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J. Biol. Chem., Vol. 279, Issue 16, 16144-16153, April 16, 2004
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-Primase Is Influenced by Its Interaction with Mcm10p*
**
From the
Program in Molecular Biology, Memorial-Sloan Kettering Cancer Center, New York, New York 10021, the ¶Department of Biology Education, Seoul National University, Seoul 151-748, Korea, the Department of Biochemistry, Ajou University School of Medicine, Suwon 442-749, Korea, and the ||UCLA School of Medicine, Microbiology and Immunology, Center for Health Sciences, Los Angeles, California 90024-1747
Received for publication, January 7, 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-primase complex in vitro by interacting specifically with the catalytic p180 subunit and stimulates DNA synthesis catalyzed by the pol -primase complex with various primed DNA templates. We investigated the mechanism by which Mcm10p activates the polymerase activity of the pol -primase complex by generating truncated derivatives of the full-length 593-amino acid Mcm10p. Their ability to stimulate pol polymerase activity and bind to single-stranded DNA and to pol were compared. Concomitant with increased deletion of the N-terminal region (from amino acids 95 to 415), Mcm10p derivatives lost their ability to stimulate pol polymerase activity and bind to single-stranded DNA. Truncated derivatives of Mcm10p containing amino acids 1–416 retained the pol binding activity, whereas the C terminus, amino acids 496–593, did not. These results demonstrate that both the single-stranded DNA binding and the pol binding properties of Mcm10p play important roles in the activation. In accord with these findings, Mcm10p facilitated the binding of pol -primase complex to primed DNA and formed a stable complex with pol -primase on primed templates. A mutant that failed to activate or bind to DNA and pol , was not observed in this complex. We suggest that the interaction of Mcm10p with the pol -primase complex, its binding to single-stranded DNA, and its activation of the polymerase complex together contribute to its role in the elongation phase of DNA replication. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The DNA polymerase -primase (pol -primase) complex also plays an essential role in initiation by synthesizing short RNA primers required to begin both leading and lagging strand DNA synthesis. The four subunit structure of pol -primase is conserved, and each subunit is essential for cell viability in yeast (5, 6). The largest subunit of pol -primase (p180) contains the DNA polymerase catalytic center (7) and elongates RNA primers of 8–12 nucleotides synthesized by primase. The catalytic center of primase is located within the p48 subunit (8), which exists as a tight complex with p58 (9, 10). The p58 subunit was shown to be required for distinct aspects of primer synthesis, including initiation and elongation (11). The fourth subunit of DNA pol -primase, p70/
, is suggested to recruit the complex onto chromatin as a result of its cell cycle-regulated phosphorylated state (12, 13). Polymerase -primase subunits have been reported to interact with a number of replication proteins including Cdc45 (14), Dna2 (15), RPA (16, 17), and viral initiator proteins (16, 18, 19), consistent with the idea that replication forks are large precisely assembled multiprotein complexes.
Mcm10p has been implicated both in the initiation and elongation steps of DNA replication. MCM10 was first identified in Saccharomyces cerevisiae using screens for mutants defective in DNA replication and for the stable maintenance of plasmids (20, 21). Initiation at replication origins is drastically reduced in the mcm10-1 mutant, and replication across origins is impeded (21, 22). Mutations in MCM10 result in a delay in the completion of DNA synthesis after cells are released from HU arrest (23), suggesting that Mcm10p is essential for continued fork progression. MCM10 mutants are suppressed by mutant MCM5 and MCM7 genes and are synthetically lethal with mutant genes of ORC, CDC45, DNA2, DPB11, and genes encoding subunits of DNA polymerase 
and 
(22–27). Seven new mutants named slm1–slm6 for synthetically lethal with mcm10 include mutations in genes that are allelic to MCM7, MCM2, CDC45, DNA2, and mutations in novel DNA repair genes represented by SLM2 and SLM6 (28). Biochemical and genetic interactions have been reported between Mcm10p and members of the Mcm2-7p family and the S. pombe Dfp1p/Dbf4p (29). These interactions lead to the stimulation of the Hsk1/Cdc7-Dfp1/Dbf4p kinase-catalyzed phosphorylation of Mcm2p and Mcm4p in vitro (30). In S. cerevisiae, Mcm10p is required for the association of Mcm2-7p complex with origins at early stages of replication (22). However, depletion of Mcm10p from Xenopus extracts affects a later step of DNA replication, blocking the chromatin association of Cdc45p, events that lead to DNA unwinding, suggesting that Mcm10p is required for progression of the pre-RC to subsequent replication stages (31). This is in agreement with recent results in S. pombe, where Cdc23 was shown to be required for Cdc45 chromatin association and did not affect earlier stages of initiation (32). Thus, Mcm10p appears to play important roles at all stages of DNA replication, like Cdc45 and the Mcm2-7 complex, and may be situated at the moving replication fork in association with the elongation machinery.
