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¹ Laboratory of Cell Regulation,
² Laboratory of Structural Biology, Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, UK

	Abstract

Top Abstract Introduction Results Discussion Experimental procedures References

 
Skp1 is a central component of the E3 ubiquitin ligase SCF (Skp1-Cullin-1-F-box). It forms an adapter bridge between Cullin-1 and the substrate-determining component, the F-box protein. In order to establish the role of Skp1, a temperature sensitive (ts) screen was carried out using mutagenic PCR (polymerase chain reaction) and 9 independent ts mutants were isolated. Mapping the mutated residues on the 3-D structure of human Skp1 suggested that the mutants would be compromised in binding to F-box proteins but not Cullin-1 (Pcu1). In order to assess the binding properties of ts Skp1, 12 F-box proteins and Pcu1 were epitope-tagged, and co-immunoprecipitation performed. This systematic analysis showed that ts Skp1 retains binding to Pcu1. However, binding to three specific F-box proteins, essential Pof1, Pof3 involved in maintaining genome integrity, and nonessential Pof10, was reduced. skp1^ts cells exhibit a G2 cell cycle delay, which is attributable to activation of the DNA damage checkpoint. Intriguingly, contrary to pof3 mutants, in which this checkpoint is required for survival, checkpoint abrogation in skp1^ts suppresses a G2 delay and furthermore almost rescues the ts phenotype. The activation mechanism of the DNA damage checkpoint therefore differs between pof3 and skp1^ts, implicating a novel role for Skp1 in the checkpoint-signalling cascade.

	Introduction

Top Abstract Introduction Results Discussion Experimental procedures References

 
The timely and selective destruction of proteins within the cell is essential for proper regulation of many processes in cell biology, in particular those requiring fast and irreversible changes in protein levels. Regulation of this kind is frequently seen in key cell cycle events and as such is highly important to the successful survival of all organisms. Ubiquitin proteolysis is a major cellular mechanism for controlling breakdown of proteins. This system relies on selection of substrates for destruction by a series of enzymes known generically as the ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin ligase (E3) (Hershko & Ciechanover 1992; Hochstrasser 1996). The E3 ligase determines the substrate specificity of the pathway. Several different E3s exist in any one organism and fall into two broad categories, the HECT or RING finger type E3s (Freemont 2000).

A multicomponent E3, the SCF (Skp1-Cullin-1-F-box), is one such RING finger type E3 (Bai et al. 1996; Deshaies 1999; Feldman et al. 1997; Patton et al. 1998a; Peters 1998) and consists of at least the 3 components listed above and a fourth, Rbx1/Roc1/Hrt1, which contains the RING finger (Kamura et al. 1999; Lammer et al. 1998). In S. cerevisiae it is suggested that a fifth component Sgt1 is also a member of the complex (Kitagawa et al. 1999). The RING finger protein is necessary for the recruitment of the E2 to the complex whilst the F-box protein, containing a 50 amino acid, loosely conserved motif, known as the F-box, is involved in binding to Skp1 (Bai et al. 1996; Patton et al. 1998b). Studies in yeast and mammals have highlighted the existence of multiple F-box proteins in each organism (Cenciarelli et al. 1999; Regan-Reimann et al. 1999; Winston et al. 1999) and these are important in the identification and selection of substrates for destruction. The BTB/POZ domain protein Skp1 has the unique function of bridging the core complex to the F-box protein and as such plays a central role in the complex (Zheng et al. 2002).

Previously we and others have identified and characterized the F-box proteins Pop1, Pop2 (Kominami et al. 1998; Kominami & Toda 1997) and Pof3 (Katayama et al. 2002). Pop1 and Pop2 are involved in the maintenance of accurate genome ploidy by regulating the timely destruction of Rum1 and Cdc18, a CKI (cyclin-dependent Kinase Inhibitor) and replication factor, respectively. Failure to degrade these substrates results in polyploid cells. We have also shown that Pof3 is involved in the maintenance of genomic integrity. Loss of Pof3 function results in activation of the DNA damage checkpoint pathway and cells lacking Pof3 show a G2 cell cycle delay and chromosomal defects such as a high rate of minichromosome loss and derepression of transcriptional silencing at heterochromatin. Consistent with this, abrogation of components of the DNA damage checkpoint in this mutant leads to a lethal ‘cut’ phenotype, reminiscent of uncontrolled cell division without chromosome segregation (Katayama et al. 2002).

