Auxin-inducible protein depletion system in fission yeast
© Kanke et al; licensee BioMed Central Ltd. 2011
Received: 9 October 2010
Accepted: 11 February 2011
Published: 11 February 2011
Inducible inactivation of a protein is a powerful approach for analysis of its function within cells. Fission yeast is a useful model for studying the fundamental mechanisms such as chromosome maintenance and cell cycle. However, previously published strategies for protein-depletion are successful only for some proteins in some specific conditions and still do not achieve efficient depletion to cause acute phenotypes such as immediate cell cycle arrest. The aim of this work was to construct a useful and powerful protein-depletion system in Shizosaccaromyces pombe.
We constructed an auxin-inducible degron (AID) system, which utilizes auxin-dependent poly-ubiquitination of Aux/IAA proteins by SCFTIR1 in plants, in fission yeast. Although expression of a plant F-box protein, TIR1, decreased Mcm4-aid, a component of the MCM complex essential for DNA replication tagged with Aux/IAA peptide, depletion did not result in an evident growth defect. We successfully improved degradation efficiency of Mcm4-aid by fusion of TIR1 with fission yeast Skp1, a conserved F-box-interacting component of SCF (i mproved-AID system; i-AID), and the cells showed severe defect in growth. The i-AID system induced degradation of Mcm4-aid in the chromatin-bound MCM complex as well as those in soluble fractions. The i-AID system in conjunction with transcription repression (off-AID system), we achieved more efficient depletion of other proteins including Pol1 and Cdc45, causing early S phase arrest.
Improvement of the AID system allowed us to construct conditional null mutants of S. pombe. We propose that the off-AID system is the powerful method for in vivo protein-depletion in fission yeast.
Schizosaccharomyces pombe is a widely used model organism for analysis of important cellular functions [1–3]. The use of conditional inactivation by mutations or depletion of proteins in vivo has been used successfully for analysis of gene functions. A conditional protein degradation system, so-called "degron", which depletes proteins from cells, is a powerful tool for analyzing the "null" phenotype of various genes. In budding yeast, a heat-inducible degron (ts-degron) system has been devised [4, 5] and used for studies of essential gene functions [6, 7]. In fission yeast, the ts- degron mutant of Bir1, a nuclear protein involved in mitotic segregation, has been reported to cause destruction of the protein, resulting in growth defects at restrictive temperature . For functional analysis, however, as this system is unable to deplete proteins sufficiently to arrest the cell cycle, it is often combined with ts- alleles of the genes of interest [9, 10].
Recently, an auxin-inducible degron (AID) system was developed for use in budding yeast and higher eukaryotic cells . This strategy involves a plant-specific mechanism that relies on response to the plant hormone auxin and a conserved poly-ubiquitination pathway involving the E3 ubiquitin ligase, SCF (S kp1, C ullin and F-box protein) complex [12, 13]. In plant cells, auxin binds to transport inhibitor response 1 (TIR1) protein [14, 15] and promotes binding of the SCFTIR1, a form of SCF containing TIR1, to Aux/IAA transcription repressors [16–18]. The Aux/IAA proteins are poly-ubiquitinated by the SCFTIR1 and then degraded by proteasomes . Except for the auxin-dependent recognition of Aux/IAA proteins by TIR1, components of the SCF and ubiquitin-proteasome pathway, especially F-box interacting protein skp1, are conserved among eukaryotes. This makes it possible to form the SCFTIR1 in non-plant cells by expression of TIR1 and to degrade Aux/IAA-tagged proteins depending on addition of auxin.
We have constructed an AID system for use in S. pombe by expressing TIR1. Although Mcm4 protein fused with Aux/IAA peptide (aid-tag) at the C-terminus (Mcm4-aid) was decreased upon addition of auxin, the strain did not show any obvious growth defect because of inefficient depletion. Here we describe an improved form of the AID system. Depletion of Mcm4-aid protein was greatly enhanced by expression of Skp1-TIR1, a fusion protein comprising plant TIR1 and fission yeast Skp1. This i-AID system (i mproved AID system) was applicable for depletion of other essential replication proteins including Orc2, Cdc45 and Pol1. Furthermore, in conjunction of the i-AID system with transcription repression (off-AID system), Cdc45- or Pol1-depleted cells showed severe replication defects resulting in cell cycle arrest. The off-AID system involving two-step gene modifications will be a powerful tool for analyzing the function of essential genes under null conditions in S. pombe.
