Nuclear distribution and chromatin association of DNA polymerase α-primase is affected by TEV protease cleavage of Cdc23 (Mcm10) in fission yeast
© Yang et al; licensee BioMed Central Ltd. 2005
Received: 04 February 2005
Accepted: 07 June 2005
Published: 07 June 2005
Cdc23/Mcm10 is required for the initiation and elongation steps of DNA replication but its biochemical function is unclear. Here, we probe its function using a novel approach in fission yeast, involving Cdc23 cleavage by the TEV protease.
Insertion of a TEV protease cleavage site into Cdc23 allows in vivo removal of the C-terminal 170 aa of the protein by TEV protease induction, resulting in an S phase arrest. This C-terminal fragment of Cdc23 is not retained in the nucleus after cleavage, showing that it lacks a nuclear localization signal and ability to bind to chromatin. Using an in situ chromatin binding procedure we have determined how the S phase chromatin association of DNA polymerase α-primase and the GINS (Sld5-Psf1-Psf2-Psf3) complex is affected by Cdc23 inactivation. The chromatin binding and sub-nuclear distribution of DNA primase catalytic subunit (Spp1) is affected by Cdc23 cleavage and also by inactivation of Cdc23 using a degron allele, implying that DNA polymerase α-primase function is dependent on Cdc23. In contrast to the effect on Spp1, the chromatin association of the Psf2 subunit of the GINS complex is not affected by Cdc23 inactivation.
An important function of Cdc23 in the elongation step of DNA replication may be to assist in the docking of DNA polymerase α-primase to chromatin.
Proteases play key roles in the regulation of many cellular processes, such as signal transduction, apoptosis, and the activation of chromosome disjunction in mitosis [1–4]. Artificial proteolytic regulation is possible using TEV protease, which recognizes a highly specific sequence  and is not deleterious when expressed in a variety of cell types [6–11]. Artificial cleavage of target proteins, engineered to contain a TEV cleavage sequence (Tcs), can thus be effected by TEV protease expression in vivo. TEV protease-mediated cleavage has been used in topological studies of protein location  and to study the role of regulatory proteolysis, such as in analysis of separase function . Proteolytic cleavage can be used to determine how removal of specific domains of a protein affects its function  and, by removing a signal that targets the protein to a specific compartment, can effect a change in a protein's cellular localization .
We investigate here the utility of TEV-mediated cleavage of nuclear proteins in fission yeast. We demonstrate that a strain expressing nuclear-targeted TEV protease has no growth defects compared to a wild-type strain and shows efficient cleavage of nuclear proteins containing a Tcs. We use TEV protease-mediated cleavage to investigate the function of Cdc23 (homologous to Mcm10 in other organisms), which is an essential replication protein, required for the initiation and elongation steps of DNA replication ([14–17], see [18, 19] for reviews). Cdc23/Mcm10 is chromatin associated, and in S. cerevisiae and vertebrate cells this association occurs during S phase [20–22], but its biochemical function is unclear. In S. cerevisiae, Mcm10 was originally implicated in pre-replicative complex formation, when Mcm2-7 proteins associate with ORC at replication origins . Subsequent studies in both yeasts and Xenopus have shown that Mcm10 is required for the later step of replication initiation, as the protein is needed for chromatin association of Cdc45 [21, 24, 25]. In vitro, fission yeast Cdc23 enhances the ability of Hsk1 (Cdc7) protein kinase to phosphorylate Mcm2 , and binds to the catalytic subunit of DNA polymerase α-primase (pol α-primase), catalytically activating its DNA polymerase activity . Very recently, Ricke and Bielinsky have shown that Mcm10 is required for the stability of the pol α catalytic subunit in S. cerevisiae .
In this paper, we use TEV protease cleavage of Cdc23 to show that removal of a 170 aa C-terminal domain of the protein, previously with no attributed functions, blocks DNA replication. Regulation of TEV protease under the thiamine-repressible nmt1 promoter in conjunction with a Tcs-containing allele of cdc23 can thus be used to inactivate conditionally the protein. We show in addition that inactivation of Cdc23 affects the chromatin binding and nuclear distribution of the DNA primase catalytic subunit (Spp1) of pol α-primase.
