Mcm10 interacts with Rad4/Cut5 TopBP1 and its association with origins of DNA replication is dependent on Rad4/Cut5 TopBP1

Abstract

Initiation of DNA replication in eukaryotes is a highly conserved and ordered process involving the co-ordinated, stepwise association of distinct proteins at multiple origins of replication throughout the genome. Here, taking Schizosaccharomyces pombe as a model, the role of Rad4 TopBP1 in the assembly of the replication complex has been examined. Quantitative chromatin immunoprecipitation experiments confirm that Rad4 TopBP1 associates with origins of DNA replication and, in addition, demonstrate that the protein is not present within the active replisome. A direct interaction between Rad4 TopBP1 and Mcm10 is shown and this is reflected in the Rad4 TopBP1-dependent origin association of Mcm10. Rad4 TopBP1 is also shown to interact with Sld2 and Sld3 and to be required for the stable origin association of these two proteins. Rad4 TopBP1 chromatin association at stalled replication forks was found to be dependent upon the checkpoint protein Rad9, which was not required for Rad4 TopBP1 origin association. Comparison of the levels of chromatin association at origins of replication and stalled replication forks and the differential requirement for Rad9 suggest functional differences for Rad4 TopBP1 at these distinct sites.