Auxin-dependent degradation of Mcm4-aid in fission yeast
NLS and Skp1-fusion to AtTIR1 enhances the efficiency of the AID system
Optimizing the expression of Skp1-AtTIR1-NLS from the genomic locus
To examine how efficiently Mcm4-aid protein was degraded in the P adh15 -skp1-AtTIR1-NLS mcm4-aid strain, the amount of Mcm4-aid in cell extracts prepared from log-phase cells in the presence or absence of auxin was analyzed by immunoblotting. The amount of Mcm4-aid protein decreased rapidly within 1 h to below 12.5% of the original amount (Figure 3C and 3D). The decrease was detected as early as 20 min after addition of auxin (Figure 3E). Under such conditions, a sharp 1C DNA peak appeared and was still evident at 8 h after addition of auxin (Figure 3F). The cell number did not increase after one cell division (Figure 3G) and cells were arrested with elongated shape containing a single nucleus (data not shown). These results show that the improved AID (i-AID) system depleted Mcm4-aid protein to cause tight cell cycle arrest at early S phase.
Degradation of Mcm4-aid protein in the chromatin-bound MCM complex
If Mcm4-aid is degraded in the chromatin-associated MCM complex, the other subunits may not remain on the chromatin. To examine whether the other MCM subunits were released upon degradation of Mcm4-aid protein, we carried out a chromatin immnoprecipitation (ChIP) assay using anti-Mcm6 antibody. Two sets of primers amplifying the ars2004 locus, an efficient replication origin , and a non-ARS1 locus, 30 kb distant from the origin, were used for real-time PCR to measure the relative amounts of precipitated DNA (Figure 4C). In HU-arrested cells without auxin (HU2h, HU3h-A), Mcm6 was enriched at ars2004 in comparison with non-ARS1, while no significant localization was observed in G2-arrested cells (G2) (Figure 4C), as shown in a previous study . Upon addition of auxin, IP recovery of ars2004 was decreased markedly to a level similar to that of non-ARS1 (Figure 4C, HU3h+A). These results indicate that Mcm4 is required for maintenance of the other MCM subunits on the chromatin. From these results, we concluded that the i-AID system induced efficient degradation of Mcm4-aid in the chromatin-bound MCM complex.
As the MCM complex is essential for progression of the replication fork, dissociation of the MCM complex will arrest DNA replication. To confirm this, we used flow cytometry to analyze the recovery of DNA replication upon removal of HU (Figure 4D). In the absence of auxin, DNA replication readily resumed after removal of HU, as DNA the content increased from 1C to 2C in 0-90 min (Figure 4D, left panel). In contrast, the DNA content of cells treated with auxin remained at 1C even at 120 min after removal of HU (Figure 4D, right panel), indicating that depletion of Mcm4 prevented progression of the replication fork. This is consistent with the above conclusion that Mcm4 in the chromatin-bound MCM complex is efficiently depleted by the i-AID system.