Signalling through the DNA damage checkpoint pathway is initiated through Rad3/ATR and results in activation of the downstream kinase Chk1. Chk1 kinase is responsible for arrest of the cell cycle by effects on Cdc2 tyrosine15 phosphorylation (O’Connell et al. 2000; Rhind & Russell 2000; Zhou & Elledge 2000). Upon Chk1 activation this inhibitory Cdc2 phosphorylation is maintained, most likely through control of both Wee1 kinase and Cdc25 phosphatase. Inhibition of Cdc2 activity results in the G2 arrest observed in cells with checkpoint activation (O’Connell et al. 2000; Rhind & Russell 2000; Zhou & Elledge 2000). Pof3 is required for genome integrity to the extent that survival of pof3 cells is entirely dependent on this G2 delay and therefore activation of the DNA damage checkpoint.

Here we show that Skp1 is also involved in the DNA damage checkpoint pathway. Mutants created in this study show activation of the checkpoint and a G2 delay similar to pof3 deletion mutants. However, contrary to pof3, skp1 mutants are substantially rescued by inability to signal the checkpoint. This suggests that Skp1 may play a role independent of its F-box protein, Pof3, in initiating the checkpoint and that activation may be spurious in skp1^ts cells. We discuss a novel role for Skp1 in the DNA damage checkpoint and the maintenance of genome integrity.

	Results

Top Abstract Introduction Results Discussion Experimental procedures References

 
Deletion of core components of the SCF

The Skp1 protein of S. cerevisiae (Bai et al. 1996) was used to identify homologues in S. pombe using a BLASTP search of the SWISS-PROT database. One homologue was identified with 50% identity and 65% similarity. The entire ORF of this gene was deleted by one-step gene replacement in a heterozygous diploid. Homologues to Rbx1 and Sgt1 were also identified and these genes were likewise disrupted by integration of a ura4⁺ cassette. Disrupted loci were detected by PCR and diploids sporulated. Tetrads of each disruptant were analysed. Two spores from each tetrad germinated and these two spores were shown to be Ura^- indicating that skp1⁺, rbx1⁺ and sgt1⁺ are essential genes (Fig. 1). Upon microscopic examination of the plates it was seen that sgt1::ura4⁺ and rbx1::ura4⁺ cells could germinate and undergo some cell divisions (Fig. 1B,C). These cells showed elongated phenotypes. skp1::ura4⁺ cells could also germinate but undergo fewer cell divisions and show a rounded phenotype (Fig. 1A). We previously showed that the fission yeast homologue of Cullin-1, Pcu1, is also essential for cell viability, and deleted spores germinate and undergo several cell divisions with increased ploidy (Kominami et al. 1998). These results indicate that all 4 SCF components genes are essential for cell viability. Apparent phenotypic differences suggest either that essential biological processes requiring each protein function are not the same among individual components or that each may play a distinct role in addition to their role in the SCF.

View larger version (91K): [in this window] [in a new window]   Figure 1  Gene disruption of ORFs encoding SCF components. Heterozygous diploids of the CHP428/429 (Table 3) strain deleted for (A) skp1(B) rbx1 and (C) sgt1 are shown. Two growing colonies are observed. These are Ura-. The locations where spores were placed but no colony growth is seen were examined by microscope. Bar: (A) 2 µm; (B) & (C) 10 µm.

The F-box hypothesis proposes that the diversity of the SCF complex relies on interchangeable F-box proteins combining with the core complex (Patton et al. 1998b). The F-box proteins of S. cerevisiae, X. laevis and H. sapiens have been investigated by a combination of database searching, two-hybrid assays and-non-denaturing protein affinity purification (Cenciarelli et al. 1999; Regan-Reimann et al. 1999; Seol et al. 2001; Winston et al. 1999). We attempted to identify novel F-box proteins of S. pombe by database searching. The F-box motif from Pop1 and Pop2 was used for BLASTP searches of the Sanger Centre gene DB. Further F-box proteins were identified using S. cerevisiae F-box protein YBR280C (Seol et al. 2001). This identified Pof9 and Pof10. The F-box protein Pof10 was used to carry out a final BLASTP search which identified Pof11, 12 and 13. Using homology searching, a total of 16 F-box protein ORFs were identified in the S. pombe genome (Table 1).

Results from deleting components of the SCF complex suggests that although the core complex is essential to cellular vegetative growth, the majority of F-box proteins are dispensable or possibly redundant at least during normal vegetative cycles. It is possible that nonessential F-box proteins may play an important role under some particular conditions such as stress response and starved circumstances, which we have not tested in a systematic fashion. Essentiality of all the 4 core components of the SCF may also imply that they have functions above and beyond their involvement in the SCF complex. In order to investigate the behaviour of the core complex further, we carried out a screen to identify ts mutants in one of the core components of the SCF complex, Skp1.

