Polθ is a key mediator of the alt-EJ pathway, which serves as a backup mechanism for DSB repair when HRR or c-NHEJ is unavailable. While alt-EJ has been studied in general, its specific role in mitosis remains unclear. M-phase presents a unique challenge for DNA repair due to condensed chromatin, lack of homologous templates, and limited checkpoint activation. This thesis aims to define Polθ’s contribution to DSB repair across the cell cycle, with a special focus on its function during mitosis.

To achieve this, we established robust synchronization protocols for G1, S, G2, and M phases in U2OS and A549 cells, both in WT and POLQ^-/- backgrounds. IR induced DSBs were introduced at defined cell cycle stages, and repair efficiency was assessed using survival assays, immunofluorescence (γH2AX, 53BP1, RPA70), and PFGE. We further incorporated specific repair inhibitors (NU7441 for c-NHEJ and B02 for HRR) to evaluate the compensatory role of alt-EJ.

Our results show that Polθ functions as a flexible backup across interphase. In G1, Polθ deficiency alone had little effect, but when c-NHEJ was blocked, POLQ^-/- cells became highly sensitive to IR, indicating alt-EJ compensates for c-NHEJ loss. In S-phase, HRR normally dominates, but Polθ becomes essential when HRR is inhibited, displaying a synthetic lethal interaction. G2-phase results echoed this, with POLQ^-/- cells showing increased IR sensitivity, especially under dual inhibition.

Most notably, Polθ plays a frontline role in mitosis. POLQ^-/- mitotic cells were significantly more IR-sensitive than WT, even at low doses. c-NHEJ also contributes to mitosis, as shown by reduced WT survival under NU7441. However, dual perturbation of Polθ and c-NHEJ resulted in near-complete cell death, indicating these are the only active repair pathways during M-phase. PFGE confirmed that POLQ^-/- cells retained more unrepaired DSBs in mitosis, especially under c-NHEJ inhibition. These findings revise the prior assumption that mitotic cells are incapable of DSB repair.

Beyond repair, Polθ also affects cell cycle dynamics. POLQ^-/- cells exited mitosis prematurely after IR and entered G1 with unresolved damage. This failure to maintain a damage-induced mitotic arrest or activate a proper G1 checkpoint indicates that Polθ is required not only for repair but also for regulating cell cycle progression in response to damage.

In summary, Polθ functions as a versatile DNA repair factor: in interphase, it acts as a backup to c-NHEJ and HRR; in mitosis, it becomes essential for survival. Its activity supports both genome integrity and proper checkpoint engagement. These findings enhance our understanding of alt-EJ as a regulated, context-dependent repair process and establish Polθ as a central component of the DDR across the cell cycle.

Therapeutically, Polθ’s selective importance in repair-deficient cells makes it an attractive target for cancer treatment. Its upregulation in HRR deficient tumors and contribution to mitotic survival suggest that Polθ inhibitors could be combined with DNA damaging agents to achieve selective tumor cell killing, while sparing normal cells. This work lays the groundwork for further development of Polθ-targeted therapies and provides new insights into how DSB repair is orchestrated during the most vulnerable phase of the cell cycle.