Application of the i-AID system for depletion of other essential proteins in fission yeast
Auxin-induced growth defects in aid-tagged strains
catalytic subunit of Polα
second largest subunit of Polα
primase subunit of Polα
catalytic subunit of Polε
second largest subunit of Polε
catalytic subunit of DDK
Efficient depletion of replication proteins by the off-AID system causes tight cell-cycle arrest
We have developed a powerful protein depletion system, "off-AID", in fission yeast by combining transcription repression with auxin-dependent protein degradation. An initial attempt that involved expression of TIR1 in strain mcm4-aid showed only a marginal defect on cell growth in the presence of auxin. However, the i-AID system consisting of TIR1 fused with fission yeast Skp1 markedly increased the efficiency of Mcm4-aid protein degradation, causing severe replication defect and cell cycle arrest. This suggested that the interaction between plant TIR1 and fission yeast Skp1 might be rate-limiting for SCFTIR1 assembly. We also showed that the level of TIR1 expression is crucial for efficient depletion of Mcm4-aid protein as observed in plant cells [14, 25]. Expression of Skp1-TIR1-NLS from the adh15 promoter resulted in severely defective growth on an auxin plate, whereas a reduced cellular concentration of Skp1-TIR1-NLS expressed from the nmt41 promoter did not result in the same phenotype (Figure 3). We noticed that the NAA concentration is also important for the AID system in fission yeast. The mcm4-i-aid strain did not show growth defect at 0.1 mM NAA, whereas NAA concentration higher than 1 mM affected the growth of untagged strain (data not shown). Since auxin may act as a signaling molecule to promote morphogenesis in budding yeast , a similar pathway may present in fission yeast. The i-AID system decreased the level of Mcm4-aid protein to about 10% of the original amount at 1 h after auxin addition (Figure 3), suggesting the utility of this system for depletion of the protein within a short period during the cell cycle. Although the i-AID system resulted in retardation of DNA replication and cell growth for more than half (11 among 15) of essential factors tested, significant defect was not observed for the mcm10-i-aid, hsk1-i-aid, ssl3-i-aid and cia1-i-aid (Figure 5 and Table 1). For the latter cases, the amount of remaining tagged protein seems to be higher (20-30% of the native amount) than those showing the defect (Figure 5C and data not shown). Difference in degradation efficiency might be caused by the affinity of TIR1 with the target proteins or the efficiency of ubiquitination in different targets. In addition, whether or not depletion of a protein causes severe phenotype may depend on the amount of protein required for its function. However, for the proteins that were not efficiently depleted by the i-AID system, the off-AID system that combines transcription repression with the i-AID system would be advantageous. The off-AID system promoted a reduction of the Cdc45-aid and Pol1-aid proteins to almost undetectable levels, causing severe defects in DNA replication. In addition, essential proteins unrelated to replication such as Ssl3 that is involved in sister chromatid cohesion and Cia1/Asf1 required for histone deposition can be depleted by the off- AID system to cause severe growth defects (Table 1). Therefore, this approach appears to be widely applicable for in vivo protein depletion in fission yeast.
In comparison of the AID system with previously published strategies, the AID system has several advantages. Comparing the flow cytometry results by depletion of the same target protein Mcm4, mcm4-i-aid cells remained with a sharp 1C DNA peak as long as 8 hr, whereas DNA content significantly increased after 3 hr incubation in mcm4ts-td cells (Figure 3F and ), suggesting that the i-AID system achieved tight cell cycle arrest in early S phase. In contrast to ts-degron, which requires high-temperature shift or sometimes temperature-sensitive allele of the target gene, the AID system promotes the degradation by addition of a synthetic auxin to the culture. This allows use of the cdc25-22 temperature-sensitive mutation for cell cycle synchronization. Furthermore, the off-AID system causes more extensive depletion than the i-AID system alone (Figure 6 and data not shown). On the other hand, there are some disadvantages of the off-AID system. It requires two-step modifications of target genes; fusion of the aid-tag sequence at the C-terminus and replacement of the promoter with the nmt81 promoter. The N-terminally aid-tagged pol1-Noff-aid, cdc20-Noff-aid and mcm10-Noff- aid strains, which were constructed by one-step replacement of the N-terminus of the gene, did not exhibit significant defect in cell growth or DNA replication (data not shown). A limitation of the AID system we noticed is that the degradation efficiency decreases at high temperature (36°C). Although use of OsTIR1 instead of AtTIR1 have overcome this problem in budding yeast and DT40 AID systems , mcm4-i-aid strain expressing P adh15 -skp1-OsTIR1 in fission yeast did not improve degradation efficiency at 36°C (data not shown).