Mutagenesis of skp1⁺ to create temperature sensitive mutants

We chose to investigate Skp1 as it is central to the SCF complex due to its unique bridging function between the Cullin-1 (Pcu1) and the F-box protein. Therefore, the creation of mutants in this protein could potentially result in a situation where interaction of the core complex with a subset of F-box proteins may be affected whilst the core complex remains intact. Alternatively mutant Skp1 may be defective in binding to Pcu1. To create ts skp1 mutants, we used a targeted approach for mutagenesis of a single gene in a random manner. skp1⁺ was mutagenized in a PCR reaction involving increased quantities of nucleotides, which lowers the fidelity of the Taq DNA polymerase (Cadwell & Joyce 1992) (see Experimental procedures). Flanking sequences on either side of the gene were then used to recombine these mutated skp1 cassettes back into the S. pombe genome. The host strain for this recombination contains a ura4⁺ gene inserted after the termination codon of the skp1⁺ gene. The homologous recombination then results in the loss of this ura4⁺ marker gene (Fig. 2A). Integrants could therefore be selected by their ability to grow on plates containing 5-FOA. 200 5-FOA resistant colonies were obtained by this method and these colonies were assessed for temperature sensitivity. 9 colonies were ts and turned dark red on plates containing the phloxine B dye, a stain which is absorbed by dead cells (Moreno et al. 1991) (Fig. 2B).

View larger version (47K): [in this window] [in a new window]   Figure 2  Isolation of temperature sensitive skp1 mutants. (A) Schematic representation of the method used to create temperature sensitive mutants. 1. integration of ura4+ into the 3' region of genomic skp1+ 2. Genomic DNA after integration of ura4+ 3. Plasmid DNA containing skp1+ and 300 base pairs of downstream genomic sequence used for a mutagenic PCR reaction using primers from the green and orange regions. 4. PCR fragment containing skp1mut and 300 base pairs of downstream sequence is recombined into the genome of skp1+–ura4+ strain indicated by the black lines. 5. End result loss of ura4+ marker and integration of skp1mut fragment. (B) skp1ts mutants grown on YE5S plates with phloxine B dye at 26 °C (left) and 36 °C (right).

Genomic DNA from each of the skp1^ts mutants was prepared and amplified with 4 primers designed to span the skp1⁺ region, in order to sequence the skp1 locus in each of the mutants. Sequencing revealed two groups of mutants, those containing point mutations and those with mutations in the termination codon of skp1⁺ (Table 2). These termination codon mutants allowed read through of the STOP codon to another a further 21 bases downstream. The STOP codon was altered in five out of the nine mutants. The remaining 4 contained point mutations. Allele 2 and Allele 6 contained the same mutation. The sequencing data revealed 3 point mutations that were clustered in a small region in the C-terminus of the Skp1 protein (Table 2 and see below).

The crystal structure of human Skp1 bound to the F-box protein Skp2 and the core complex has been previously determined (Schulman et al. 2000; Zheng et al. 2002). Due to the high level of homology between S. pombe and human Skp1, we were able to model the S. pombe Skp1 structure on that of human Skp1 using 3D-jigsaw software (Bates et al. 2001). From this model we established that residues I110 and L113 are buried within the Skp1 hydrophobic core at the end of helix 5 and beginning of helix 6 (Fig. 3A,B). Accessible residues on both these helices form critical contacts with the F-box domain. The Skp1^tsA3 (L113F) and Skp1^tsA7 (I110T) point mutants created in skp1^ts mutants therefore introduce bulky residue (L113F) or a small polar residue (I110T), respectively, into the Skp1 hydrophobic core and are likely to indirectly perturb the interaction with the F-box domain by local alteration of the Skp1 conformation.

View larger version (47K): [in this window] [in a new window]   Figure 3  Mutation sites in skp1ts mutants. (A) Amino acid sequence alignment of S. pombe, S. cerevisiae and H. sapiens Skp1. Known S. cerevisiae skp1 temperature sensitive mutants are marked in pink with arrows (Bai et al. 1996; Connelly & Hieter 1996). skp1ts mutants in S. pombe are marked in red with asterisks. Identical residues are shown in black, similar residues in grey. The helices and domains determined from the crystal structure of human Skp1 (Schulman et al. 2000) are shown underneath the alignment and F-box contact residues are indicated with a purple dot. (B) Schematic representation of a model for S. pombe Skp1 (light blue) bound to an F-box domain derived from the structure of human Skp1-Skp2 complex (Schulman et al. 2000; pdb code 1FQV [PDB] ). Point mutations A2, A3 and A7 described in this paper cluster near helix 5 and helix 6 highlighted within the black box. The right hand panel shows a close up of this region indicating the position of the mutated residues and selected secondary structural elements.

