Requirements for the interaction of mouse Polκ with ubiquitin and its biological significance

Polκ protein is a eukaryotic member of the DinB/Polκ branch of the Y-family DNA polymerases, which are involved in the tolerance of DNA damage by replicative bypass. Despite universal conservation through evolution, the precise role(s) of Polκ in this process has remained unknown. Here we report that mouse Polκ can physically interact with ubiquitin by yeast two-hybrid screening, glutathione S-transferase pulldown, and immunoprecipitation methods. The association of Polκ with ubiquitin requires the ubiquitin-binding motifs located at the C terminus of Polκ. In addition, Polκ binds with monoubiquitinated proliferating cell nuclear antigen (PCNA) more robustly than with non-ubiquitinated PCNA. The ubiquitin-binding motifs mediate the enhanced association between monoubiquitinated PCNA and Polκ. The ubiquitin-binding motifs are also required for Polκ to form nuclear foci after UV radiation. However, the ubiquitin-binding motifs do not affect Polκ half-life. Finally, we have examined levels of Polκ expression following the exposure of mouse cells to benzo[a]pyrene-dihydrodiol epoxide or UVB radiation.