Expression of TEV protease in S. pombe and cleavage of Mcm7-Tcs-GFP
Inactivation of Cdc23 by TEV protease cleavage
To determine if removal of a C-terminal domain of the Cdc23 replication factor affects the function of the protein, we inserted a Tcs just after serine 424 of a CFP-tagged version of Cdc23, 170 aa from the C terminus, thus generating Cdc23S424::Tcs, (Fig 2A). Amino acids 1-424 of Cdc23 contains most domains with ascribed functions and it was of interest to determine how cleavage after this region would affect the function of the protein. A poorly conserved region was chosen for the Tcs insertion to minimize the possibility that this modification alone would inactivate the protein. The expression level of Cdc23S424::Tcs is the same as the wild-type protein (Fig. 2C). A strain containing this insertion is viable at all temperatures in the absence of TEV protease, implying that the Tcs insertion does not affect Cdc23 function, but induction of the TEV protease prevents growth of this strain (Fig. 2B). Unexpectedly, this effect shows temperature-sensitivity, in that the strain grows at temperatures up to about 32°C, but is not viable at 37°C. The cleavage site used generates a C-terminal fragment with a stabilizing N-terminal amino acid (serine) as predicted by the N-end rule , and we could detect two fragments of the expected size after induction of the protease (Fig. 2C). The proportion of full-length protein is reduced at 37°C on TEV protease expression, consistent with the temperature-sensitivity of the strain. The effect is unlikely to be due to an increase in TEV protease activity, since the enzyme is more active at 32°C than 37°C  and we observed efficient cleavage of a different substrate (Mcm7-GFPN760::Tcs) at 32°C (Fig. 1). Instead, the Tcs in Cdc23 is presumably more accessible to the protease at higher temperatures, perhaps due to a change in conformation of Cdc23, allowing more efficient cleavage to occur.
Nuclear localization or chromatin binding of Cdc23 is not specified by the C-terminal region of the protein
Cleavage of Cdc23 affects the chromatin association and nuclear distribution of DNA polymerase α-primase
Cleavage of the Cdc23S424::Tcs protein in a strain also expressing Spp1-GFP was effected by inducing the TEV protease at 25°C, and then shifting to 37°C. In a wild-type strain, this temperature shift has no effect on Spp1-GFP chromatin binding (data not shown). In contrast to the effect of HU arrest on Spp1 chromatin binding, Cdc23S424::Tcs cleavage caused a reduction in the proportion of cells retaining chromatin-associated Spp1 (from 14% to 3%; Fig. 5F). A reduction was also seen in thiamine-containing medium, probably reflecting leaky expression of TEV protease from the nmt1 promoter under repressing conditions, leading to some Cdc23 inactivation. We also observed a reduction in Spp1 chromatin binding when TEV protease was induced at 37°C in the absence of a temperature shift (data not shown). In addition to an effect on Spp1 chromatin binding, we observed a striking increase in the percentage of cells containing 1–2 bright foci of Spp1-GFP after Cdc23 inactivation (from 2% to 23%; Fig. 5F). These were generally nuclear or peri-nuclear but not necessarily co-localizing with DAPI-staining chromatin regions and may reflect formation of insoluble aggregates of Spp1 after Cdc23 inactivation. These foci were not observed during an HU-induced S phase arrest (Fig. 5C), so they appear to be a specific response to Cdc23 inactivation.
Chromatin association of pol α-primase has been reported to be dependent on Cdc45 [37, 39]. Since Cdc23/Mcm10 can affect Cdc45 chromatin association [21, 24, 25], the effect on pol α-primase could be indirectly mediated by Cdc45. We therefore examined chromatin association of Cdc45-YFP after TEV protease inactivation of Cdc23S424::Tcs. In contrast to the effect on Spp1, Cdc45 chromatin association was not prevented by TEV protease cleavage of Cdc23 and in fact there was an increase in the percentage of cells with chromatin-bound Cdc45 (Fig. 5G). This is similar to what is observed in an HU arrest [25, 40], presumably reflecting accumulation of cells blocked in S phase. This result suggests that the essential function of the C-terminal region of Cdc23 is not connected to Cdc45 chromatin association and may be more directly related to pol α-primase function.