A remarkable feature of the i-AID system was that it selectively degraded Mcm4-aid protein from the MCM complex without depleting the other subunits (Figure 4). This feature would make the system suitable for functional analysis of single components of a large complex. This property is attributable to the ubiquitin-mediated protein degradation system, as reported for the ts-degron system in budding yeast . It was also notable that the i-AID system degraded Mcm4-aid protein not only in the soluble fraction but also in the chromatin-bound MCM complex (Figure 4). In HU-arrested cells, where the replication machinery stalls near the replication origin, depletion of Mcm4-aid protein caused dissociation of Mcm6 from chromatin, probably along with the other subunits. Consistently, replication did not resume after removal of HU, because the MCM complex is required as a replicative helicase for fork progression (Figure 4). Therefore, depletion of a component of the chromatin-bound complex, such as the replication fork, would be a powerful tool for analyzing the functions of chromatin proteins at specific stages of the reaction. It is likely that cytosolic proteins in fission yeast are also susceptible to auxin-dependent degradation, as has been described , although we did not test them in the present study.
The mcm4-i-aid strain exhibited the most marked defects in growth and replication upon addition of auxin among the aid-tagged strains tested (Table 1). This is probably because a significantly large number of MCM complexes need to be loaded onto several hundred replication origins on the chromosomes  within the short G1 phase in the fission yeast cell cycle. Once the MCM complexes on chromatin have dissociated through degradation of Mcm4-aid protein in S phase, cells are unable to resume DNA replication because loading of the MCM complex onto chromatin is strongly inhibited after onset of S phase in order to avoid re-replication .
We provided an improved auxin-indusible degron system for fission yeast. The i-AID system, where TIR1 is fused with fission yeast Skp1, greatly enhanced degradation efficiency of Mcm4-aid protein, and the off-AID system, which combines the i-AID system with transcription repression, successfully depleted Cdc45-aid and Pol1-aid proteins causing arrest at early S phase. The off-AID system is a powerful method for depletion of specific proteins within fission yeast cells.
Strains and media
Fission yeast strains used in this study
h - ade6X ura4-D18
h - ade6::ade6 + -P nmt41 -AtTIR1-9myc ura4-D18
h - ade6::ade6 + -P nmt41 -AtTIR1-9myc mcm4::mcm4-2HA-IAA17
h - mcm4::mcm4-2HA-IAA17
h + mcm4::mcm4-2HA-IAA17 leu1-32
h - mcm4::mcm4-2HA-IAA17 ade6X
h - ade6::ade6 + -P adh15 -skp1-AtTIR1-2NLS-9myc ura4-D18
h - ade6::ade6 + -P adh15 -skp1-AtTIR1-2NLS-9myc mcm4::mcm4-2HA-IAA17
h - ade6::ade6 + -P adh81 -skp1-AtTIR1-2NLS-9myc mcm4::mcm4-2HA-IAA17
h - ade6::ade6 + -P nmt41 -skp1-AtTIR1-2NLS-9myc mcm4::mcm4-2HA-IAA17
h - ade6::ade6 + -P adh15 -skp1-AtTIR1-2NLS-9myc mcm10::mcm10-IAA17
h - ade6::ade6 + -P adh15 -skp1-AtTIR1-2NLS-9myc cdc20::cdc20-IAA17
h - ade6::ade6 + -P adh15 -skp1-AtTIR1-2NLS-9myc pol1::pol1-IAA17
h - ade6::ade6 + -P adh15 -skp1-AtTIR1-2NLS-9myc orc2::orc2-IAA17
h - ade6::ade6 + -P adh15 -skp1-AtTIR1-2NLS-9myc orc6::orc6-IAA17
h - ade6::ade6 + -P adh15 -skp1-AtTIR1-2NLS-9myc cdc45::cdc45-IAA17
h - cdc45::cdc45-IAA17
h - ade6::ade6 + -P adh15 -skp1-OsTIR1-natMX6-P adh15 -skp1-AtTIR1-2NLS ura4-D18