The addition of 7 extra amino acids on to the C-terminus of Skp1 due to read through of the STOP codon would interfere with the variable Skp1/F-box interface of helix 8 and also affect the binding of Skp1 to F-box proteins. None of the mutations created in this screen are predicted to compromise the binding of Skp1 to Pcu1 as the binding to Pcu1 occurs at the N-terminal region of Skp1 (Fig. 3B). The modelling of the Skp1^ts proteins therefore suggests that all the mutant Skp1 proteins are specifically defective in binding to F-box proteins, but not to Pcu1.

G2delay phenotype of skp1^ts mutants

We now examined the physiological effects of mutation of skp1⁺ on S. pombe cells. Cells of each point mutant, skp1^tsA2, skp1^tsA3 and skp1^tsA7 grown at 36 °C in liquid media for 8 h were examined. The most characteristic phenotype in all cases was cell elongation (Fig. 4A). Loss of the F-box proteins Pop1 and/or Pop2 causes a large cell phenotype with polyploid DNA content (Kominami et al. 1998; Kominami & Toda 1997). In order to assess if the phenotype exhibited by skp1^ts point mutants was related to that seen in a pop1 or pop2 mutant we carried out FACS analysis to establish the DNA content of skp1^ts cells. S. pombe is a haploid organism but spends the majority of its cell cycle in G2 (Moreno et al. 1991). An asynchronous culture of S. pombe cells would therefore be expected to have a 2C DNA content. The skp1^ts cells also contain a 2C DNA content suggesting there is no polyploidy despite the elongated cells observed (Fig. 4B).

View larger version (65K): [in this window] [in a new window]   Figure 4  The G2-M phase delay of skp1ts mutants. (A) Cells grown for 8 h at 36 °C are shown from each of the point mutants, showing the elongation phenotype of skp1ts cells. Bar indicates 10 µm. (B) FACS analysis of wild-type, skp1tsA7 and skp1tsA7 rum1 cells. Samples taken every two hours were analysed by propidium iodide staining in a Beckman-Dickinson FACS scan. The positions of 2C and 4C peaks are shown. (C) Rum1-HA levels in skp1tsA7 cells at 0 and 4 h at 36 °C. Cell extract was prepared from a wild-type untagged strain and AL158 strain and immunoblotted with anti-HA antibodies. Levels of -tubulin are shown to demonstrate equal loading.

A7rum1

Interactions of Skp1^ts protein with Cullin-1 and individual F-box proteins

The position of the mutations in skp1 and the evidence that skp1^tsA7 can degrade a known SCF substrate suggested to us that the core complex of the SCF remained intact in this mutant, and that the mutant phenotype observed may be mediated through an effect on one or more F-box proteins. As Rum1 is a known substrate of the F-box protein Pop1 (Kominami & Toda 1997), we would expect that this complex is intact. In order to verify this and to examine the effects of mutation on Skp1 interactions we carried out a binding analysis of mutant Skp1^tsA7 with F-box proteins and Pcu1 by immunoprecipitation. Each F-box protein was tagged with a myc epitope at its C-terminus. Immunoprecipitations were carried out using a polyclonal anti-Skp1 antibody. In cases where the C-terminus could not be tagged due to loss of protein function, GFP (green fluorescence protein) was used to tag the N-terminus of the protein under the thiamine repressible nmt promoter (e.g. GFP-Pof6 and GFP-Fdh1).

Pcu1-myc was found to associate just as strongly with Skp1^tsA7 as with wild-type Skp1 (Fig. 5A, lanes 3 and 4). However the F-box proteins, Pof1, Pof3 and Pof10 all showed reduction in their interactions with Skp1^tsA7 (lanes 7, 8, 9,10 19 and 20). The amount of binding was quantified (Experimental procedures) and in the case of Pof3 the reduction in binding between wild-type and Skp1^tsA7 was almost 50%, whilst 30% reduction in binding was observed in Pof1 and Pof10 (Fig. 5C). The interactions of other F-box proteins with Skp1^tsA7 were not reduced, though the degree of binding between Pop1 and Skp1 is difficult to judge as very little Pop1 proteins were co-precipitated in either wild-type or skp1^tsA7 cells (Fig. 5B,C). In summary, Skp1^tsA7 protein is defective in its binding to 3 F-box proteins, Pof1, Pof3 and Pof10 but maintains full binding to the other 8 F-box proteins and the Cullin-1, Pcu1.