The GINS complex has been shown to be loaded onto chromatin at initiation in S. cerevisiae and Xenopus [42–45] and we examined whether this complex is also affected by disruption of Cdc23 function. We first determined whether GINS shows cell cycle dependent changes in detergent extractability. A strain where the Psf2 subunit of GINS is C-terminally tagged with YFP is viable and shows nuclear localization throughout the cell cycle (Fig. 6E). After detergent extraction, Psf2 is only retained in binucleate cells, and is resistant to detergent extraction in HU-arrested cells (Fig. 6B, E), suggesting that the protein is chromatin associated in S phase similar to results obtained with Spp1. After shifting the cdc23tstd strain to the non-permissive temperature, Psf2 becomes resistant to detergent extraction, similar to what is observed in an HU arrest (Fig. 6C, E). Similar results were obtained after TEV protease cleavage of Cdc23 (data not shown). This suggests Cdc23 is not required to maintain Psf2 chromatin association and execution of the Cdc23 function is needed for displacement of Psf2 in the course of S phase.
In this paper we demonstrate that in vivo cleavage by TEV protease can be used to inactivate a modified version of the Cdc23 replication factor, engineered to contain a cleavage site for the protease. We infer that the C-terminal 170 aa region is essential for the DNA replication function of Cdc23. Since N- and C-terminal fragments appear to be stable after cleavage, the C-terminal region must require covalent linkage to the rest of the protein for its function. Although no clear sequence motifs have been identified in the C-terminal region, this part of the protein could have a discrete function that requires linkage to the N-terminal domain. Alternatively, interaction between the N and C-terminal regions could simply be necessary for the function of the protein in a manner which is affected by TEV protease cleavage. This function does not appear to involve nuclear localization or chromatin binding, since nuclear localization of the C-terminal fragment is lost after Cdc23 cleavage (Fig. 4).
We also show that inactivation of Cdc23, either by proteolytic cleavage or using a ts degron allele, affects the chromatin association and nuclear distribution of Spp1 primase subunit (Figs. 5, 6), suggesting that an essential role of Cdc23 is related to recruitment of pol α-primase to chromatin. Cdc45 has also been implicated in binding of pol α-primase to chromatin [37, 39], but in this context it may not be relevant. Although complete inactivation of Cdc23/Mcm10 can block chromatin association of Cdc45 [21, 24, 25], TEV protease cleavage of Cdc23 does not prevent Cdc45 chromatin binding. One interpretation of these results is that Mcm10/Cdc23 has two functions, one related to pol α primase function, perhaps related to the C-terminal domain cleaved off by TEV protease, and the other related to Cdc45 chromatin binding.
These results are consistent with in vitro effects of S. pombe Cdc23 on pol α-primase activity , and evidence that S. cerevisiae Mcm10 promotes chromatin binding of the large primase subunit (Pri2) independently of effects on Cdc45, based on histone cross-linking experiments . The observations we have made for Spp1-GFP may also apply to the three other subunits of pol α-primase (p49/Spp2, p70/B-subunit, p180/Pol1). However, given the requirement for S. cerevisiae Mcm10 for stability of the p180 polymerase catalytic subunit  and the constitutive chromatin binding of at least a fraction of S. pombe p70 , further work will be needed to determine the fate of other pol α primase subunits on Cdc23 inactivation.
In contrast to effects on Spp1, chromatin association of the Psf2 subunit of GINS is seen in cells arrested in S phase by using a degron allele of cdc23 (Fig. 6), similar to the result obtained by an HU arrest of DNA replication. This suggests that maintenance of GINS chromatin association is independent of Cdc23 function and that Psf2 displacement requires Cdc23 function in the elongation step of replication. At first glance, this result is inconsistent with previous work linking Cdc45 with GINS function. Specifically, chromatin association of Sld5 in Xenopus extracts is blocked by Cdc45 depletion , and in S. cerevisiae, the Sld3 partner of Cdc45 is essential for Psf1 origin association . However, degradation of Cdc23 using the cdc23tstd allele used in our experiments is inefficient in cycling cells  and Cdc45 chromatin binding is not blocked under conditions of the Psf2 experiment shown in Fig. 6 (see Additional file: 1).
In this and a related study  we have demonstrated the utility of TEV protease for analysis of protein function in fission yeast. TEV protease can be used to determine the role of specific domains of the protein and this method can be used for topological analysis of protein function by restricting TEV protease expression to specific compartments. For this approach to be useful, the target protein must tolerate the insertion of a Tcs, which was an issue with our related study on Pol3 function  and the Tcs must be accessible to TEV protease in the folded protein. In this study, cleavage of Cdc23S424::Tcs was more efficient at higher temperatures, possibly reflecting a conformational change in Cdc23 which makes the Tcs more accessible to the protease. Alternatively, the interaction of Cdc23 with other factors might be changed by temperature in a manner which affects access to the Tcs.