In the course of studies with S. pombe Mcm10p/Cdc23p, we detected an interaction between Mcm10p and the pol -primase complex. This interaction was specific for the catalytic subunit of the polymerase complex and prompted us to examine the influence of Mcm10p on the catalytic activity of pol -primase. In this report, we demonstrate that Mcm10p can markedly stimulate the DNA polymerase activity of the complex and appears to do so by interacting with both single-stranded DNA and the p180 subunit, thus facilitating the binding of the polymerase complex to primed DNA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-primase complex were constructed by subcloning the cDNAs encoding full-length proteins into pFastBac1 (Invitrogen). pFastBac-p180 was constructed by PCR amplification of pREP81-p180 (kindly provided by T. S. Wang, Stanford University, Palo Alto, CA) using the primers p180-1 (5'-TGT GCG CGC AAT CAT GAG AAA GAG AAA CGC-3') and p180-2 (5'-AAA CTG CAG TCA CGA TGA AAA TAT CAG TCC-3') to obtain the non-tagged p180 clone. Primers p180-1 and p180-3 (5'-AAA CTG CAG CGA TGA AAA TAT CAG TCC CAT-3') were used to obtain the C-terminal His6-2Flag-tagged p180 clone. These products, as well as those described below, were subcloned into the BssHII and PstI sites of pFastBac1. pFastBacp70 was constructed by PCR amplification using primers p70-1 (5'-TGT GCG CGC AAC CAT GGA ATT TCC TAT CGA TGA TG-3') and p70-2 (5'-AAA CTG CAG CTA ACG GTC TAT GGT AGA C-3') to obtain the untagged p70 clone. Primers p70-1 and p70-3 (5'-AAA CTG CAG TAG TTT TAA AAT TTC TGC CTT G-3') were used to construct the C-terminal His6-2Flag-tagged p70 clone. pFastBac-p58 was formed by PCR amplification of pREP81-spp2 using primers pSpp2-1 (5'-TGT GCG CGC AAC CAT GTT CAG AAC GAC CAA-3') and pSpp2-2 (5'-AAA CTG CAG AGC TTA TGA TTC TAA ACT AAG TTG-3') to obtain the untagged p58 clone, or primers pSpp2-1 and pSpp2-3 (5'-AAA CTG CAG CGA TGA AAA TAT CAG TCC CAT-3') to obtain the C-terminal His6-2Flag-tagged p58 clone. pFastBac-p48 was prepared as described for pFastBac-p58, except that the PCR primers used were pSpp1-1 (5'-TGT GCG CGC AAC CAT GAC TGT TCA AAT CGA TGA ATT G-3') and pSpp1-2 (5'-AAA CTG CAG AGC TTC CAT CCT TTC ATG-3') to synthesize the non-tagged p48, or primers pSpp1-1 and pSpp1-3 (5'-AAA CTG CAG AAA TTC CAA ATT CTC ATG TTT TCT TTT-3') to obtain the C-terminal His6-2Flag-tagged p48 clone.
Expression and Purification of S. pombe pol -Primase Subunits and Complexes—Sf9 cells were grown in Grace's medium, supplemented with 10% fetal calf serum. Co-infections with different combinations of viruses were used to prepare the pol -primase subcomplexes (Fig. 1). The four-subunit pol -primase complex was isolated from Sf9 cells infected at a multiplicity of infection of 10 in a 2-liter flask (250 ml of 2.0 x 106 cells/ml). Cells were harvested, lysed in 20 ml of Buffer L (50 mM Hepes-KOH, pH 7.4, 0.1 M sodium glutamate, 10 mM MgC12, 1 mM KC1, 0.5% Nonidet P-40, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM benzamidine), incubated on ice for 15 min, followed by Dounce homogenization (10 strokes with a B pestle), sonication (three times each for 1 min), and centrifugation at 16,000 x g for 30 min at 4 °C. The supernatant (20 ml, 21.5 mg/ml) was mixed with 2 ml of Ni-NTA beads, the mixture rotated at 4 °C for 3 h, and bound proteins eluted in Buffer E (50 mM Hepes-KOH, pH 7.4, 0.1 M sodium glutamate, 0.2 M imidazole, 1 mM MgC12, 10 mM KC1, 0.1% Nonidet P-40, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM benzamidine). Approximately 8 mg of protein was obtained from this step. To obtain homogeneous pol -primase, the Ni-NTA fraction (350 µg) was subjected to glycerol gradient sedimentation on a 15–40% gradient. Approximately 23 µg of protein of the pure complex was recovered in peak fractions.
