscore the broader impact of TMEJ, illustrating its role in both adaptive survival mechanisms and pathological mutation accumulation.