Protein Phosphatase 1 Recruitment by Rif1 Regulates DNA Replication Origin Firing by Counteracting DDK Activity

Summary The firing of eukaryotic origins of DNA replication requires CDK and DDK kinase activities. DDK, in particular, is involved in setting the temporal program of origin activation, a conserved feature of eukaryotes. Rif1, originally identified as a telomeric protein, was recently implicated in specifying replication timing in yeast and mammals. We show that this function of Rif1 depends on its interaction with PP1 phosphatases. Mutations of two PP1 docking motifs in Rif1 lead to early replication of telomeres in budding yeast and misregulation of origin firing in fission yeast. Several lines of evidence indicate that Rif1/PP1 counteract DDK activity on the replicative MCM helicase. Our data suggest that the PP1/Rif1 interaction is downregulated by the phosphorylation of Rif1, most likely by CDK/DDK. These findings elucidate the mechanism of action of Rif1 in the control of DNA replication and demonstrate a role of PP1 phosphatases in the regulation of origin firing.

Flag -+ -+ + + + + + + + + + + w-t Figure S1. Related to Figures 1 and 4. Expression levels of mutant Rif1 proteins.         A. Analysis of protein levels of budding yeast wild-type Rif1 and Rif1-PP1 (referring to Figure 1) tagged at their C-terminus with 1 copy of the Flag epitope, from exponentially growing cultures. The same gel was also blotted for Pgk1, as a loading control. C-terminally tagged versions of the protein product of the rif1-PP1 allele were found to be present at levels comparable to the wild-type tagged allele. B. Alignment of the relevant regions of Rif1 proteins from 5 Saccharomyces species (referring to Figure 4A). The RVxF and SILK type motifs are indicated in purple and green, respectively.
Putative DDK sites are indicated in orange: these can be 'intrinsic' (SE/SD/TE/TD), or 'phosphorylation generated' where the negative charge C-terminal to the serine or threonine is provided by a prior phosphorylation event (for example by CDK). Finally, putative CDK sites are indicated in blue. C.
Analysis of protein levels for the budding yeast Rif1 allele bearing changes to alanine at positions 116,118,147,148,. Although S. cerevisiae bears two additional SILK and RVxF type motifs further downstream from the first two (at positions 222 and 316), these putative motifs are embedded within the Ankyrin repeat region of the protein and are not well-conserved within the Saccharomyces genus. We found that the rif1-3xPP1 allele (bearing mutations in the GILR motif at position 222 in addition to those within the first two motifs) was expressed at lower levels, possibly indicative of folding problems. D. Analysis of the protein levels of S. cerevisiae Rif1-9D, bearing the changes indicated in Figure 4A. E. Analysis of protein levels of fission yeast wild-type Rif1 and Rif1-PP1 (referring to Figure 1) tagged at their C-terminus with 10 copies of the Myc epitope, from exponentially growing cultures. The same gel was also blotted for tubulin, as a loading control. Cterminally tagged versions of the protein product of the rif1-PP1 allele were found to be present at levels comparable to the wild-type tagged allele. F. Sequence alignment of the N-terminal region of the rif1 gene in four Schizosaccharomyces species. RVxF (purple) and SILK (green) motifs, and putative DDK (orange) and CDK (blue) sites as above are indicated, in addition to Mec1/Tel1 sites (grey). G. Analysis of the protein levels of S. pombe Rif1-12D, Rif1-7A, and Rif1-7APP1, bearing the changes indicated in Figure 4D. Replication intermediates were then separated on alkaline denaturing agarose gels and analyzed by southern blotting using probes for ARS305 (left panel) and ARS603 (right panel). Probes were generated by PCR using oligos DO1787/1788 and DO2498/2499 respectively. Analysis of temperature sensitive alleles of fission yeast cdc2 combined with RIF1 deletion. 10-fold serial dilutions of log-phase cultures of the indicated genotypes were spotted on rich medium and incubated for 3 days at temperatures ranging from 25°C to 36°C. D. FACS analysis of samples in Figure 3C. Samples were harvested from the same cell cultures analysed by western blotting in Figure 3C and fixed in 70% ethanol before being stained with Propidium iodide. The distribution of DNA content was then measured using FACS and the profiles aligned using Cell Quest software (Becton Dickinson).     In the strains bearing the Gal-HO cassette there is no HO site in the genome (the endogenous site at the MAT locus has been deleted) and therefore no HO cleavage is induced, and no effect on cell cycle progress is elicited.
pAB942 introduces an ADE2 marker and a stretch of telomeric repeats at the subtelomere of VII-L, with no effects on the other loci and telomeres analyzed. pAB704 (present in strains YAB1410 and YAB1706) is similar to pAB942 but carries an HO site: this plasmid will introduce a DSB at the ADH4 locus, with no consequences on cell cycle progression or replication timing of other loci, as the break is flanked by telomeric arrays.