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-D-galactopyranoside and incubated for 7 h at 16 °C. Cells were harvested and resuspended in 35 ml of lysis buffer (50 mM Tris-HC1, pH 8.0, 0.5 M NaC1, 0.5% Triton X-100, 10% glycerol, 1 mM PMSF, 10 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM benzamidine). Cells were incubated for 1 h on ice in lysis buffer with 50 µg/ml lysozyme, disrupted by sonication, and centrifuged at 16,000 x g for 30 min at 4 °C. The supernatant was mixed with 3 ml of Ni-NTA beads (Invitrogen) pre-equilibrated with Buffer H/0.5 (50 mM Hepes-KOH, pH 8.0, 0.5 M NaC1, 10% glycerol, 10 mM MgC12, 1 mM PMSF, 10 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM benzamidine) and incubated at 4 °C for 3 h with rocking. The beads were washed three times with 10 bead volumes of Buffer H/0.5 containing 25 mM imidazole, and bound proteins were eluted with an equal bead volume of Buffer H/0.5 containing 0.2 M imidazole, yielding 
6 mg of Mcm10p. This material was diluted 2.0-fold in Buffer H plus 0-0.25 M NaC1 and applied to a heparin-agarose column (0.9 cm x 1.5 cm). The column was washed with two column volumes of Buffer H, 0.3 M NaC1, and bound proteins eluted with Buffer H, 0.6 M NaC1. The eluted protein peak (2 mg of protein) was pooled and an aliquot (250 µg of protein) was applied to a Sephadex-200 column (HR 10/30) in Buffer H, 0.4 M NaC1 to yield 100 µg of purified protein. Mcm10p preparations from the heparin-agarose step were used in immunoprecipitation, and nitrocellulose DNA binding reactions and Sephadex-200 column preparations were used in the replication and formation of DNA-protein complex assays described below. Truncated Mcm10p derivatives, Mcm10p-(96–593) containing amino acids 96–593, Mcm10p-(295–593), Mcm10p-(1–497), Mcm10p-(1–303), and Mcm10p-(1–230) were also cloned into a pET28 vector, expressed in E. coli BL21(DE3), and purified in a similar manner. The Mcm10p-(416–593) derivative did not bind to heparin-agarose, and for this reason this step was replaced by Q-Sepharose chromatography in Buffer H, 0.05 M NaC1 and Mcm10p-(416–593) was eluted with Buffer H, 0.15 M NaCl.
Interactions between pol -Primase Subunits and Mcm10p—Purified proteins (2 pmol each), shown in Fig. 1, were combined and incubated in 0.5 ml of Buffer B (50 mM Tris-HCl, pH 7.4, 100 mM NaC1, 10 mM MgC12, 1 mM KC1, and 0.1% Nonidet P-40) at 4 °C for 1 h and then mixed with 30 µl of anti-Flag M2 antibody-agarose beads in the absence or presence of 0.1 mg/ml Flag peptide. After incubation for 1 h at 4 °C, beads were collected by centrifugation, washed three times with 0.3 ml of Buffer B, resuspended in SDS sample buffer, and subjected to SDS-PAGE and immunoblotting using anti-Mcm10p. In Mcm10p immunoprecipitation experiments, 2.5 µl of Mcm10p polyclonal antibodies or pre-immune serum were added to an incubated mixture of Mcm10p and pol -primase. After 1 h at 4 °C, 30 µl of protein A-agarose beads were added, and proteins bound were analyzed by SDS-PAGE and immunoblotting with anti-Flag.
In vitro transcription/translation-immunoprecipitation experiments with Mcm10p derivatives were performed (see Fig. 5C). Radiolabeled, in vitro translated full-length and truncated derivatives of Mcm10p were incubated with 2 pmol of purified pol -primase complex in an immunoprecipitation experiment as described above, except that the omission of pol -primase was used as a negative control. After incubation and washing, Laemmli loading dye was added and the proteins were subjected to SDS-PAGE and quantitated with a Fuji imager.
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DNA Replication Assays—DNA polymerase activity was measured using three different DNA templates: hairpin A, hairpin C, and the primed 89-mer TG40 repeat. Reaction mixtures (20 µl) contained 50 mM Hepes buffer, pH 7.0, 10 mM magnesium acetate, 75 mM KCl, 50 mM sodium glutamate, 1 mM DTT, 0.1 mg/ml BSA, 0.05% Nonidet P-40, 20 µM dNTPs required to support DNA synthesis (i.e. with hairpin A, dATP and [-32P]dTTP; with hairpin C, dATP and [-32P]dGTP; with the TG40 repeat, dCTP and [-32P]dATP), 10 pmol of substrate (unless otherwise specified), and the indicated amounts of enzyme in the presence or absence of Mcm10p as specified. After incubation for 60 min at 37 °C, an aliquot of the reaction mixture was spotted on DE-81 paper, washed three times with 0.5 M sodium phosphate, and nucleotide incorporation measured. The remaining reaction mixture was treated with 10 mM EDTA, 0.5% SDS, 0.1 mg/ml proteinase K and incubated at 37 °C for 15 min, then extracted with phenol-chloroform-isoamyl alcohol (25:24:1) and subjected to 10% denaturing PAGE analysis.