View larger version (85K): [in this window] [in a new window]   Figure 5  Binding profile of wild-type Skp1 and Skp1tsA7 to individual F-box proteins. (A) Immunoprecipitations of 13myc tagged F-box proteins. The myc tagged F-box is indicated above the lane. Upper panel is the protein input. Immunoprecipitation was carried out using rabbit polyclonal anti-Skp1 antibodies and immunoblotting with monoclonal 9E10 anti-myc antibody (middle panel) and polyclonal anti-Skp1 antibody (lower panel). Protein extracts were prepared from strains with skp1+ backgrounds grown at 36 °C for 4 h, or cells carrying a skp1tsA7 mutation grown at 36 °C for 4 h. (B) Immunoprecipitation of GFP-Fdh1. Protein input is shown on the left, Immunoprecipitation on the right. Protein extracts were prepared from strains with skp1+ backgrounds or cells carrying a skp1tsA7 mutation, grown at 36 °C for 4 h. Immunoprecipitation carried out using anti-Skp1 antibodies as above. Immunoblotting with monoclonal anti-GFP antibodies (upper right) and anti-Skp1 antibodies (lower right). (C) NIH image quantification of the bands from (A) and (B). Bands were quantified (see Experimental procedures) and normalized for background. Quantity of binding is given as a ratio, where strains containing skp1+ alleles are taken to have a binding ratio of one.

From our earlier analysis (Table 1) we were aware that the deletion of either Pof3 or Fdh1, an F-box protein containing a DNA helicase in its C-terminus (Kim et al. 2002) gave rise to G2 cell cycle delay, which appeared similar to defective phenotypes seen in skp1^ts cells. Biochemical analysis suggested that the interaction of Pof3 is affected in a skp1^tsA7 strain but that Fdh1 remains bound to Skp1^tsA7 at normal levels (Fig. 5A–C). This suggests that in skp1^tsA7 mutants Pof3 function is already defective, whilst the function of Fdh1 remains intact. We confirmed this interpretation by examining the effect of deletions of fdh1⁺ and pof3⁺ in a skp1^tsA7 strain. When cells were grown at the restrictive temperature for the skp1^ts mutant and the amount of cell elongation measured, we found that the elongation phenotype of fdh1skp1^tsA7 was additive, with cells sometimes reaching up to 25 µm in length in the double mutant, whilst in the single mutants they averaged around 14 µm in length (Fig. 6). In pof3skp1^tsA7 cells, however, the length rarely exceeded that seen in the pof3 or a skp1^tsA7 strain alone. We also attempted to construct pof3fdh double mutants and found that double mutants are inviable. Microscopic observation of lethal spores indicated that spores can germinate, but arrest with elongated morphology (data not shown). This suggests that although a G2 delay phenotype occurs in skp1^ts cells, it is mediated only through effects on the F-box protein Pof3 and not through Fdh1, and that Pof3 and Fdh1 function in an independent manner.

View larger version (19K): [in this window] [in a new window]   Figure 6  fdh1 phenotype is additive with skp1ts but pof3 is not Cell length measurements of 200 cells grown at 36 °C for each of the strains stated.

Pof3 has previously been seen to be involved in integrity of the genome and a deletion shows a G2 delay phenotype with activation of the DNA damage checkpoint (Katayama et al. 2002). As skp1^tsA7 cells show a G2 delay phenotype and a 50% reduction in Pof3 binding to mutant Skp1^tsA7, we decided to investigate the state of the DNA damage checkpoint in skp1^tsA7 cells. In order to assess whether the DNA damage checkpoint was activated we examined the phosphorylation status of Chk1. In circumstances where the checkpoint is activated Chk1 becomes phosphorylated (Walworth & Bernards 1996). A strain carrying chk1⁺-myc in a skp1^tsA7 background was grown at 36 °C for 4 h after which the phosphorylation status of Chk1-myc was examined. The protein showed a mobility shift associated with phosphorylation, which was not evident when the cells were grown at 26 °C (Fig. 7A). Therefore, the DNA damage checkpoint is activated in skp1^tsA7 cells under restrictive conditions without exposure of exogenous DNA damaging agents.

View larger version (92K): [in this window] [in a new window]   Figure 7  The DNA damage checkpoint is activated in skp1ts mutants. (A) Chk1 phosphorylation. Samples of protein were prepared from wild-type cells, and wild-type cells that had been irradiated with UV, skp1tsA7 cells at 26 °C and skp1tsA7 cells after 4 h at 36 °C. Immunoblotting for Chk1-myc epitope was carried out using an anti-myc 9E10 monoclonal antibody. (B) Cells deleted for the checkpoint signalling protein Rad3 do not show cell elongation in skp1tsA7 background. Pictures show skp1tsA7, skp1tsA7rad3, and skp1tsA7rad3 cells transformed with a pREP1 plasmid carrying rad3+. Bar indicates 10 µm. (C) Rescue of the ts phenotype in skp1tsA7 rad3 or skp1tsA7chk1. Spot test dilutions of skp1tsA7 strains carrying different checkpoint gene deletions is shown. Suppression of skp1tsA7 is stronger in rad3 than in chk1. Strains were spotted on to YE5S rich media plates beginning with a dilution of 105 cells and decreasing 10 fold per spot. Plates were incubated at 26 °C or 36 °C for 3 days and then photographed.

