Mitotic Hela cells expressing GFP-Mitotic Hela cells expressing GFP-Polθ vs Mitotic Hela cells expressing GFP-Polθ treated with Volasertib vs Mitotic Hela cells expressing GFP-Polθ treated with Volasertib

We show that Polθ is recruited to mitotic Double-strand breaks (DSBs) to slow down cell cycle progression and allow DNA repair. Because Polθ is one of the only repair protein to forms repair foci during mitosis, we investigated its regulation during mitosis. We performed immunoprecipitation (IP) of Polθ and assessed phosphorylation by immunoblot analysis (using pan phospho antibodies). We observed a phosphorylation signal corresponding to the size of Polθ when IP was performed from mitotic cell extracts. This phosphorylation signal was abolished when cells where treated with two different PLK1 inhibitors (PLK1i), indicating that PLK1 is responsible for Polθ phosphorylation in mitosis. In order to elucidate the regulation of mitotic Polθ activity, we performed mass spectrometry (MS)-based phosphorylation analysis of Polθ in mitosis with or without the PLK1 inhibitor Volasertib. We found 5 phosphorylated residues. To assess the functional consequences of Polθ phosphorylation by PLK1, we mutated some of the identified residues and found that the phospho dead mutant of Polθ fails be recruited to DSBs in mitosis. This indicates that PLK1-mediated regulation of mitotic Polθ repair is essential for its proper functioning