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

Pol kappa protein is a eukaryotic member of the DinB/Pol kappa 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 Pol kappa in this process has remained unknown. Here we report that mouse Pol kappa can physically interact with ubiquitin by yeast two-hybrid screening, glutathione S-transferase pulldown, and immuno-precipitation methods. The association of Pol kappa with ubiquitin requires the ubiquitin-binding motifs located at the C terminus of Pol kappa. In addition, Pol kappa 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 Pol kappa. The ubiquitin-binding motifs are also required for Pol kappa to form nuclear foci after UV radiation. However, the ubiquitin-binding motifs do not affect Pol kappa half-life. Finally, we have examined levels of Pol kappa expression following the exposure of mouse cells to benzo[a] pyrene-dihydrodiol epoxide or UVB radiation.