Interestingly, deletion of components of the checkpoint pathway, such as rad3 or chk1, but not cds1, resulted in a substantial rescue of the temperature sensitive phenotype of skp1^tsA7 cells (Fig. 7C). This is in direct contrast to a pof3 deletion, which when combined with deletion of any of the DNA damage checkpoint components suffers a lethal ‘cut’ phenotype (Katayama et al. 2002). Also skp1^tsA7pof3rad3 triple mutants are inviable with a ‘cut’ phenotype at 27 °C (data not shown). It is therefore possible that although there is a reduction of binding to Pof3 in skp1^tsA7 cells, there may be other defects that result in the induction of the DNA damage checkpoint independent of Pof3.

	Discussion

Top Abstract Introduction Results Discussion Experimental procedures References

 
In this study we have shown that Skp1 is capable of binding to the identified F-box proteins of S. pombe. We have created 9 ts mutants in skp1⁺ that show a G2 delay and cell elongation. The G2 delay seen in these cells is caused by activation of the DNA damage checkpoint and the skp1^ts phenotype is rescued substantially by inactivating this checkpoint. The Skp1^tsA7 protein shows a reduction in its capacity to bind to certain F-box proteins, one of which, Pof3, is known to be involved in the DNA damage checkpoint (Katayama et al. 2002). This evidence further corroborates a role for the SCF complex in the DNA damage checkpoint, a crucial guardian of genome integrity and cell division.

Insight into mutated Skp1 proteins from structural analysis

Determination of the nucleotide sequence of the skp1^ts genes and assignment of mutated amino acid residues in the 3-D structure of the human Skp1-Skp2 complex have provided valuable information as to how the ts Skp1 proteins may have altered functions. From this we predicted that ts Skp1 proteins would not be defective in binding to cullin-1 (Pcu1), instead interactions with F-box proteins would be compromised in a subtle manner. In order to investigate this idea, we performed binding assays of Pcu1 and the F-box proteins with the Skp1^tsA7 protein. As predicted the binding between Pcu1 and Skp1^tsA7 is not impaired. Furthermore we have found that the interactions of 3 F-box proteins, Pof1, Pof3 and Pof10, are specifically affected. It is of note that the binding between the Skp1^tsA7 protein and these 3 F-box proteins is only partially compromised, 50% reduction in Pof3 and 30% reduction in Pof1 and Pof10. This is again consistent with the prediction that binding between Skp1^tsA7 and F-box proteins would not be completely abolished, instead being partially defective. In order to address the question of why these particular F-box proteins are affected we have compared F-box sequences of Pof1, 3 and 10 with those from the other F-box proteins to look for common characteristics in the sequence of these 3 F-box proteins. The F-box sequences of Pof1, 3 and 10 contain an F-box closer to the canonical F-box sequences, rather than a divergent one. However, we have not noticed any other particular sequences in common in these 3 F-box domains.

Understanding the phenotype of skp1^tsA7 cells

skp1^tsA7 cells display a robust G2 delay phenotype at the restrictive temperature. How does this phenotype arise? Pof10 is a nonessential protein that shows a pop1-like polyploid phenotype upon over-expression due to competitive inhibition of Pop1 binding to Skp1 (Ikebe et al. 2002). Deletion of pof10⁺, however, has no effect on cells. Therefore, it seems unlikely that the reduction of binding of this protein contributes to the skp1^tsA7 phenotype. This leaves the remaining F-box proteins, Pof1 and Pof3 as candidates effectors of the skp1^ts phenotypes. Pof1 is an essential protein and its reduction in binding to Skp1^tsA7 seems likely to be responsible for some of the temperature sensitive phenotypes of these cells. To address this possibility, we have been investigating a physiological role for Pof1 by creating ts mutants (C.H. and T.T. unpublished observation).

The F-box protein that shows the most pronounced reduction in binding to Skp1^tsA7 is Pof3. Given the phenotypic similarities of skp1^tsA7 cells and pof3, such as the activation of the DNA damage checkpoint accompanied with Chk1 phosphorylation and cell elongation, it seems highly likely that the majority of skp1^tsA7 phenotypes are due to the accumulation of Pof3 substrates. This idea is complemented by the finding that a skp1^tsA7 pof3 strain does not show additive phenotypes, the average cell length between pof3 and skp1^tsA7 pof3 is indistinguishable. Another F-box protein that we considered a likely candidate for causing the skp1^tsA7 phenotype of checkpoint activation and cell elongation is Fdh1, which contains both an F-box and a DNA helicase. Deletion of fdh1⁺ also results in a G2 delay with activation of the DNA damage checkpoint (Kim et al. 2002) (A.L. and T.T., unpublished observation). However, fdh1 phenotypes appear not to contribute significantly to those of skp1^tsA7 cells, as the combined effects of skp1^tsA7fdh1 are additive, in contrast to those of skp1^tsA7pof3. Furthermore, binding between Skp1^tsA7 and Fdh1 shows no obvious impairment.