DNA Primer Templates—The 94-mer hairpin C (5'-(C)36 TTT CCC TGT GCC CTT CGT ATA CGA TGG GTT TTT CCC ATC GTA TAC GAA GGG CAC AGG G-3'), the 94-mer hairpin A (5'-(A)36 TTT CCC TGT GCC CTT CGT ATA CGA TGG GTT TTT CCC ATC GTA TAC GAA GGG CAC AGG G-3'), the 89-mer TG40-repeat (5'-(TG)19 TTGG TTG GCC GAT CAA GTG CCC AGT CAC GAC GTT GTA AAA CGA GCC CGA GT-3'), and the 50-mer primer (5'-CAC TCG GGC TCG TTT TAC AAC GTC GTG ACT GGG CAC TTG ATC GGC CAA CC-3') were synthesized by International Technology (IDT), and purified by denaturing 6% SDS-PAGE. To anneal the 50-mer primer to the 89-mer TG40-repeat template DNA, the two oligomers were mixed in a 2:1 (primer:template) molar ratio in 0.1 M NaC1, 10 mM Tris-HC1, pH 7.5, and 1 mM EDTA, pH 8.0, heated to 95 °C for 5 min, and slowly cooled to 37 °C.
Isolation of a Mcm10p and pol -Primase Complex on Primed DNA— The protein-DNA complex containing the pol -primase and Mcm10p was isolated by affinity capture on an immobilized DNA template containing a 5'-biotin group, which is bound to streptavidin-agarose beads. To form the immobilized complex, a DNA replication reaction mixture (20-µl reaction volume) containing Buffer F (50 mM Hepes-KOH buffer, pH 7.0, 10 mM magnesium acetate, 75 mM KCl, 50 mM sodium glutamate, 0.1 mg/ml BSA, 1 mM DTT, 0.05% Nonidet P-40), 5 pmol of 5'-biotinylated hairpin C DNA template, 100 µM dATP, varying levels of pol -primase (indicated) in the presence or absence of 2 pmol of Mcm10p was incubated at room temperature for 30 min with occasional agitation. Streptavidin-agarose (Pierce) beads (10 µl) were added, and the mixture was rotated at room temperature for 20 min. The mixture was then chilled on ice, and the beads washed three times with 400 µl of ice-cold wash buffer (40 mM Hepes-KOH, pH 7.5, 10 mM magnesium acetate, 20% glycerol, and 0.02% Nonidet P-40). The washed beads were resuspended in 30 µl of SDS-PAGE sample, the beads were boiled, and proteins were separated by SDS-PAGE and analyzed by Western blotting.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Primase—S. pombe pol -primase subunits comprise p180, , Spp2, and Spp1, homologues of the human pol -primase p180, p70, p58, and p48 subunits, respectively. In conformity with the terminology used for the human proteins, we will refer to the S. pombe subunits of the pol -primase as p180, p70, p58, and p48. The apparent molecular masses of S. pombe p180, p70, p58, and p48 subunits in SDS-PAGE are 180, 70, 53, and 56 kDa, respectively (Fig. 1, lanes 1–4).
Four recombinant baculoviruses, each encoding a specific subunit of pol -primase, were constructed and used to infect Sf9 cells. Both untagged and C-terminal His-Flag-tagged derivatives of the p70, p58, and p48 subunits expressed well in Sf9 cells, whereas expression of the full-length p180 subunit was low possibly as a result of protease degradation. Coexpression of the p180 and p70 subunits, however, increased the recovery of the full-length p180 subunit (data not shown). Combinations of untagged and tagged subunits were tested to determine the optimal combinations for the isolation of the four-subunit and other subcomplexes. Proteins were purified by either consecutive binding to Ni-NTA resin followed by anti-Flag monoclonal antibody binding and elution, or, as in the case of the four-subunit enzyme, Ni-NTA binding followed by glycerol gradient sedimentation. These procedures resulted in the isolation of highly purified individual subunits (Fig. 1, lanes 1–4), the p180-His-Flag/p70 complex (Fig. 1, lane 5), the p58-His-Flag/p48 (Fig. 1, lane 6) subunit complex, and the holoenzyme complex (Fig. 1, lane 7). The purified cloned pol -primase complex contained polymerase activity (7.0 µmol of deoxynucleotide incorporated in 30 min/mg of protein) and primase activities (13 µmol of deoxynucleotide incorporated in 30 min/mg of protein, as performed by Schneider et al.) (10), comparable with those of the pol -primase complex isolated from extracts of S. pombe cells (data not shown). His-tagged spMcm10p (called Cdc23p in S. pombe) was cloned and expressed in E. coli and purified as described under "Materials and Methods." The purified material was more than 90% pure as shown in Fig. 1 (lane 8).
Mcm10p Interacts with the pol -Primase—The binding of Mcm10p to chromatin and its association with a number of proteins involved in the initiation and elongation of DNA replication prompted us to examine its interactions with purified pol -primase complex using co-immunoprecipitation procedures (Fig. 2). The four-subunit pol -primase associated with the Mcm10p (Fig. 2A). When individual Flag-tagged subunits of the pol -primase were examined, strong interaction was noted only with the p180 subunit (Fig. 2B, lanes 2–5). These experiments were carried out by immunoprecipitation with Flag antibodies followed by Western blotting with anti-Mcm10p. The interaction between Mcm10p and p180 was also observed in reciprocal immunoprecipitation experiments using Mcm10p polyclonal antibodies (Fig. 2C), whereas interaction with p70 was not observed.
