DNA damage, including double-strand breaks (DSBs), must be efficiently repaired to preserve genomic stability and prevent the development of cancer. The Family A DNA Polymerase Theta (Pol Theta, encoded by POLQ) is a replicative and repair enzyme that facilitates repair of DSBs through an error-prone alternate pathway known as microhomology-mediated end joining (MMEJ). We have identified an acral melanoma tumor sample from a patient in the Yale SPORE database with the Y239C missense mutation. Predictive software SIFT and PolyPhen-2 algorithms predict this substitution to be damaging to the function of the enzyme. Previous studies have explored truncated Pol Theta function through E.coli expression studies but have missed the coordinated function between the complex domains including the helicase, polymerase and disordered. Therefore, we will express and purify full-length in eukaryotic insect cells. We hypothesize that the Y239C variation located within the helicase domain of Pol Theta may affect the helicase structure, the MMEJ activity, and/or the helicase activity, such as its role in strand annealing of DNA templates for subsequent processing by the polymerase domain of Pol Theta. According to Alphafold predictions, there is a disruption in the alpha-helices conformation in the helicase domain. Using biochemical methods, we will characterize the functional effects of the full-length Pol Theta Y239C mutation including the ability of the variant to bind DNA, perform MMEJ, and its fidelity of function. If our hypothesis is correct, this change in amino acids may promote genomic instability, support cancer progression, and contribute to resistance to cancer treatment.

Pol Theta (POLQ) plays a crucial role in DNA repair and is an error-prone enzyme, resulting in about 2.4 incorrect nucleotide insertions per 1000 bases. Its error-prone nature results in mutations that potentially cause genomic instability. In past studies, upregulation of Pol Theta has been found to be connected to breast cancer with poor clinical outcomes in patients. It has also been demonstrated that patients deficient in tumor suppressor genes such as BRCA experience increased genomic instability due to inefficient Pol Theta repair, which becomes the dominant pathway for double-strand breaks. To better understand the mechanistic link between Pol Theta-mediated repair and cancer, we’ve identified a POLQ variant from the cBIO portal in a patient diagnosed with stage II invasive breast carcinoma that metastasized to the bones and chest walls despite previous cancer treatment. Although the patient did not have a BRCA mutation, a POLQ mutation, E2370K, was identified. We hypothesize that the E2370K mutation may further reduce DNA repair, increasing genomic instability and cancer progression in this patient. E2370K was expressed and purified from E. coli to assess its DNA repair activity, including its ability to bypass 8-oxo-G and Cisplatin damage. A POLQ-knockdown MCF-7 breast cancer cell line was generated via CRISPR to introduce that mutation and evaluate repair with Cisplatin. Preliminary results show Pol Theta E2370K can bypass damaged DNA despite common breast cancer therapy, indicating that mutated patients might develop chemoresistance and worse clinical outcomes.