Rad4TopBP1 associates with Srr2, an Spc1 MAPK-regulated protein, in response to environmental stress.

Rad4(TopBP1) is a scaffold in a protein complex containing both replication proteins and checkpoint proteins and plays essential roles in both replication and checkpoint responses. We have previously identified four novel fission yeast mutants of rad4+(TopBP1) to explore how Rad4(TopBP1), a single protein, can play multiple roles in genomic integrity maintenance. Among the four novel mutants, rad4-c17(TopBP1) is a thermosensitive mutant. Here, we characterized rad4-c17(TopBP1) and identified a rad4-c17(TopBP1) allele specific suppressor named srr2+ (suppressor of Rad4(TopBP1) R2 domain). srr2+ has previously been identified as an environmental stress-responsive gene (GenBank accession number AL049644.1, locus spcc191.01). srr2+ null cells are sensitive to hydroxyurea (HU) at elevated temperatures. Deletion of srr2+ in rad4-c17(TopBP1) exacerbates the HU sensitivity of the mutant. Overexpression of srr2+ suppresses the rad4-c17(TopBP1) mutant sensitivity to temperature and HU and restores the compromised ability of rad4-c17(TopBP1) to activating Cds1 kinase in response to HU treatment. Furthermore, stress-activated MAPK, Spc1 (also known as StyI or Phh1), induces the expression and phosphorylation of the Srr2 protein. Significantly, environmental stress induces co-precipitation of Srr2 protein with Rad4(TopBP1), and the co-precipitation is compromised in the rad4-c17(TopBP1) mutant. These results have led us to propose a model; Rad4(TopBP1) exists in a large protein complex to coordinate genomic perturbations with checkpoint responses to maintain genomic integrity. In addition, when cells experience environmental stress, Rad4(TopBP1) associates with Srr2, an Spc1 MAPK-responsive protein, to survive the stress, potentially by providing a link of the Spc1 MAPK response to checkpoint responses.