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Activity—We next examined whether the Mcm10p-p180 interaction influenced the enzymatic activities associated with pol -primase. The template used in the replication reaction (Fig. 3A) was a hairpin molecule containing a 25-bp duplex region and a single-stranded 5' tail of 3 dTMPs followed by 36 dCMP residues (hairpin C). Reactions with this template were carried out in the presence of dATP and [-32P]dGTP. In the absence of dATP, dGMP incorporation was not detected (data not shown). We found that the addition of Mcm10p increased the polymerase activity 3–5-fold. The stimulation of the polymerase activity by Mcm10p was maximized by lowering the pH of the reaction (from 7.4 to 7.0) and by adding potassium chloride, sodium glutamate, and Nonidet P-40. Under these conditions, the activity of pol was reduced 5-fold without affecting the polymerase activity observed in the presence of Mcm10p. Using these conditions, the influence of a fixed concentration of Mcm10p on the rate of DNA synthesis by varying levels of the pol -primase complex was examined (Fig. 3A). As shown, Mcm10p stimulated DNA synthesis and this effect was most dramatic at low concentrations of pol -primase. Reactions containing 40 or 200 fmol of pol -primase were stimulated 25- and 4-fold by Mcm10p, respectively. Marked stimulation of the polymerase activity by Mcm10p was also observed with all DNA templates described under "Materials and Methods."
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activity was examined in replication assays carried out in the presence of a constant amount of pol -primase and varying levels of Mcm10p (Fig. 3B). Reaction mixtures contained 10 pmol of the primed 89-mer TG40 repeat template and 10 fmol of the pol -primase, conditions that supported DNA synthesis poorly in the absence of Mcm10p (Fig. 3A). Maximal stimulation was observed with 1 pmol (50 nM) Mcm10p, and 1/2 Vmax was detected at 10 nM Mcm10p. These results indicate that the level of Mcm10p required to stimulate the polymerase activity maximally was 50–100 times higher than the level of pol -primase added. On the other hand, maximal activation by Mcm10p occurred at a molar level considerably lower than the DNA template added. Under the conditions described in Fig. 3A, 0.8 pmol of DNA was elongated in the presence of a 0.2 pmol of Mcm10p (and 10 fmol of pol -primase complex). Thus, although the stimulation of the polymerase activity required a large molar excess of Mcm10p, the molar ratio of Mcm10p added to DNA elongated was 1:4, suggesting that Mcm10p acted catalytically relative to the chains of DNA synthesized.
The stimulatory effects of Mcm10p, described in Fig. 3, were also observed with the pol -primase isolated from S. pombe cells, the cloned p180-p70 complex, and the cloned p180 subunit alone (data not shown). The polymerase activity of the p180-p70 complex was almost identical to that of the cloned four-subunit complex, but the activity of the p180 subunit alone was 20% that of the four-subunit complex (data not shown).
The primase activity of the pol -primase complex, measured in the coupled Klenow assay (10), was stimulated up to 8-fold in the presence of increasing levels of Mcm10p (maximally in the presence of 500 fmol). However, increasing concentrations of Mcm10p markedly inhibited this activity, likely as a result of the binding of Mcm10p to the single-stranded poly(dT) used in the assay (data not shown). The DNA binding properties of Mcm10p are described in more detail below.
The nature of the products synthesized by pol -primase in the presence and absence of Mcm10p were examined using the 89-mer template hybridized to a 50-mer primer in which the single-stranded template region contained a 40-TG repeat sequence (Fig. 3C). As shown in lanes 1 and 2, the 5'-32P-labeled 50-mer primer in the duplex structure (lane 2) was fully elongated to the expected size (89-mer) by the Klenow fragment (lane 1). In the presence of Mcm10p, relatively low levels of pol -primase supported the full extension of the primer (lanes 8–12). In contrast, in the absence of Mcm10p the same levels of the polymerase complex were poorly active (lanes 3–5). In the absence of Mcm10p, full-length extension was observed with 25 fmol of pol -primase (lane 6); full-length extension in the presence of Mcm10p was evident with the lowest levels of the polymerase complex added (1.5 fmol, lane 8). In the absence of Mcm10p, under the conditions used, marked sigmoidicity in DNA synthesis was observed as a function of polymerase complex with all DNA substrates used, as shown in Fig. 3A.
