Abstract
The Swi1-Swi3 replication fork protection complex and Mrc1 protein are required for stabilization of stalled replication forks in fission yeast. Hsk1 kinase also plays roles in checkpoint responses elicited by arrested replication forks. We show that both Swi1 and Swi3, the abundance of which are interdependent, are required for chromatin association of Mrc1. Co-immunoprecipitation experiments show the interactions of Swi1-Swi3, Mrc1 and Hsk1. Mrc1 interacts with Swi3 and Hsk1 proteins through its central segment (378-879) containing a SQ/TQ cluster, and this segment is sufficient for checkpoint reaction. The SQ/TQ cluster segment (536-673) is essential but not sufficient for the interactions and for resistance to replication inhibitor hydroxyurea. Mrc1 protein level is increased in hsk1-89 cells due to apparent stabilization, and we have identified a potential phosphodegron sequence. These results suggest that interactions of the Swi1-Swi3 complex and Hsk1 kinase with Mrc1 may play a role in cellular responses to stalled replication forks in fission yeast.