h - cdc25-22 ade6::ade6 + -P adh15 -skp1-AtTIR1-2NLS-9myc mcm4::mcm4-2HA-IAA17
h - ade6::ade6 + -P adh15 -skp1-AtTIR1-2NLS pol1::hphMX6-P nmt81 -pol1-IAA17
h - cdc45::hphMX6-P nmt81 -cdc45-IAA17
h - cdc25-22 cdc45::hphMX6-P nmt81 -cdc45-IAA17 mcm10::6flag-mcm10 ade6::ade6 + -P adh15 -skp1-OsTIR1-natMX6-P adh15 -skp1-AtTIR1-2NLS
h - cdc25-22 pol1::hphMX6-P nmt81 -pol1-IAA17 mcm10::6flag-mcm10 ade6::ade6 + -P adh15 -skp1-OsTIR1-natMX6-P adh15 -skp1-AtTIR1-2NLS
h + mcm4::mcm4-2HA-IAA17 leu1-32 pREP41D
h + mcm4::mcm4-2HA-IAA17 leu1-32 pREP41D-AtTIR1-9myc
h + mcm4::mcm4-2HA-IAA17 leu1-32 pREP41D-AtTIR1-2NLS-9myc
h + mcm4::mcm4-2HA-IAA17 leu1-32 pREP41D-skp1-AtTIR1-9myc
Construction of Pnmt41-AtTIR1 strain
To introduce AtTIR1 gene under control of Pnmt41, AtTIR1-9myc sequence was introduced between the nmt41 promoter and terminator sequences  on pUC-nmt41 to generate pKM15. Then a 4.4 kb Not I fragment from pKM15 containing P nmt41 -AtTIR1-9myc-T nmt1 from pKM15 was inserted into the Not I site downstream of ade6+ in pKM17 to generate pKM21. pKM21 was digested by Eco RI and used for transformation of TNF47 (h-ade6X ura4-D18) to gain HM1813 (h-P nmt41 -AtTIR1-9myc ura4-D18). Integration of the fragment was confirmed by PCR.
Construction of mcm4-aid strains
Primers used in this study
Construction of plasmids carrying modified TIR1
The AtTIR1-9myc and AtTIR1-NLS-9myc genes were inserted between the nmt41 promoter and terminator sequences  on pREP41-Dual to generate pKM7 and pKM45, respectively.
To fuse fission yeast Skp1 at the N-terminus of AtTIR1, the skp1 gene lacking introns was PCR-amplified from a cDNA library (donated by H. Nojima) with a 16-amino-acid linker (Gly-Ile-Pro-Asp-Leu-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Asp-Leu-Thr-Ser) at the C-terminal of the protein using the primers skp1-F and skp1-R (Table 3), then cloned into the Cla I-Eco RI site of pBluescriptII SK(+), resulting in pKM44. The skp1 fragment from pKM44 together with the AtTIR1-9myc fragment was inserted between the nmt41 promoter and terminator sequence on pREP41-Dual to create pKM71.
pREP41-Dual, pKM7, pKM45, and pKM71 plasmids were introduced into HM1910 (h+mcm4-2HA-aid leu1-32) by electroporation to gain MKF53, MKF54, MKF55 and MKF60, respectively .
Construction of TIR1 integrant strains
For integration of the skp1-AtTIR1-NLS-9myc gene under control of the nmt41 promoter into the ade6+ locus, the skp1 fragment together with an AtTIR1-NLS-9myc fragment was inserted between the nmt41 promoter and terminator sequences on pUC-nmt41, resulting in pKM82. Then Not I fragment containing P nmt41 -skp1-AtTIR1-NLS-9myc-T nmt1 from pKM82 was inserted into the Not I site downstream of ade6+ in pKM17 to generate pKM84.
For construction of the skp1-AtTIR1-NLS gene under control of weak derivatives of the adh1 promoter, the nmt41 promoter of pKM84 was replaced by promoter fragments from pRAD15 and pRAD81 (provided by Y. Watanabe), resulting in pKM104 and pKM105, respectively. Then pKM84, pKM104 and pKM105 were digested by Eco RI and used for transformation of HM2423 (h-mcm4-2HA-aid ade6X) to generate HM2491 (h-mcm4-2HA-aid ade6+-P nmt41 -skp1-AtTIR1-NLS-9myc), HM2473 (h-mcm4-2HA-aid ade6+-P adh15 -skp1-AtTIR1-NLS-9myc) and HM2475 (h-mcm4-2HA-aid ade6+-P adh81 -skp1-AtTIR1-NLS-9myc), respectively. Integration of skp1-AtTIR1-NLS-9myc at the ade6+ locus was confirmed by genomic PCR.