Mcm10p Binds ssDNA and dsDNA—Mcm10p is associated with chromatin during all stages of the cell cycle. Although genetic and biochemical studies indicate that Mcm10p interacts with a number of proteins involved in pre-RC formation and the elongation reaction, its DNA binding properties have not been reported. A nitrocellulose binding assay was used to examine the DNA binding activity of Mcm10p, which was compared with the DNA binding properties of RPA (Fig. 4A). For this purpose, single-stranded 5'-32P-labeled 89-mer, and double-stranded 5'-32P-labeled 89-mer DNA substrates were used. As reported, RPA specifically bound to ssDNA but not dsDNA (33). In contrast, Mcm10p bound both ssDNA and dsDNA, although the binding to ssDNA was 20 times more effective. The apparent association constants of RPA and Mcm10p for ssDNA were calculated to be 2 x 109 and 5 x 107 M-1, respectively.
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activity with other ssDNA-binding protein, RPA and E. coli SSB (Fig. 4B). In these assays, 10 pmol of the primed DNA template hairpin A was used, which contained a 40-nucleotide single-stranded, oligo(dA)40 region. As previously reported (33), 2 molecules of RPA are required to completely coat a single strand region of 40 nucleotides. Both human and spRPA stimulated the polymerase activity but only at concentrations significantly higher than Mcm10p, as shown. Equimolar amounts of RPA and template, or 10 pmol of RPA in assays containing 10 pmol of template, were required to fully stimulate pol activity to the same extent observed with 1pmol of Mcm10p (Fig. 4B). E. coli SSB did not stimulate pol activity at any level.
As shown in Fig. 4A, 250 fmol of Mcm10p bound 25 fmol of the ssDNA (89 mer). If DNA binding were the only means by which Mcm10p activated pol -primase, a much higher molar ratio of Mcm10p to template would be required for full activation of pol activity in reactions described in Figs. 3B and 4B. However, as shown in these figures, full activation of pol required a low molar ratio of Mcm10p to DNA template. Thus, the mechanism by which Mcm10p stimulates pol activity seems to be different than that of RPA, and is not solely the result of its ability to bind to ssDNA. As described below, the region of Mcm10p that binds to ssDNA overlaps with the region required to interact with the p180 subunit of the pol complex and to activate the pol polymerase activity. It is likely that both of these properties are required for the activation of the pol polymerase activity.
We examined whether RPA influenced the Mcm10p activation of the polymerase activity. In the absence or presence of 1 pmol of RPA (it would take 10–20 pmol of RPA to fully coat the 10-pmol primer region (40-nucleotide ssDNA region), there was no change in the level of Mcm10p required to fully activate pol (data not shown). These data indicate that Mcm10p and RPA do not cooperate in this reaction.
Studies with Truncated Derivative of Mcm10p—To identify regions in Mcm10p that contribute to its ssDNA binding activity and activation of pol , N- and C-terminal truncated derivatives of Mcm10p were prepared (Fig. 5A). DNA fragments encoding the proteins shown in Fig. 5A were cloned into a pET28 vector, and the proteins were expressed in E. coli and purified as described under "Materials and Methods." The amino acids present in the truncated derivative are specified. The proteins isolated were 80–90% pure.
DNA Binding Activity of Mcm10p and Its Truncated Derivatives—We used the nitrocellulose binding assay to examine the in vitro ssDNA binding activity of the Mcm10p and its truncated derivatives (Fig. 5B). The DNA binding activity of Mcm10p and its truncated derivatives fell into four groups. The full-length Mcm10p and the truncated derivative Mcm10p-(1–497) bound equally well to the ssDNA, Mcm10p-(1–303) bound 2–3-fold less well than full-length Mcm10p, Mcm10p-(295–593) bound only at relatively intermediate and high levels of protein (500–1000 fmol), and Mcm10p-(416–593) failed to bind DNA.
Identical results to those described above were obtained from examination of the Mcm10p-DNA complexes bound to streptavidin-agarose beads (data not shown). In these experiments, a 5'-biotinylated hairpin C DNA was incubated with varying levels of Mcm10p or the truncated derivatives, protein-DNA complexes formed were bound to streptavidin-agarose beads, the beads were washed extensively, proteins bound to the beads were subjected to Western blot analyses, and the data were quantitated with the Fuji imager. These two different approaches yielding identical results indicate that there are two regions important for DNA binding: the N-terminal region, residing between amino acids 1 and 303, as well as the region containing the zinc-finger domain (298–324), residing between amino acids 295 and 416 (see Fig. 8).
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-primase, we examined interactions of pol -primase complex with radiolabeled Mcm10p and its truncated derivatives expressed in rabbit reticulocyte lysates as determined by co-immunoprecipitation analysis (Fig. 5C). Full-length Mcm10p bound most efficiently to the pol -primase complex (17.2% of input Mcm10p), whereas Mcm10p-(1–303) and Mcm10p-(295–593) bound less well (11.2 and 5.6%, respectively) and Mcm10p-(416–593) did not interact. These data indicate the regions required for DNA binding overlap with those required to interact with pol -primase complex.
