Four germline POLH variants, including two found in skin tumors, impair DNA polymerase η function and cellular tolerance to UV radiation and cisplatin.

DNA polymerase (pol) η is vital for accurately replicating DNA opposite ultraviolet light (UV)-induced cyclobutane pyrimidine dimers and cisplatin-induced intrastrand purine crosslinks. While human POLH deficiency is linked to the disease xeroderma pigmentosum variant, the functional consequences of germline and somatic POLH variants remain largely unexplored. We characterized nine nonsynonymous POLH germline variants, five of which have also been found in various tumors. Enzyme activity was first assessed using recombinant pol η (residues 1-432) proteins. Variants F17S, C227Y, and R356X displayed substantially reduced or nearly abolished polymerase activity opposite cis-syn cyclobutane thymine dimer (CTD) compared to the wild-type. Cellular effects were then evaluated in POLH-deficient human embryonic kidney (HEK) 293 cells. Unlike cells transfected with wild-type POLH, cells transfected with F17S, R81C, C227Y, or R356X variants failed to rescue UV- and cisplatin-sensitivity. Interestingly, the R81C variant protein was undetectable in transfected cells. Further steady-state kinetic analysis revealed that the F17S, C227Y, and R356X variants had 3- to 5000-fold reductions in k/K values for correct dATP insertion opposite CTD, while the R81C variant exhibited kinetics comparable to the wild-type enzyme. CRISPR/Cas9-mediated knock-in of the R81C variant in HEK 293T cells was associated with significantly impaired pol η protein expression and increased cisplatin sensitivity. Notably, R81C and R356X mutations have been reported in skin cancer samples. These findings suggest that R81C, F17S, C227Y, and R356X POLH variants-underexpressed or hypoactive-may be insufficient to protect cells from UV radiation and cisplatin, highlighting their potential implications for individual susceptibility to UV and cisplatin damage.