Rad4TopBP1, a scaffold protein, plays separate roles in DNA damage and replication checkpoints and DNA replication

Rad4(TopBP1), a BRCT domain protein, is required for both DNA replication and checkpoint responses. Little is known about how the multiple roles of Rad4(TOPBP1) are coordinated in maintaining genome integrity. We show here that Rad4(TopIP1) of fission yeast physically interacts with the checkpoint sensor proteins, the replicative DNA polymerases, and a WD-repeat protein, Crb3. We identified four novel mutants to investigate how Rad4TopBP1 could have multiple roles in maintaining genomic integrity. A novel mutation in the third BRCT domain of rad4+(TopBP1) abolishes DNA damage checkpoint response, but not DNA replication, replication checkpoint, and cell cycle progression. This mutant protein is able to associate with all three replicative polymerases and checkpoint proteins Rad3(ATR)-Rad26(ATRIP), Hus1, Rad9, and Rad17 but has a compromised association with Crb3. Furthermore, the damaged-induced Rad9 phosphorylation is significantly reduced in this rad4(TopBP1) mutant. Genetic and biochemical analyses suggest that Crb3 has a role in the maintenance of DNA damage checkpoint and influences the Rad4(TopBP1) damage checkpoint function. Taken together, our data suggest that Rad4(TopBP1) provides a scaffold to a large complex containing checkpoint and replication proteins thereby separately enforcing checkpoint responses to DNA damage and replication perturbations during the cell cycle.