In the approach used here, the Tcs generates a C-terminal fragment with serine as the N-terminal amino acid, which is predicted to generate a stable protein. However, TEV protease also cleaves sequences such as ENLYFQ∨R [5, 46], which generates a C-terminal fragment with a destabilizing N-terminal amino acid according to the N-end rule. Such a module can be inserted at the N-terminus of the target protein, which may be more tolerated and accessible than insertion of the Tcs at internal sites. We are currently investigating whether this strategy provides a simpler approach to effect rapid inactivation of target proteins.
We show that TEV protease can be used in S. pombe to inactivate target proteins engineered to contain a protease cleavage site. Using this approach we show that the C-terminal domain of the Cdc23/Mcm10 replication factor, previously with no attributed functions, is essential for DNA replication. Inactivation of Cdc23/Mcm10 affects the chromatin binding and nuclear distribution of pol α-primase, suggesting it may help to attach the polymerase to the replication fork. In contrast, Cdc23/Mcm10 inactivation does not prevent maintenance of chromatin binding of the GINS elongation factor, as judged by analysis of the Psf2 subunit.
Fission yeast strains
S. pombe strains used
ade6-M210 leu1-32 ura4-D18 h -
spp1-GFP::kan r ade6-M120 leu1-32 ura4-D18 h -
cdc45(sna41)-YFP::ura4 + ade6-M210 leu1-32 ura4-D18 h -
cdc45(sna41)-YFP::ura4 + nmt1*cdc23-1E2-td::kan r h -
mcm7-GFP N 760 ::Tcs ::ura4 + ade6-M210 leu1-32 ura4-D18 h - [pREP3X-TEV-NLS(LEU2)]
spp1-GFP::kan r nmt1*-cdc23-1E2-td::kan r leu1-32
mcm7-GFP N 760 ::Tcs ::ura4 + ade6-M210 leu1-32 ura4-D18 h -
ade6-M210 leu1-32 ura4-D18 h - [pREP3X-TEV-NLS(LEU2)]
cdc23-CFP S 424 ::Tcs::ura4 + ade6-M210 leu1-32 ura4-D18 h -
cdc23-CFP S 424 ::Tcs::ura4 + ade6-M210 leu1-32 ura4-D18 h - [pREP3X-TEV-NLS(LEU2)]
cdc23-CFP S 424 ::Tcs::ura4 + leu1-32 ura4-D18 h - [pREP3X-TEV-NLS(LEU2)]
cdc45(sna41)-YFP::ura4 + cdc23-CFP S 424 ::Tcs::ura4 + leu1-32 ura4-D18 ade6 [pREP3X-TEV-NLS(LEU2)]
psf2-YFP::kan r ade6-M210 leu1-32 ura4-D18 h -
psf2-YFP::kan r nmt1*-cdc23-1E2-td::kan r
spp1-GFP::kan r cdc23-CFP S 424 ::Tcs::ura4 + leu1-32 ura4-D18 ade6 [pREP3X-TEV-NLS(LEU2)]
Construction of a plasmid expressing TEV protease under nmt1 regulation(pREP3X-TEV-NLS)
A fragment encoding NLS-myc9-TEV-NLS-NLS was amplified from plasmid YIp204 (GAL-NLS-myc9-TEV proteinase-NLS-NLS, ) using oligonucleotide primers 5'XhoITEV204 (TTTCTCGAGAGAAAAAACCCCGGATCTATGCCAA) and 3'SmaITEV204 (AAACCCGGGGCCAAGCTTGCATGCCTGCAGTTAATC) and cloned into XhoI and SmaI sites of the plasmid pREP3X , to generate the pREP3X-TEV-NLS plasmid.
Construction of mcm7 and cdc23 alleles containing TEV cleavage sites
To construct mcm7-GFP N760 ::Tcs, we first constructed a plasmid pSMUG2+mcm7 by subcloning the ApaI-SmaI fragment from pSMUC2+mcm7  into pSMUG2+ . The sense oligonucleotide 5'EspI-TEV-KasI-F (CCGGGTTACTTCTTTAGAAGTTGGTCGTCGCTTTTCTCCTGATGAAAATTTGTACTTCCAATCTGGCGC) and anti-sense oligonucleotide 5'KasI-TEV-EspI-R (CCGGGCGCCAGATTGGAAGTACAAATTTTCATCAGGAGAAAAGCGACGACCAACTTCTAAAGAAGTAAC) were annealed and cloned into the XmaI site of pSMUG2+mcm7. This was used to tag the endogenous mcm7+ gene by cutting with MluI and transforming into strain P138 to generate P1288. The sequence inserted after the terminal amino acid of Mcm7, before the GA linker-GFP module is RVTSLEVGRRFSPDENLYFQS (underlined sequence is the Tcs).