Activation of the Pol Activity by the Truncated Mcm10p Derivatives—The region in Mcm10p required to activate pol -primase was examined. As shown in Fig. 6, stimulation of the polymerase activity by the truncated proteins decreased as deletions of the N-terminal region were increased. Thus, in the presence of 0.5 pmol of protein, stimulation of pol activity by Mcm10p-(295–593) and Mcm10p-(416–593) was 18 and 3%, respectively, as compared with the stimulation observed with full-length Mcm10p. In contrast, a truncation that removed up to 50% of the C terminus of Mcm10p, Mcm10p-(1–303), had 80% the polymerase stimulatory activity of wild type Mcm10p. These data indicate that the N-terminal region of Mcm10p is critical for the activation of pol activity.
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-Primase Bind to an Affinity-tagged Primed Template—Based on the Mcm10p activation of the pol activity shown in Fig. 3, we surmised that pol -primase would bind to primed DNA substrates more effectively in the presence of Mcm10p than in its absence. To test this prediction, an assay that measured the binding of both pol -primase and Mcm10p to 5'-biotinylated hairpin C DNA was used. After binding, the template was immobilized by streptavidin-agarose adsorption, the beads were washed to remove unbound proteins, and the remaining proteins eluted with SDS and visualized by Western blot analysis. In this experiment, increasing amounts of pol -primase were added to binding reaction mixtures containing dATP, in the presence or absence of Mcm10p. We found that the binding of the pol -primase complex to template DNA (measured by Western blot analysis of the p48 and p180 subunits) was stimulated by the presence of Mcm10p (Fig. 7A). Importantly, Mcm10p-dependent binding of the pol -primase complex to the template was observed in the presence of 31, 62, and 125 fmol of the pol -primase complex, corresponding to levels of enzyme that were stimulated significantly by Mcm10p (Fig. 3B). The Mcm10p-dependent recruitment of pol -primase was not observed in the absence of dATP or in the presence of both dATP and dGTP (data not shown). As the hairpin C DNA template contains 3 T residues followed by 36 C residues at the 5' end, the addition of both dATP and dGTP, which leads to complete extension of the primer end, would be expected to reduce the amount of primer ends and single-stranded regions required for pol -primase binding. The requirement for the correct incoming nucleotide, dATP, provides further evidence for the specificity of protein interactions with the immobilized DNA template, and indicates that the polymerase needs to incorporate or bind one nucleotide to form a stable complex.
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-primase, 40 and 20% of the input full-length Mcm10p and of truncated derivative (1–303), respectively, were associated with the DNA, reflecting their ssDNA binding properties noted previously. In the presence of added pol -primase, this association increased to 81 and 57% for full-length Mcm10p and Mcm10p-(1–303), respectively. The truncated protein (416–593), which does not activate pol -primase or bind to ssDNA, was not associated with the template in this assay in the presence or absence of pol -primase. In addition, the truncated Mcm10p-(416–593) did not interact with pol -primase, whereas the full-length Mcm10p and Mcm10p-(1–303) did interact, as determined by co-immunoprecipitation analysis (Fig. 5C). These data suggest that Mcm10p must bind to both DNA and pol -primase to facilitate recognition of a primed template by pol -primase. |
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-primase during replication, in keeping with its putative role in elongation.
In this report, we have demonstrated the direct interaction between spMcm10p, over-expressed and purified from E. coli, and the p180 subunit of the S. pombe pol -primase complex, expressed and purified from the baculovirus-insect cell system. This interaction was detected with the p180 subunit alone, the p180 subunit complexed to p70, and the p180 subunit in the four subunit pol -primase complex. A number of Mcm10p truncated derivatives were isolated, and their interaction with the p180 subunit was examined. Removal of the Mcm10p N-terminal 70% resulted in a derivative, Mcm10p-(416–593), that did not interact with the p180 subunit, whereas two derivatives containing amino acids 295–593 and 1–303 interacted, albeit at lower efficiency than full-length Mcm10p. The sum of their interaction with the p180 subunit was equal to the amount of full-length Mcm10p that interacted with pol -primase. These findings suggest that multiple regions within Mcm10p contribute to this interaction.
In addition to interacting with the p180 subunit of pol , Mcm10p was shown to bind ssDNA more effectively than dsDNA. The ssDNA binding activity of Mcm10p was unaffected by variations in the base sequence of the ssDNA substrate or by the use of a number of different primed DNA templates (those described under "Materials and Methods") containing either a 5' or 3' recessed end. Comparison of the ssDNA binding properties of Mcm10p and its truncated derivatives indicated that C-terminal deletions hardly affected the ssDNA binding activity. In contrast, deletions of the N terminus decreased its ssDNA binding activity.
