The study of DNA polymerase θ (Pol θ) reveals important insights about its role as a low-fidelity enzyme responsible for DNA double-strand break repair and translesion synthesis, with significant consequences for genomic stability. Through advanced cryo-electron microscopy, researchers captured detailed structures of Pol θ interacting with multiple base pairs, laying bare the molecular strategies it employs to incorporate mismatched nucleotides during DNA synthesis.
Pol θ operates within the theta-mediated end joining (TMEJ) pathway, where it contributes to repairing DNA damage and circumventing certain obstacles during DNA replication. A central focus of this research is how Pol θ showcases the unusual capability of synthesizing DNA with higher error rates compared to more traditional polymerases, which are tasked with ensuring accuracy during replication. This proclivity for error incorporation, characterized by its ability to add incorrect nucleotides, is believed to play pivotal roles not only in DNA repair but also within the contexts of somatic hypermutation and evolutionary processes.
The study conducted by C. Li, L.M. Maksoud, and Y. Gao, published on March 1, 2025, details the process of elucidation through which Pol θ was examined. The researchers utilized cryo-electron microscopy to obtain high-resolution images of Pol θ bound to various nascent base pairs, including mismatched T:G and T:T pairs. Their observations were supported by extensive mutagenesis studies and fluorescence assays, which were utilized to offer additional clarity on Pol θ’s structural adaptations.
Central to the findings was the discovery of how Pol θ uniquely accommodates mismatched nucleotides within its active site. Unlike canonical high-fidelity polymerases, the finger domain of Pol θ remains closed when interacting with mismatched base pairs, facilitating the continued addition of nucleotides. This behavior stands out starkly against high-fidelity enzymes, which tend to reject mismatches by adopting alternate conformations. The authors wrote, "These observations highlight Pol θ’s distinct molecular mechanism in accommodating mismatches relative to high-fidelity polymerases within the A-family."
Key residues surrounding Pol θ's active site were identified to be instrumental to its ability to stabilize misincorporated base pairs. Unique hydrophilic residues such as Q2380 and Q2384 were shown to interact directly with nucleobases, reaffirming Pol θ's predisposition toward synthesis with reduced fidelity. Importantly, the authors highlighted, “Pol θ facilitates the looping-out of primer and template during mismatch extension, leading to insertions and deletions.” These findings elucidate the importance of specific structural features which may underlie the polymerase's tolerance for mismatches.
The significance of Pol θ becomes even more apparent when considering its therapeutic potential. Cancer cells deficient in standard DNA repair pathways, such as BRCA1 and BRCA2, may rely on Pol θ for survival, making it an attractive target for cancer therapies. Various small-molecule inhibitors, directed against either the helicase domain or the polymerase domain, have been developed to exploit this synthetic lethality phenomenon witnessed within related cancer types.
Reflecting on the broader scientific scope, the study emphasizes how Pol θ’s distinct mechanisms of error-prone synthesis serve not only as key insights for cancer treatments but also provide illuminating perspectives on the fundamental principles of DNA replication and repair. Ongoing investigations aim to explore how exact substrate contacts influence the enzyme’s behavior, particularly during instances of high-stakes DNA replication when errors could propagate mutations and facilitate various diseases.
Research efforts are likely to expand upon these foundational insights, decoding how the structure and biochemistry of Pol θ contribute to genome evolution and stability. With the increasing relevance of such findings to therapeutic strategies and our foundational knowledge of cellular mechanisms, the dialogue surrounding Pol θ is poised to be central to upcoming developments in molecular and cancer biology.