Abstract
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.
Highlights
MARCH 23, 2007 VOLUME 282 NUMBER 12 response to DNA damage, fission yeast Rad3ATR phosphorylates the checkpoint clamp protein Rad9 at Thr412/Ser423
Our previous studies have shown that rad4-c17TopBP1 mutant exhibits reduced Cds1 kinase activity in response to HU treatment at the restrictive temperature of 36 °C when cultured in rich media but is fully proficient in activation of the Chk1 damage checkpoint at its permissive temperature of 30 °C in response to CPT treatment [1]
Rad4TopBP1 Associates with Srr2 Protein in Response to Environmental Stress, and the Association Is Compromised by the Mutation in rad4-c17TopBP1—Studies have shown that nucleotide depletion induced by HU treatment or heat shock is sensed by the Spc1 MAPK pathway [19]
Summary
MARCH 23, 2007 VOLUME 282 NUMBER 12 response to DNA damage, fission yeast Rad3ATR phosphorylates the checkpoint clamp protein Rad9 at Thr412/Ser423. Disruption of srr2ϩ in rad4-c17TopBP1 overtly exacerbated the sensitivity of the double mutant to HU at 32 °C, rad4-c17TopBP1—To investigate what factors can affect the indicating that Srr2 is required for maintaining the viability of thermosensitivity of rad4-c17TopBP1, we devised a screen to rad4-c17TopBP1 when cells experience HU treatment at the identify high copy number suppressors as described under semipermissive temperature (Fig. 3B, upper panel). The ability of restoring the Cds1 kinase activation in rad4-c17TopBP1 in minimal media at 30 °C suggests a role of Srr2 in stress response.
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