Supplementary
Experimental Procedures

Drug Sensitivity and Cytotoxicity

Acute HU treatment was performed as reported (Enoch et al. 1992). To examine the cytotoxicity of Cds1 expressed in E. coli, the MBP tag in the pMAL-c2x vector (NEB) was replaced withthe cds1+ gene. E. coli harboring the resulting construct was inoculated onto plates containing IPTG.

Western and Far-Western

The HA, myc, and FLAG epitope tagged proteins were detected by Western blotting using monoclonal antibodies 12CA5 (Boehringer Mannheim), 9E10 (Dako cytomation), and anti-FLAG M2 (Stratagene), respectively. Polyclonal antibody against Mrc1 was generated in rabbits (Covance) using bacterially purified 6his-Mrc1 as the antigen and purified by affinity chromatography. The Cds1-T11 phospho-specific antibody was generated in rabbits using the peptide biotin-EEPEEATQATPQEAPLHVSQ (residues 2-20 of Cds1) as the antigen (Covance). The same peptide was used for affinity purification.

For the Far-Western analysis, Mrc1 was tagged at the C-terminus with 6hisHA. About 6  108 cells were harvested and broken in a mini-bead beater in 6 M GnHCl buffered by Tris:phosphate at pH 8.0. After centrifugation at 14,000 rpm for 5 min at 4°C, Mrc1 in the extracts was bound to 15µl TALON metal affinity resin (BD Biosciences). The resin was washed once with 8 M urea and once with 4 M urea. The bound Mrc1 was recovered by boiling the resin in 5x SDS gel loading buffer, resolved in a 7% SDS PAGE and transferred to a nitrocellulose membrane. After blocking the membrane with 5% dry milk in TBS-T [20 mM Tris:HCl (pH 7.6), 0.8% NaCl, 0.1% Tween-20], 0.5 µM GST-FHA was added and incubated with continuous shaking at 4˚C for 12 h. The membrane was washed three times in TBS-T and the bound GST-FHA was detected by anti-GST-HRP conjugate (Amersham).

BIAcore Peptide Binding Assay

The following phosphorylated and nonphosphorylated peptides containing a biotin at the N-terminus were synthesized by the Microchemistry Core Facility at the Sloan Kettering Institute and purified by reverse phase HPLC. Cds1 T11 peptide (EEPEEATQATpQEAPLHVSQ, Cds1 residues 2-20), Mrc1 T653 peptide (TQLDSTIPTpQIDSVQ, Mrc1 residues 645-659), Mrc1 S604 peptide (SLYVQNSQPSASpQLTIVDAT, Mrc1 residues 593-612) and Mrc1 TQ repeats-P (QVDSLVPTQLDSTIPTpQIDSVQ, Mrc1 residues 638-659) and Mrc1-repeats-2P (QVDSLVPTpQLDSTIPTpQIDSVQ, Mrc1 residues 638-659). Concentrations were verified by UV spectrometry (Waddell 1956). Peptides were bound to the surface of a streptoavidin-coated SA sensor chip in the BIAcore 2000 at ~10 RUs. A series of dilutions of purified GST-FHA in HBS-EP buffer [10 mM HEPES (pH 7.6), 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20] were allowed to flow over the peptide coated surface for 3 min. Binding of GST-FHA was measured by SPR. The surface of the chip was regenerated between injections by a pulse injection of 3.8 M MgCl2 followed by extensive washing with the HBS-EP buffer.

Purification of Cds1

To purify activated Cds1 from S. pombe, Cds1 tagged at the C-terminus with 6hisHA was expressed from a plasmid under the control of the cds1+ promotor. Two liters of cells were treated with 25 mM HU at 30˚C for 3 h, harvested by centrifugation, and resuspended in an equal volume (v/w) of 2xlysis buffer [50 mM Tris:HCl (pH 7.6), 150 mM NaCl, 2 mM EDTA, 1 mM Na3VO4, 10 mM pyrophosphate, 50 mM NaF, 60 mM ß-glycerophosphate, 0.1% NP-40, and proteinase inhibitors]. The cell suspension was frozen in liquid nitrogen and ground with dry ice in a coffee mill. The resulting lysate was clarified by centrifugation for 20 min at 4˚C, 43,000x g and loaded onto a 10 ml econo-column (Biorad) containing 500 µl anti-HA antibody beads. The column was incubated at 4˚C for 2 h, washed 5 times with wash buffer [25 mM Tris:HCl (pH 7.6), 250 mM NaCl] containing 0.1% Tween-20 and eluted in elution buffer [20 mM Tris:HCl (pH 7.5), 150 mM KCl, 0.5 mM DTT, 10% glycerol] containing 1 mg/ml HA peptide.