Construction of aid-tagged derivatives of replication factors
The mcm10+ gene was C-terminally tagged with IAA17 peptide using PCR. The integration cassette was amplified by two-step PCR amplification. The first PCR amplified fragments from the 972 (h-, wild type) genome containing the C-terminal region of mcm10+ linked with part of the aid-tag, and the 3'-UTR of mcm10+ linked with part of the selection marker (ura4+) gene, using the primer sets mcm10C-F and mcm10-IAA, and mcm10-ura and mcm10-dw-R (Table 3), respectively. The second PCR amplified the integration cassette from pKM40 with the primers mcm10C-F and mcm10-dw-R, and the two short fragments made by the first PCR reactions. The products of the second PCR were then used for transformation of HM2468 (h-P adh15 -skp1-AtTIR1-NLS-9myc ura4-D18). Transformants were selected on an EMM plate, and the integration of the aid-tag was confirmed by genomic sequencing. The resulting strain, HM2550 (h-P adh15 -skp1-AtTIR1-NLS-9myc mcm10-aid), showed wild-type growth, suggesting that the aid-tagged Mcm10 was functional.
HM2551 (h-P adh15 -skp1-AtTIR1-NLS-9myc cdc20-aid), HM2552 (h-P adh15 -skp1-AtTIR1-NLS-9myc pol1-aid), HM2572 (h-P adh15 -skp1-AtTIR1-NLS-9myc orc2-aid), HM2575 (h-P adh15 -skp1-AtTIR1-NLS-9myc orc6-aid), and HM2578 (h-P adh15 -skp1-AtTIR1-NLS-9myc cdc45-aid) were constructed similarly using pKM40 and the following primers (Table 3). HM2551: cdc20C-F and cdc20-IAA; cdc20-ura and cdc20-dw-R, HM2552: pol1C-F and pol1-IAA; pol1-ura and pol1-dw-R, HM2572: orc2C-F and orc2-IAA; orc2-ura and orc2-dw-R, HM2575: orc6C-F and orc6-IAA; orc6-ura and orc6-dw-R, and HM2578: cdc45C-F and cdc45-IAA; cdc45-ura and cdc45-dw-R. All the strains showed wild-type growth, except for HM2578, which grew slightly more slowly in the absence of auxin.
Construction of the double-TIR1 strain
For integration of the skp1-OsTIR1-NLS-9myc gene under control of the adh15 promoter into the ade6+ locus, a 1.7-kb Xba I-Sal I fragment containing the OsTIR1 gene from pNHK33 and a 0.4 kb Sal I-Sma I fragment containing 9myc were introduced into the Spe I-Sma I sites of pKM104 to generate pKM111. pKM111 was digested by Eco RI and used for transformation.
To remove the 9myc-tag at the C-terminus of AtTIR1, the C-terminal region of AtTIR1-NLS without myc-tag was PCR-amplified using the primers TIR1C-BmSc and TIR1-NLS-R (Table 3), and the products obtained by Bam HI digestion were used to generate pKM132, which contains the C-terminal region of AtTIR1-NLS and the transcription terminator T nmt . The C-terminal region of skp1-AtTIR1-NLS-9myc was replaced by the Nsi I-Sma I fragment from pKM132, to generate pKM136 carrying P adh15 -skp1-AtTIR1-NLS-T nmt1 .
To construct the double-TIR1 strain, the Spe I-Not I fragment from pKM132 containing the C-terminal region of AtTIR1-NLS and T nmt1 was inserted into pKM126 containing the 3'-UTR of ade6+, resulting in pKM135. Then the Xho I-Sal I fragment containing the OsTIR1 gene was introduced into the Sal I site of pKM135 to create pKM143, carrying OsTIR1-T nmt1 and the 3'-UTR of ade6+. Then the selection marker gene, natMX6, cloned from pFA6a-natMX6 and the P adh15 -skp1-AtTIR1-NLS-T nmt1 gene from pKM136 were inserted into the Not I site of pKM143 to generate pKM151. An 8.2 kb Sac II fragment from pKM151 containing OsTIR1-T nmt1 , natMX6, P adh15 -skp1-AtTIR1-NLS-T nmt1 and the 3'-UTR of ade6+ was introduced into cells harboring P adh15 -skp1-OsTIR-9myc at the ade6+ locus to construct the double-TIR1 strain. A NatR transformant that grew on an EMM plate containing clonNAT (100 μg/ml) was obtained, and integration of the fragment was confirmed by genomic PCR and southern hybridization.