Two rounds of PCR were performed to construct the cdc23 S424 ::Tcs allele. Initially, two PCR reactions were set up, using plasmid pSMUC2+Cdc23 as DNA template, using the primers: (i) 5'ApaIMCM10 (AAAGGGCCCGAACCA AGAAAGGAAGAGGAGCGATAA) and 3'MCM10tevAclI1010 (CCCGTAGAG AGGTTATTCGACTGGAAGTACAACGTTTCGGAAGCGCAACATCGC); (ii) 5' MCM10tevAclI1010 (GCGATGTTGCGCTTCCGAAACGTTGTACTTCCAGTC GAATAACCTCTCTACGGG) and 3' ATGCYfp-screen (ACAGCTCCTCGCCCTTGCTCACCAT). Products from the two PCR reactions were mixed and boiled and used for a secondary PCR reaction using primers 5'ApaIMCM10 and 3' ATGCYfp-screen. The PCR product was cloned into ApaI and HindIII sites of plasmid pSMUC2-Cdc23 . This was used to tag endogenous cdc23+ gene by cutting with SpeI and transforming into strain P138 to generate P1322. All constructs were verified by sequencing.
Construction of strains expressing fluorescently-tagged Spp1 and Psf2
The catalytic subunit of DNA primase (Spp1) was tagged with GFP using the oligos p48F1 (TGTACTTTAAATCGTTCAGTAGCCAGCTTTTTAAAGAAACAGTAGGAAATAAAAGAAAACATGAGAATTTGGAATTTCGGATCCCCGGGT) and p48R1 (GATTTAACGGAAAACAATATGCTCCACGTAAATAGAAGTCAGAGCTTCCATCCTTTCATGAGTAAATGTATGCCCAAATGAATTCGAGCTCGTTTAAACT) using pFA6a-GFP-kanMX6 as template  to generate a PCR product that was used to tag the endogenous Spp1 gene by transforming strain P138. Psf2 was tagged with YFP using the oligos 5'ApaI-Psf2 (CTTGGGCCCATACGAGATGATGAATTGGAAAACG) and 3'XhoIPsf2 (TTTCTCGAGTTCTTCTTGGGAAACTTGAACAAT) which was cloned into pSMUY2+ ; cleavage of this plasmid with EcoRV was used to tag the endogenous psf2+ gene in strain P138 with YFP, generating strain P1411. All constructs were verified by sequencing. Strains containing Spp1-GFP or Psf2-YFP showed tagged proteins with the expected molecular weights when analyzed by western blotting, using anti-GFP antibody (data not shown).
Chromatin binding assay
Chromatin binding assay and image analysis was carried out as previously described for Psf2 [35, 50]. For analysis of Spp1, a low-salt extraction buffer was used with the following composition: 20 mM Pipes-KOH pH 6.8, 0.4 M sorbitol, 10 mM KAc, 0.5 mM spermidine, 0.15 mM spermine, 1 mM EDTA. Extraction with higher salt buffers results in loss of Spp1 from S phase cells (data not shown). Data shown are the averages of at least two experiments. Nuclear retention of Spp1-GFP and Psf2-YFP after detergent extraction was abolished after digestion of chromatin with micrococcal nuclease (data not shown). Chromatin binding assays were performed at least twice and error bars show the statistical range. At least 100 cell were counted for each data point.
Flow cytometry was carried out using sytox green (1 μm) as previously described .
Protein extracts for Western blotting were made by trichloroacetic acid extraction as previously described . For western blot analysis, antibodies against full-length Cdc23 (from S. Aves ), GFP (3E1 monoclonal), TEV protease (from M. Ehrmann), and α-tubulin (Sigma T5168) were used. Detection was performed using the chemiluminescence procedure.
This work was supported by grants from the BBSRC (43/G15095) and Cancer Research-UK. We are grateful to Michael Ehrmann and Steve Aves for generous gifts of antibody and Frank Uhlmann for plasmids. We are grateful to Lynne Larkman for technical support and to Shao-Win Wang for comments.
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