Inactive Cds1 tagged at the C-terminus with 6his3myc was purified from ∆rad3∆tel1, ∆rad3∆mrc1, or ∆rad3∆cds1S. pombe as indicated. Cell extracts were prepared as described above except that EDTA was omitted from the lysis buffer. Extracts were added to 1 ml TALON resin and incubated at 4˚C for 1 h. After washing five times with wash buffer containing 0.1% NP-40 and 10 mM imidazole, Cds1 was eluted in elution buffer containing 150 mM imidazole.

Kinase dead Cds1(D312E) tagged at the N-terminus with MBP and at the C-terminus with 6his was expressed in E. coli and purified with maltose resin (NEB) following manufacture’s instruction. After cleavage with factor Xa (NEB), Cds1(D312E) was further purified with TALON resin. FKBP-Cds1cat containing Cds1 kinase domain (150-460aa, Cds1cat) fused to FKBP (ARIAD Pharmaceuticals, Inc.) was expressed using the pET28b vector (Amersham Biosciences). The FKBP-Cds1cat contains 6his at the N-terminus and a FLAG tag at the C-terminus for protein purification and Western analysis. A point mutation M159I was introduced to prevent expression of a truncated protein resulting from internal initiation. Bacteria containing the fusion construct in pET28b were grown 20˚C and incubated with 0.4 mM IPTG at 20˚C for 30 min. The FKBP-Cds1cat protein was purified on TALON resin. An aliquot (0.52 mg) was treated with 6000 units of -phosphatase (NEB) in 300 µl of 1x phosphatase buffer (NEB) at 30˚C for 40 min to remove residual phosphoryl groups. The dephosphorylated protein was purified again on TALON resin.

Figure legends for supplemental figures

Figure S1. Mrc1 is regulated by Cdc10.

(A)Expression of mRNAs corresponding to S. pombe genes SPAC694.06C and SPAP14E8.02 is dependent upon Cdc10. Cdc10 is a component of the transcription factor MBF (MluI-binding factor) and is necessary for the G1-S phase transition in fission yeast (Lowndes et al. 1992). MBF binds to a cis-acting promotor element ACGCGT known as the MCB (MluI cell-cycle box). We carried out a genome-wide search for genes with MCBs immediately upstream of coding sequences. Our search uncovered 25 such genes of which 13 were genes of unknown function. By comparison of mRNA levels at the permissive and non-permissive temperatures in Cdc10ts cells, we observed that the expression of two such genes, SPAC694.06C and SPAP14E8.02, were dependent upon Cdc10. Figure S1A shows the relevant data. An S. pombecdc10ts strain was incubated at 25˚C (left lane) or 36.5˚C for 4 h (right lane), after which mRNA was prepared and fractionated by gel electrophoresis. The mRNAs corresponding to two candidate genes (SPAC694.06C and SPAP14E8.02) identified in a preliminary screen by real-time PCR (data not shown) were detected by Northern blotting and quantified in a PhosphorImager. Data were normalized using actin mRNA as the loading control. Similar Northern blots for cdc18, a known Cdc10-regulated gene, are shown on the left. The sizes of the ORFs of SPAC694.06C and SPAP14E8.02 and the relative mRNA levels (in percentages of the mRNA levels in permissive temperature) are shown on the bottom. We observed that both SPAC694.06C and SPAP14E8.02 are not essential for normal cell cycle but SPAC694.06C was required for DNA replication checkpoint (data not shown). SPAC694.06C was subsequently named mrc1+ by other groups (Alcasabas et al. 2001; Tanaka and Russell 2001).

(B)Expression of Mrc1 and SPAP14E8.02 mRNAs peaks in G1/S. An S. pombecdc25ts strain was blocked in G2 phase by incubation at 36.5˚C for 4 h and then released into the cell cycle at 25˚C. Total RNA was prepared from samples collected at 20 min intervals and fractionated by electrophoresis in a 1% agarose gel. Mrc1 and SPAP14E8.02 mRNA were detected by Northern blotting (lower panel). Cell cycle progression was monitored by counting septated cells (top panel).

(C)Expression of Mrc1 and SPAP14E8.02 proteins peaks in G1/S. Mrc1 and SPAP14E8.02 were tagged with 3HA at their C-termini in a cdc10ts strain. Cells were blocked in G1 by incubation at 36.5°C and released into the cell cycle at 25˚C. Aliquots were removed at 20 min intervals for analysis by flow cytometry (top panel) and Western blotting with anti-HA antibody (lower panel). The two bars on the left of the flow cytometry data indicate two sequential S phases.

