RAD54 suppresses theta-mediated end joining in non-homologous end joining-compromised FaDu tumor cells

Cells maintain genomic integrity through multiple DNA repair pathways. DNA double strand breaks (DSBs) are particularly hazardous, leading to cell death or genomic instability if not properly repaired. Three major DSBs repair pathways exist: non-homologous end joining (NHEJ), homologous recombination (HR), and theta-mediated end joining (TMEJ). However, it is unclear whether tumor cells can efficiently engage all three pathways in response to ionizing radiation (IR). We investigated DSB repair in FaDu human head and neck squamous cell carcinoma cells by generating knockout cells deficient in NHEJ protein DNA Ligase 4 (LIG4), HR protein RAD54, or TMEJ protein Polymerase Theta (Pol Ɵ), respectively and assessed IR sensitivity and Cas9-induced mutational signatures. NHEJ-deficient cells displayed increased IR sensitivity and delayed 53BP1 foci resolution, revealing strong reliance on NHEJ for DSB repair. RAD54 deficient cells showed no increased IR sensitivity, but TMEJ-mediated mutations increased, especially when NHEJ was inactive. These results indicate that RAD54 channels repair toward high-fidelity HR while suppressing mutagenic TMEJ. This study underscores the critical role of NHEJ in DSB repair and provides insights into the compensatory use of mutagenic repair pathways in human tumor cells when HR is compromised.