Identification of Plk1 interacting proteins by XChIP-MS

Polo-like kinase 1 (Plk1) is an important protein kinase for checkpoint recovery and adaptation in response to DNA damage and replication stress. However, although Plk1 is present in S phase, little is known about its localization and function during unperturbed DNA replication. Using Xenopus laevis egg extracts, mimicking early embryonic replication, we developed a Plk1 chromatin immunoprecipitation approach followed by a proteomic shotgun analysis (XChIP-MS) to identify Plk1 interactions with chromatin and with specific nuclear proteins. We purified and cross-linked nuclei after pre-RC assembly before the start of DNA replication, or later during DNA replication. The DNA was fractionated by sonication and immunoprecipited using anti Plk1 or control antibodies then proteins were analyzed by nanoLC/MS/MS.