Figure S2. Mrc1 has additional roles in survival of DNA damage and acute replication blocks.

(A)Sensitivity of Mrc1 mutants to acute treatment with HU. The data presented in the body of the paper indicate that the role of Mrc1 in mediating survival of chronic HU treatment is channeled through phosphorylation of T645 or T653. In particular, the sensitivity of S. pombe to chronic HU exposure is similar in the mrc1 and the T645A-T653A mutants. To determine whether the same is true during acute exposure to HU, exponentially growing S. pombe cells (2x106 cells/ml) were incubated at 30˚C in EMM6S medium containing 10 mM HU. At each time point, a 10 µl sample was diluted 1000 fold in sterile water, and 200 cells were spread onto EMM6S plates for 3 days at 30˚C. The survival curves indicate the percentage of surviving cells relative to the untreated sample (Enoch et al. 1992). The data indicate that the T645A-T653A mutant is quite sensitive to HU under these conditions, but not as sensitive as mrc1. As in the case of chronic exposure to HU, the sensitivity of a mutant lacking all of the sites in the Mrc1 SQ cluster was slightly less than that of the T645A-T653A mutant. These findings suggest that activation of Cds1 through phosphorylation of Mrc1 is not the only means by which Mrc1 protects cells from the effects of acute replication blocks..

(B)MMS Sensitivity. To examine the possible role of Mrc1 in the response to DNA damage, wild-type and mutant S. pombe cells, serially diluted at 1:5, were spotted on EMM6S plates or EMM6S plates containing 0.008% or 0.012% MMS and incubated at 30˚C for 3 days. The data indicate that the mrc1 mutant is sensitive to MMS, but elimination of either the Mrc1 TQ repeats or the Mrc1 SQ cluster has little effect on MMS sensitivity. Thus, Mrc1 plays a role in the DNA damage response that does not require the phosphorylation-dependent Cds1 activation described in the body of the paper. Elucidation of the role of Mrc1 in the DNA damage response will require additional studies.

Figure S3. Activation of Cds1 requires that all three functional domains reside in the same molecule. Two S. pombe expression vectors with LEU2 and ade6 markers, respectively, were used to co-express the indicated mutant Cds1 molecules under the control of the cds1+ promotor in ∆cds1 cells. The cells were serially diluted 1:5 and spotted on HU plates.

Figure S4. Overexpression of wild-type Cds1 in S. pombe induces cell cycle delay that is independent of Rad3 and Mrc1.

(A)Phenotype of cells overexpressing Cds1. Wild-type or mutant Cds1 was cloned into expression vectors containing the thiamine repressible nmt1+ promotor and introduced into ∆rad3∆mrc1 cells. After incubation for 20 h at 30˚C in the presence (top panel) or absence (lower panel) of thiamine (20 µg/ml) the cells were photographed under phase-contrast microscopy. Black bar: 40 µm.

(B)Cds1 kinase activity. Cultures the of S. pombe strains in (A) were incubated for 20 h, and Cds1 kinase activity was measured in the standard kinase assay using MyBP as substrate. The gel was analyzed by autoradiography (top panel) and Coomassie staining (bottom panel).

(C) Estimation of the Cds1 concentration in wild-type S. pombe. Protoplasts prepared from cells growing in log phase were lysed in SDS gel loading buffer and protein samples were resolved in a 10% SDS PAGE. The NB2118 strain was obtained from Dr. P. Russell and the YJ374 strain was made in this laboratory. After transferring proteins to a nitrocellulose membrane, Cds1 was detected by Western blotting. The bacterially purified kinase dead Cds1 mutant protein was separated in the same gel and used as the standards. Assuming 0.25 OD600 cells contain approximately 500 pg Cds1, the concentration of Cds1 inside a S. pombe cell is approximately 83 nM.

Figure S5. Activation of Cds1 kinase activity by induced dimerization of the catalytic domain requires ATP. The FKBP-Cds1cat fusion protein (50 nM) was first incubated with AP20187 at the optimal molar ratio for dimerization (0.63) in the presence (+ATP) or absence (-ATP) of 50 µM unlabeled ATP at 30˚C for 40 min. Samples were then split into two halves: 1 µM AP20187 (+) was added to one half in order to promote dissociation of dimers that had formed in the first incubation, and no additional AP20187 (-) was added to the second in order to maintain the preformed dimers. All samples were then incubated with MyBP and 50 µM [-32P]ATP at 30˚C for another 20 min under standard kinase assay conditions. Kinase activities after the 2nd addition of AP20187 are shown in percentages (bottom) as compared with that of the 1st step of the reactions.

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