Construction of Pnmt81-pol1-aid and Pnmt81- cdc45-aid strains
For construction of the P nmt81 -pol1-aid strain, the 5'-UTR of pol1+ was amplified by PCR using the primers pol1up-F-Spe and pol1up-R-Bgl (Table 3), and the Spe I-Bgl II-digested product was cloned into the Spe I-Bam HI sites upstream of hphMX6 to generate pKM154. The N-terminal region of pol1+ was PCR-amplified using the primers pol1N-nmt-F and pol1dw-R-Bm (Table 3), and the product was digested by Nde I and Bam HI, and then cloned into the Nde I-Bam HI sites downstream of P nmt81 to create pKM158. Then Sac I fragment of pKM154 containing the 3'-UTR of pol1+ and hphMX6 was introduced into the Sac I site of pKM158 to form pKM159. pKM159 was digested by Eco RV and used for transformation of HM2552 (h-P adh15 -skp1-AtTIR1-NLS-9myc pol1-aid) to create HM3150 (h-P adh15 -skp1-AtTIR1-NLS-9myc P nmt81 -pol1-aid). The insertion was confirmed by genomic PCR.
A strain expressing Cdc45-aid from P nmt81 was constructed as described below. The N-terminal fragment of cdc45+ was cloned from pGAD-Cdc45 into the Nde I-Eco RV sites of pKM158 to create pKM163. The 5'-UTR of cdc45+ was PCR-amplified using the primers cdc45up-F-Spe and cdc45up-R-Bm (Table 3), and the product digested by Spe I and Bam HI was cloned into the Spe I-Bam HI sites upstream of hphMX6, resulting in pKM164. The 2.0-kb Sac I fragment containing the 3'-UTR of cdc45+ and hphMX6 was inserted into the Sac I site of pKM163 to create pKM165. pKM165 was used for transformation of HM2580 (h-cdc45-aid) to generate HM3325 (h-P nmt81 -cdc45-aid), and the integration was confirmed by genomic PCR.
Preparation of cell extracts and immunoblotting
S. pombe cells (1 × 108 cells) were fixed with 20% TCA and suspended in 0.1 ml of urea solution (50 mM NaPi [pH 8.0], 8 M urea, 1 mM DTT, 0.1% Nonidet P-40). Cells were disrupted with acid-washed glass beads using a Micro Smash (TOMY) three times for 45 sec each time. Proteins in the extracts were separated by SDS-PAGE and transferred onto PVDF membrane (Immobilon, Millipore Corp). The membranes were incubated for 1 h at room temperature in PBST (10 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl, 1.76 mM KH2PO4, 0.1% Tween 20) containing 5% skim milk and reacted in PBST containing 1% skim milk overnight at 4°C with rabbit anti-Mcm4 antibody , rabbit anti-Mcm2 antibody , rabbit anti-Mcm3 antibody , rabbit anti-Mcm5 antibody , rabbit anti-Mcm6 antibody , rabbit anti-Mcm7 antibody , rabbit anti-Mcm10 antibody (will be published elsewhere), rabbit anti-Cdc45 antibody , rabbit anti-IAA17 antibody , mouse anti-Myc antibody 9E11 (NeoMarkers) or mouse anti-TAT1 antibody  at dilutions of 1:2,000, 1:1,000, 1:3,000, 1:1,000, 1:3,000, 1:1,000, 1:2,000, 1:2,000, 1:2,000, 1:1,000 and 1:500, respectively. HRP-conjugated anti-mouse or anti-rabbit immunogloblin G was used as the secondary antibody (1:10,000; Jackson). Binding was visualized with West Pico Chemiluminescent Substrate and Femto Maximum Sensitivity Substrate (Thermo).
