Dbf4‐dependent kinase and the Rtt107 scaffold promote Mus81‐Mms4 resolvase activation during mitosis

DNA repair by homologous recombination is under stringent cell cycle control. This includes the last step of the reaction, disentanglement of DNA joint molecules (JMs). Previous work has established that JM resolving nucleases are activated specifically at the onset of mitosis. In case of budding yeast Mus81‐Mms4, this cell cycle stage‐specific activation is known to depend on phosphorylation by CDK and Cdc5 kinases. Here, we show that a third cell cycle kinase, Cdc7‐Dbf4 (DDK), targets Mus81‐Mms4 in conjunction with Cdc5—both kinases bind to as well as phosphorylate Mus81‐Mms4 in an interdependent manner. Moreover, DDK‐mediated phosphorylation of Mms4 is strictly required for Mus81 activation in mitosis, establishing DDK as a novel regulator of homologous recombination. The scaffold protein Rtt107, which binds the Mus81‐Mms4 complex, interacts with Cdc7 and thereby targets DDK and Cdc5 to the complex enabling full Mus81 activation. Therefore, Mus81 activation in mitosis involves at least three cell cycle kinases, CDK, Cdc5 and DDK. Furthermore, tethering of the kinases in a stable complex with Mus81 is critical for efficient JM resolution. Synopsis DNA joint molecule resolution by the Mus81‐Mms4 nucleases is tightly controlled during mitosis. Dbf4‐dependent kinase (DDK), best known for its replication roles, now joins previously implicated cell cycle kinases CDK and Cdc5/Plk in this regulation. The budding yeast Cdc7‐Dbf4 kinase regulates the Mus81‐Mms4 resolvase in mitosis. DDK and Cdc5 form a complex that targets Mms4 and leads to mitotic Mus81 activation. Scaffold protein Rtt107 promotes DDK and Cdc5 activity towards Mus81‐Mms4 and is required for full Mus81 activation. DDK, Cdc5 and Rtt107 target Mms4 interdependently, suggesting that they form a signal amplification loop. Graphical Abstract Cdc7‐Dbf4 kinase joins CDK and Cdc5/Plk cell cycle kinases to exquisitely time resolution of DNA joint molecules before cell division.