Structure/function analysis of the essential protein Rad4TopBP1 in S. pombe

Rad4TopBP1, a multiple BRCT domain protein, is essential for initiation of replication and participates in checkpoint responses following genotoxic treatment during the S and G2 phases of the cell cycle. Rad4 interacts with several proteins that are involved in initiation of DNA replication (i.e. Sld2) or activation of Chk1 and/or Cds1 checkpoint kinases (i.e. Rad9, Crb2, and Rad3). However, it remained unclear how Rad4 and its homologues are regulated to coordinate these diverse functions. This PhD project presents a comprehensive structure/function analysis of the fission yeast Rad4TopBP1. In order to obtain separation of function mutants of Rad4, a hydroxylamine random mutagenesis genetic screen was performed. However, we were not able to separate a checkpoint activation function from replicative function. Rad4 being phosphorylated in a Cdc2-dependent manner, the role of Rad4 phosphorylation by Cdc2 was investigated. A mutant strain containing multiple mutations at Rad4 Cdc2 consensus phosphorylation sites does not exhibit significant sensitivity to DNA damage or HU. In addition, Rad4 Cdc2 phosphorylation sites do not play a role in DNA re-replication. There is no significant phenotypic effect observed after DNA damage in S. pombe strains expressing a Rad4 protein deleted for a putative domain (RXL motif) interaction with cyclin, or harboring mutations in putative sumoylation motifs, or C-terminus truncation. In higher eukaryotes, TopBP1 binds and activates the ATR-ATRIP complex via an ATR-activating domain (AAD) in order to activate a checkpoint function. We identified a potential AAD in C-terminal of Rad4 in S. pombe. I show that Rad4 physically associates with Rad3 in vitro in an AAD-dependent manner. S. pombe strains mutated in the AAD show a slight sensitivity to DNA damage and HU. The rad4 AAD mutants do not completely prevent Rad3-mediated G2/M checkpoint activation after DNA damage. The sensitivity in a rad4 AAD mutant increases when damage occurs in S-phase, when histone H2A phosphorylation is defective. I established an artificial checkpoint induction system in the absence of exogenous lesions by targeting checkpoint proteins onto chromatin in S. pombe. Interestingly, Rad4 AAD is essential for checkpoint activation in this system. Because this checkpoint activation is independent of ssDNA-RPA formation, the data suggest that the AAD plays a role in chromatin-mediated checkpoint maintenance/amplification. Altogether, this pathway seems to play an important role in S-phase when DSBs resection is limited.