Cell cycle synchronization
To synchronize the cell cycle, the thermosensitive mutation cdc25-22 was used for G2/M arrest . Derivatives carrying cdc25-22 were incubated at 36°C for 3 h for arrest at the G2/M boundary and then released at 25°C. To repress transcription, thiamine was added at a final concentration of 10 μg/ml at the indicated time points before G2/M arrest (see Figure legends). Synthetic auxin, NAA (1-naphthaleneacetic acid) (Nacalai Tesque), was added at a concentration of 0.5 mM, 1 h before the release from G2/M to induce protein degradation.
To arrest cells in early S phase, cells released from the G2/M boundary were cultured for 3 h in the presence of 12 mM hydroxyurea (HU, Sigma), which depletes the cellular dNTP pools. NAA was added 1 h before the release from HU arrest.
Chromatin immunoprecipitation assay
ChIP assays were performed as described previously  with some modifications. Derivatives of cdc25-22 grown in EMM medium at 25°C for 15 h to 0.6 × 107 cells/ml were arrested at the G2/M boundary by incubation at 36°C for 3 h and then released at 25°C in the presence of 12 mM HU to retard replication forks. The cells (3 × 108) were fixed in 1% formaldehyde (Sigma) for 15 min and then in 125 mM glycine for 5 min at room temperature with gentle shaking. After being washed once with cold water, the cells were suspended in 450 μl of breaking buffer (50 mM Hepes-KOH [pH 7.4], 1 mM EDTA, 140 mM NaCl, 0.1% sodium deoxycholate, 0.1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 0.1% proteinase inhibitor cocktail [Sigma]) and disrupted with glass beads using a Micro Smash (TOMY) five times for 30 s each time. The cell extracts were recovered by centrifugation at 3,000 rpm for 10 s. After addition of 50 μl of 10% Triton X-100, the samples were sonicated four times for 10 s each time. The supernatant obtained by centrifugation at 15,000 rpm for 10 min was used for immunoprecipitation with magnet beads (Dynal) conjugated with rabbit anti-Mcm6 antibody (1:400). After incubation of the cell extracts with the beads at 4°C for 2 h, the immunoprecipitates were rinsed with ChIP lysis buffer (50 mM Hepes-KOH [pH 7.4], 1 mM EDTA, 140 mM NaCl, 0.1% sodium deoxycholate, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride) once, ChIP lysis buffer containing 640 mM NaCl twice, ChIP wash buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 250 mM LiCl, 0.5% sodium deoxycholate, 0.5% Nonidet P-40) twice and TE1 (10 mM Tris-HCl [pH 8.0], 1 mM EDTA) once, and then the obtained protein-DNA complex was eluted with TES (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 1% SDS) by incubation at 65°C for 30 min. To reverse the cross-linking, the eluates were incubated at 65°C for 15 h. The remaining proteins were digested with Proteinase K (Merck) and the DNA was purified by extraction with phenol and chloroform. The DNA recovered by ethanol precipitation was suspended in TE. DNA prepared from whole-cell extracts or immunoprecipitated fractions was analyzed by real-time PCR using SYBR green I in a 7300 real-time PCR System (Applied Biosystems). The primer sets used for real-time PCR were ars2004 region-273F and ars2004 region-338R for ars2004, and nonARS1 region-514F and nonARS1 region-583R for the non-origin region (Table 3).
Cells were fixed with 70% ethanol and incubated with 0.5 μg/ml Propidium iodide and 50 μg/ml RNaseA in 50 mM sodium citrate for 1 h at 37°C. Samples were then measured using a FACScan (BECTON DICKINSON).
Deposit of strains
Strains HM2468, HM2473 and HM2985 and plasmid pKM40 are deposited to the National BioResource Project http://yeast.lab.nig.ac.jp/nig/ and will be available upon request.
We thank Drs. Haruhiko Takisawa for valuable discussion and Yoshinori Watanabe and Hiroshi Nojima for kindly providing derivatives of the adh1 promoter and S. pombe cDNA library, respectively. We also thank Mr. Tetsuya Handa, Ryota Ueda, Makoto Yoshida, Ooi LingFan and Ms. Shuqi Yan for construction of AID strains. This study was supported by a Grant-in-Aid from the Ministry of Education, Science, Technology, Sports, and Culture, Japan, to H.M and by a Grant-in-Aid for JSPS Fellows to M.K.
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