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. 2014 Mar 24;9(3):e92936.
doi: 10.1371/journal.pone.0092936. eCollection 2014.

Rad4 mainly functions in Chk1-mediated DNA damage checkpoint pathway as a scaffold protein in the fission yeast Schizosaccharomyces pombe

Ming Yue  1 , Li Zeng  2 , Amanpreet Singh  1 , Yongjie Xu  1
Affiliations

Rad4 mainly functions in Chk1-mediated DNA damage checkpoint pathway as a scaffold protein in the fission yeast Schizosaccharomyces pombe

Ming Yue et al. PLoS One. .

Erratum in

Abstract

Rad4/Cut5 is a scaffold protein in the Chk1-mediated DNA damage checkpoint in S. pombe. However, whether it contains a robust ATR-activation domain (AAD) required for checkpoint signaling like its orthologs TopBP1 in humans and Dpb11 in budding yeast has been incompletely clear. To identify the putative AAD in Rad4, we carried out an extensive genetic screen looking for novel mutants with an enhanced sensitivity to replication stress or DNA damage in which the function of the AAD can be eliminated by the mutations. Two new mutations near the N-terminus were identified that caused significantly higher sensitivities to DNA damage or chronic replication stress than all previously reported mutants, suggesting that most of the checkpoint function of the protein is eliminated. However, these mutations did not affect the activation of Rad3 (ATR in humans) yet eliminated the scaffolding function of the protein required for the activation of Chk1. Several mutations were also identified in or near the recently reported AAD in the C-terminus of Rad4. However, all mutations in the C-terminus only slightly sensitized the cells to DNA damage. Interestingly, a mutant lacking the whole C-terminus was found resistant to DNA damage and replication stress almost like the wild type cells. Consistent with the resistance, all known Rad3 dependent phosphorylations of checkpoint proteins remained intact in the C-terminal deletion mutant, indicating that unlike that in Dpb11, the C-terminus of Rad4 does not contain a robust AAD. These results, together with those from the biochemical studies, show that Rad4 mainly functions as a scaffold protein in the Chk1, not the Cds1(CHK2 in humans), checkpoint pathway. It plays a minor role or is functionally redundant with an unknown factor in Rad3 activation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Genetic screen of novel rad4 mutants with enhanced sensitivities to HU and MMS in the rad4+ shut-off strain.
A, thiamine-controlled cell growth of the shut-off strain (nmt-rad4) in which the endogenous promoter of rad4+ was replaced with a thiamine repressive nmt81 promoter. Logarithmically growing cells were diluted in fivefold steps and spotted on EMM6S plates with (+) or without (-) thiamine. The plates were incubated at 30°C for 3 days before being photographed. B, thiamine-regulated expression of Rad4 was examined by Western blotting. Untagged Rad4, Rad4 with the deletion of whole C-terminus (ΔC) and HA-tagged Rad4 were expressed on a vector in the shut-off strain. Expression of Rad4 in the presence (+) or absence (-) of thiamine was examined by using anti-Rad4 antibodies (Top). The same membrane was stripped and blotted with anti-HA antibody (bottom). Asterisks indicate the cross-reacting materials. C, molecular architecture of Rad4 with relative locations of the newly isolated (solid circles) and previously reported (open circles) mutations and the AAD domain. The four BRCT repeats are marked by roman numerals. The regions covered by mutational PCRs in the genetic screening are also shown. D, sensitivities of the rad4 mutants to HU and MMS were assessed by spot assay in the shut-off strain. The newly screened (top part) and the previously reported (lower part) mutations are marked on the right. Wild type cells, Δrad3 mutant, and the shut-off strain carrying an empty vector or the vector expressing wild-type Rad4 were used as controls.
Figure 2
Figure 2. Drug sensitivities of S. pombe with the integrated rad4 mutations.
A, the two-step marker switching method used for integration of the mutations. The genomic locus of rad4+ is shown on top. B, sensitivities of the cells with integrated rad4 mutations to HU or MMS were examined by spot assay. The newly identified (upper part) and the previously reported (lower part) mutants are marked on the right. C, expression of Rad4 in the cells with the integrated rad4 mutations was detected in whole cell lysates by Western blotting using anti-Rad4 antibodies. The asterisk denotes the cross-reactive material. Note: like the T45M mutant, A60P is a ts mutant.