A mechanism for checkpoint activation in skp1^tsA7 cells

In order to explain how skp1^tsA7 cells activate the checkpoint we postulate that a substrate of the SCF accumulates, and this accumulation either results in DNA damage with ensuring checkpoint activation or direct activation of the checkpoint. Our results show that the checkpoint activation may be detrimental to the cell's survival in skp1^tsA7 mutants, in contrast to pof3 cells, which are dependent on checkpoint activation for survival. This suggests that the DNA damage checkpoint has been activated unnecessarily in skp1^tsA7 cells. Although there is no previous precedent for such a scenario in the DNA damage checkpoint, it could be explained in a manner analogous to the spindle assembly checkpoint activation in mph1⁺ over-expressing cells. Accumulation of Mph1 results in a metaphase arrest without spindle damage (Hardwick et al. 1996). Importantly, in the absence of downstream components, such as Mad2, this metaphase arrest is alleviated. Given this analogy, it is feasible that in skp1^tsA7 cells the SCF substrate that accumulates, through reduced activity of Pof3 or another pathway distinct from Pof3 such as Pof1, activates the checkpoint without actual damage to DNA (Fig. 8).

View larger version (16K): [in this window] [in a new window]   Figure 8  A scheme to show how accumulation of an SCF substrate could lead to checkpoint activation. (A) Accumulation of the spindle checkpoint signalling protein Mph1 can activate the spindle assembly checkpoint in conditions where there is no spindle damage resulting in metaphase arrest. By analogy accumulation of some protein (denoted as X) could induce activation of the DNA damage checkpoint regardless of DNA damage but resulting in G2 arrest and cell elongation. (B) When Mad2 is deleted in an Mph1 overproducing cell the checkpoint is now no longer activated. In a similar manner when the DNA damage checkpoint is abrogated through rad3 or chk1 deletion cells no longer elongate or arrest at G2 as the DNA damage checkpoint cannot be activated.

Conserved SCF functions

The SCF complex is highly conserved throughout evolution. Its role as a regulator of the G1/S phase boundary is conserved from yeast, where the SCF^Cdc4 degrades Sic1 (Bai et al. 1996; Feldman et al. 1997), through to humans where the SCF^Skp2 degrades the equivalent CKI, p27^Kip1 (Lisztwan et al. 1998). The DNA damage checkpoint is likewise conserved from yeast to humans and its functional significance in terms of prevention of untimely cell division, resulting in DNA defects that are implicated in many human diseases including cancers, is clear. Thus, an interaction between the SCF and the DNA damage checkpoint pathway is once again likely to be conserved from yeast to humans. The further elaboration of this interaction may provide new components of the ever-expanding DNA damage checkpoint network.

	Experimental procedures

Top Abstract Introduction Results Discussion Experimental procedures References

 
Strains, media, genetic methods and nomenclature

Stains used in this study are listed in Table 3. YE5S was used as rich media and modified synthetic EMM2 as minimal media. Standard techniques were used as described (Moreno et al. 1991) for growth and maintenance of strains. Gene disruptions are indicated by the gene name followed by a symbol, e.g. fdh1. Temperature sensitive mutations of genes are annotated by a ^ts symbol and the allele number given as A, e.g. skp1^tsA7. Proteins are denoted by a capital letter, e.g. Skp1 and the mutant form Skp1^tsA7.

As described (Katayama et al. 2002) F-box ORFs were identified by searches of the Sanger Centre S. pombe geneDB (Sanger Centre, Hixton, UK).

Gene disruption and tagging

All genes were disrupted using PCR generated fragments (Bahler et al. 1998). The 1.8 kb ura4⁺ or 1.6 kb kan^r gene was amplified with flanking sequences corresponding to the 5'- and 3' ends of the relevant genes. These fragments were transformed into a ura4/ura4 diploid. Ura⁺ or G418^r colonies were selected on minimal plates lacking uracil or rich plates containing G418, respectively. Correct disruption of genes of interest was verified by colony PCR using two primers, one from the ura4⁺ or kan^r marker gene and the other from the deleted gene. These heterozygous diploids were then sporulated on plates lacking nitrogen. (Burke et al. 2000) and germination of haploids monitored.