Strains and plasmids
All budding yeast strains were generated in the W303 background . A complete list of the strains used is reported in Table S1.
Standard budding yeast handling and growth conditions were used. Rich medium was YPAD, and drop-out media were made using pre-made mixes from United States Biological.
Transformations were performed using a modified version of the protocol described by Bahler (Bahler et al., 1998): cells were incubated with plasmid DNA, carrier DNA and 40% PEG/LiAc/TE solution for 2 hrs at 30°C, before a 10 min heat shock at 42°C; transformations were subsequently plated out directly onto plates without centrifugation.
The hsk1-89 strain was gratefully received from Tony Carr.

Immunoprecipitation of ScRif1
Cultures of exponentially growing budding yeast cells (100 ml of 1 x 10 7 cells/ml) were lysed in 15 mM Hepes pH 7.6, 150 mM NaCl, 0.5 % NP-40 with three 20 s pulses in a Beadbeater. The lysate was clarified by centrifugation and the supernatant was incubated first with anti-myc 9E10 monoclonal antibody for 2 hours at 4°C and then with Protein G Dynabeads for 1 hour at 4°C. Bound proteins (eluted by boiling in Laemmli buffer) and input samples were separated on a 8% (top panel) and 10% (bottom panel) SDS gel and Western blotting was performed using anti-Flag (M2, Sigma) or anti-Myc antibody (homemade 9E10).

Immunoprecipitation of SpRif1
Whole cell extracts from 2x10 8 fission yeast were prepared by resuspending in 500 µl of chilled lysis buffer (50 mM NaCl, 50 mM Tris pH 7.5, 10% glycerol, 4 mM B-mercaptoethanol, 1 mM EDTA) containing 1 complete protease inhibitor tablet (Roche) per 7 ml of buffer. Following lysis in a beadbeater for 3 min, 1 µl Benzonase Nuclease (Novazyme) and 50 µl 10% NP40 were added to the lysate and incubated for 30 min on ice. After centrifugation for 10 min, 30 µl of the cleared lysate was boiled with 10 µl 4x Laemmli buffer and kept aside as Input. Protein G Dynabeads (Invitrogen) were blocked for 30 min in lysis buffer + 5% BSA. 25 µl of the beads were used to pre-clear the remaining lysate for 30 min at 4°C on a rotating wheel. 1 µl rabbit anti-GFP antibody (Invitrogen) was added to the supernatant obtained after magnetic separation of the beads. The samples were then incubated with rotation at 4°C for 1 hr. 25 µl of Dynabeads were added before further incubation at 4°C for 2 hrs.
Following magnetic separation and removal of the supernatant, the beads were resuspended in 40 µl 1x Laemmli buffer and boiled for 5 min.

Synchronization of budding yeast cultures
Budding yeast cells were grown in 100 ml overnight cultures in the appropriate drop-out SC medium containing 4% raffinose. The cultures were then diluted into 300 ml of YPA 4% raffinose and grown for 2 hours with 0.025 µM α-factor to arrest the cells in G1 phase of the cell cycle. Cells at a density of 1 x 10 7 cells/ml were then switched to YPA 4% galactose for 4 hours at 30°C, while maintaining the arrest with 0.025 µM α-factor. Cells were released into S-phase by washing twice with water and switching the cells to YPA+GAL containing 0.125 mg/ml pronase at 18°C.

Quantification of budding and fission yeast DNA
Fission yeast strains containing the cdc25-22 allele were grown to mid-log-phase at 25°C, arrested in G2 for 3h at 36°C and subsequently released into medium containing 25 mM HU at 25°C for 140 min. Samples were collected for G2-arrested cells and S-phase-arrested cells (140 min in HU) by cross-linking cells in 1% formaldehyde.