Figure 3
Figure 3. Rad4 mainly functions in Chk1-mediated DNA damage checkpoint pathway.
A, wild type cells or cells with the integrated mutations were treated with (+) or without (−) MMS at 30°C for 1 hour. Phosphorylation of Chk1 was monitored by the mobility shift assay as described in the Materials and Methods. A section of the Ponceau S stained membrane is shown for the loading (bottom). B, Rad3-dependent phosphorylation of Cds1-Thr11 was examined by phosphor-specific antibody (top panel). Loading of Cds1 is shown in the lower panel. C, sensitivity of the cells to acute HU (left) or MMS (right) treatment. Cells were treated with the drugs in YE6S medium at 30°C. At each time point, an aliquot of the culture was removed and spread on YE6S plates for the cells to recover. Colonies were counted and viability was presented as percentages of the untreated cells. Each data point represents an average of three independent experiments for each mutant. D, synthetic effects of the double mutants containing Δcds1 or Δchk1 with the indicated rad4 mutations were examined by spot assay.
Figure 4
Figure 4. The C13Y and K56R mutations abolish the scaffolding function of Rad4 in vitro.
A, schematic diagram of the Rad4 fragments used in this experiment. Each fragment was fused with a GST tag for protein purification and detection in the binding assay using anti-GST antibodies. B, preferential binding of the N- and C-terminal pair of BRCT repeats of Rad4 to Crb2 and Rad9, respectively. Recombinant GST or GST-tagged Rad4 fragments were incubated with immunopurified Crb2 (middle panels) or Rad9 (right panels) bound to the anti-HA antibody beads. The bound proteins were released from the beads in gel loading buffer and analyzed by Western blotting. The membrane was striped and reprobed with anti-HA antibody for Crb2 and Rad9 (bottom panels). Aliquots of the binding reaction were analyzed separately by SDS PAGE as the input (left panel). C, equal amount of the Rad4 fragment b with or without the C13Y-K56R mutation was incubated with Crb2 bound on beads. The input and the Rad4 protein bound to Crb2 were analyzed as in B. D, fragment e with or without the E368K mutation was similarly tested for binding to phosphorylated Rad9. Phosphorylation of Rad9-Thr412 was detected by blotting of the same membrane with the phospho-specific antibody.
Figure 5
Figure 5. The C13Y and K56R mutations abolish the scaffolding function of Rad4 in vivo.
A, Rad9 was IPed from the lysates of MMS-treated cells with the indicated mutation (bottom panel). Wild type or mutant Rad4 (top two panels) Co-IPed with Rad9 was detected by Western blotting using anti-Rad4 antibodies. Phosphorylated Rad9-Thr412 was shown in the third panel from the top. B, Co-IP of Rad4 containing the indicated mutations with phosphorylated Rad9 from HU-treated cells. C, Co-IP of Rad4 and Rad9 with Crb2 from MMS-treated cells.
Figure 6
Figure 6. In vitro kinase assay using purified Rad3-Rad26 and Cds1(D312E) as the substrate.
A, time course of the in vitro kinase reaction. Rad3-Rad26 was affinity-purified from S. pombe using anti-Flag antibody resin, eluted with Flag peptide, and incubated with the kinase-inactive Cds1(D312E) as the substrate in the standard kinase buffer containing 200 μM ATP. At each time point, a small aliquot of the reaction was taken out and analyzed by SDS PAGE followed by Western blotting to examine the phosphorylation of Cds1-Thr11. Rad3 and Rad26 in the same samples were detected by using anti-HA and anti-flag antibody, respectively. Loading of the substrate was shown by Ponceau S staining. The kinase dead Rad3(D2249E)-Rad26 was similarly prepared and used as the control (last lane on the right). B, phosphorylation of Cds1-Thr11 was examined in the presence of increasing concentrations of Rad3-Rad26. The reaction was carried out at 30°C for 30 min. C, full-length Rad4 was purified from S. pombe. Removal of the N-terminal GST tag was analyzed by SDS PAGE (left) and confirmed by Western blotting (right) using anti-Rad4 antibodies. Asterisks indicate the degradation products of Rad4. D, the Rad3 kinase reactions were carried out at 30°C for 20 min in the absence or presence of increasing amount of purified Rad4 and analyzed as in A.
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This work was supported in part by the Emerging Science Seed Grant from Wright State University Boonshoft School of Medicine and a grant from Ohio Cancer Research Associates, Inc. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.