Thiamine repressible promoters with epitope tags 3HA, or GFP were integrated into the genome in front of the initiator ATG codon of genes by a PCR-based gene targeting method (Bahler et al. 1998). C-terminal tagging of genes with 3HA, 13myc or GFP eptiopes was also carried out using PCR generated fragments as described (Bahler et al. 1998). All tagging was confirmed by PCR and Western blotting with specific antibodies.

Nucleic acid preparation and manipulation

Yeast genomic DNA was prepared based on the method previously described (Burke et al. 2000). Cycle sequencing performed in a Peilter Thermal Cycler-200 using ABI prism dye terminator cycle sequencing ready reaction kit and followed by automated read out using a Perkin Elmer sequencer, ABI prism 377.

Mutagenic PCR

PCR under modified conditions was used to create fragments containing randomised mutations. This was based upon methods previously described to attain approximately one mutation per fragment (Cadwell & Joyce 1992; Leung et al. 1989). Generation of mutated PCR fragments was performed with Taq DNA polymerase. An increased amount of one purine and one pyrimidine were used to prevent mutational bias. These were used as follows: dCTP and dTTP at a final reaction concentration of 1 mM and dATP and dGTP at a final concentration of 0.2 mM. Template was supplied at 50 ng per reaction and primers at 100 ng per reaction. In addition 5 mM Mg²⁺ ions were supplied for each reaction. The total reaction volume was 100 µL and contained 5 units of enzyme. The step-wise method for creation of ts skp1 genes is described in Fig. 2.

Immunochemical assays

For immunoprecipitation analysis 2 mg of soluble protein extract was prepared using glass beads disruption in RIPA lysis buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) following the method previously described (Moreno et al. 1991). Following lysis with glass beads the extract was clarified and the protein concentration measured using the Bradford Assay (Bio-Rad, Hercules, CA, USA). 2.5 µg of polyclonal anti-Skp1 antibody (see below) was incubated with the protein extract at 4 °C for 1 h before the addition of protein A Sepharose beads (Affi-prep ® Bio-Rad) prewashed in RIPA buffer. Beads and extract were further incubated at 4 °C for an hour. The protein A beads were then washed 8 times in RIPA buffer.

Mouse monoclonal anti-myc (9E10) antibodies were purchased from BAbCO (Richmond, CA, USA) as were mouse monoclonal anti-HA (12CA5) antibodies. Mouse monoclonal anti-GFP antibodies were purchased from Roche (Roche molecular diagnostics, Indianapolis, IN, USA). Horseradish peroxidase conjugated goat anti-rabbit IgG and goat anti-mouse IgG (Bio-Rad) and a chemiluminescence system (Enhanced Chemiluminescence Amersham plc, Little Chalfont, Bucks, UK) were used to detect bound antibodies. Phosphorylation of Chk1-13myc was examined using a 10% SDS-polyacrylamide gel (SDS-PAGE) in which the ratio of acrylamide:bis-acrylamide is 200 : 1 (Katayama et al. 2002).

Preparation of polyclonal anti-Skp1 antibody cDNA containing the entire ORF of fission yeast skp1⁺ was amplified using an S. pombe cDNA library as template (Clontech, Palo Alto, CA, USA). Amplified skp1⁺ was cloned into expression vector pET-14b (Novagen, Madison, WI, USA). 6-His tagged Skp1 fusion protein was purified on Ni² ± NTA beads (QIAGEN, Valencia, CA, USA) as recommended by the manufacturer. Crude anti-Skp1 serum was affinity purified using Skp1 fusion protein immobilized on nitrocellulose filters.

Quantification of immunochemical assays

The computer program NIH image was used to quantify the intensity of bands resulting from Western blots after immunoprecipitation. A scanned image is analysed for the pixel density of any one area. A rectangular area of band on the Western blot was chosen and mean pixel density within that area calculated. All pixel densities were normalized for background. In order to verify the quantification, two to three ECL images, which were obtained from different exposure times, were examined.

	Acknowledgements

 
We thank Drs Tony Carr, Keith Gull and Paul Nurse for strains and plasmids. We thank Dr Judith Murray-Rust for helping structural modelling of Skp1^ts proteins and Dr Julie Promisel Cooper for critical reading of the manuscript. S. K. was supported by JSPS Postdoctoral Fellowships for Research Abroad. This work is supported by Cancer Research UK and the Human Frontier Science Program Research Grant.

	Footnotes

 
Communicated by: Mitsuhiro Yanagida

Present address: ^aAnalytical Research Centre for Experimental Sciences, Saga University, Saga 840-8502 Japan,

^bCentre for Gene Science, Hiroshima University, 1-4-2 Kagamiyama, Higashi-Hiroshima 739-8537, Japan

^* Correspondence: E-mail: toda{at}cancer.org.uk

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Received: 29 December 2003
Accepted: 1 February 2004

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