The interactions between Mcm10p and the p180 subunit of pol lead to a marked stimulation of the polymerase activity of the pol -primase complex measured with a variety of primed DNA templates. The increase in activity was most pronounced at low levels of pol , and on a molar basis the amount of Mcm10p required for this effect was much greater than the level of pol -primase added. We suggest that this stimulation is dependent on both the interaction of Mcm10p with pol -primase and the binding of Mcm10p to ssDNA regions of the template. Both binding reactions would be expected to increase the recruitment of pol -primase complex to primer ends. In support of this, truncated Mcm10p derivatives that bound to ssDNA more effectively stimulated the pol activity at lower protein levels than derivatives that bound to ssDNA with low efficiency (Figs. 5 and 6). We did not isolate any Mcm10p derivative that retained ssDNA binding activity but lacked the ability to bind to pol or vice versa. Such mutants could help distinguish functional domains required for ssDNA binding and pol binding, and might help ascertain the importance of these functions in the stimulation of pol activity.
RPA, like Mcm10p, interacts with ssDNA and with the p180 subunit of pol (16, 17), and in addition with T-antigen. In the SV40 replication reaction the interactions between human (h) RPA, hpol -primase, and T-antigen play a key role in the T-antigen-dependent initiation reaction. We have shown that spMcm10p (Cdc23p) interacts with the hpol -primase complex and, as shown with the fission yeast pol -primase, activated the polymerase activity of the human complex (data not presented), suggesting that the interactions between the S. pombe and Homo sapiens proteins are likely to be the same. As shown in Fig. 4B, both hRPA and spRPA increased the polymerase activity of the S. pombe pol -primase complex. However, maximal increase in pol activity was achieved with nearly 10-fold higher levels of RPA than Mcm10p, suggesting that their interactions with template DNA and pol differ. The apparent association constant of RPA for ssDNA is 40-fold more efficient than Mcm10p. Mcm10p stimulated the formation of the pol -primase-biotinylated primed DNA complex, which was stable to adsorption to streptavidin beads and extensive washings. Formation of this complex required the addition of the correct incoming nucleotide dictated by the template strand. We suspect that interaction of the pol -Mcm10p complex with ssDNA regions of the primer-template are transient, and recycle on the ssDNA region. We propose that the binding of pol to sites close to the 3' primer terminus, facilitated by Mcm10p, leads to the activation of the polymerase activity. The presence of the correct incoming nucleotide (dATP in the experiment described in Fig. 7A) would be expected to increase the association of pol with the primer end of the DNA template. Interestingly, the level of Mcm10p required to activate the pol activity was not decreased by the presence of RPA. The precise mechanism by which Mcm10p activates pol , however, remains unclear but differs from that used by RPA.
Analysis of the primary amino acid sequence indicates that multiple regions of Mcm10p appear conserved between S. cerevisiae, S. pombe, D. melanogaster and H. sapiens. Functional conservation has been demonstrated as the S. cerevisiae MCM10 gene can complement a fission yeast cdc23 mutant (34). A number of different motifs within spMcm10p for which functions have been defined are summarized in Fig. 8. A putative nuclear localization sequence, located at the N terminus of spMcm10p, is in keeping with its nuclear localization and association with chromatin (22). The presence of a Walker A (P-loop, amino acids 77–83) and B (DEXX box, amino acids 146–155) motifs in spMcm10p suggests that spMcm10p may bind and hydrolyze NTPs. The Walker A and B motifs seem to be unique to the spMcm10p and were not found in the Mcm10p of other species. In keeping with the presence of these motifs, spMcm10p had NTPase activity (10 pmol/min/pmol of spMcm10p at 30 °C), which copurified through a number of standard ion exchange chromatographic steps, co-sedimented through glycerol gradients, and co-eluted from sizing columns with spMcm10p (data not shown). This activity was unaffected by DNA or RNA. Mutant spMcm10p, containing an altered Walker A box (GKT to GAT) was devoid of NTPase activity. The biological role of this NTPase activity is presently unclear. Plasmids expressing truncated derivatives of spMcm10p in which the GKT motif was deleted (spMcm10p containing amino acids 96–593) or full-length spMcm10 with the GKT motif mutated to GAT supported growth of the cdc23-M36 mutant at non-permissive temperatures (data not shown).
We have noted an additional conserved region within the spMcm10p, located between amino acids 219 and 265, which is 40% identical to the conserved C-terminal domain of the subunit of the prokaryotic DNA polymerase III family of proteins. In spMcm10p, this region included the sequence 236QLECFLF242, which is similar to the proposed prokaryotic -clamp binding motif QLXXXLF (35). A closely related sequence (327QCVSLFLF334) is present in hMcm10p. This conserved motif is involved in a number of protein-protein interactions that tether the sliding clamp to the replicative polymerase conferring processivity during replication, as well as in other proteins that bind the -clamp (36–38). These considerations prompted us to examine the interaction of spMcm10p with S. pombe PCNA and S. pombe pol. Stable interactions were not detected. However, spMcm10p stimulated the elongation of a singly primed ssM13 DNA template at low concentrations of S. pombe pol (data not presented). The possibility that Mcm10p may interact with other proteins that support replication through this prokaryotic DNA pol III-like domain requires further investigation.
In addition to its interaction with pol -primase, Mcm10p interacts with the Mcm2-7p complex, the putative replicative fork helicase. Three temperature-sensitive mutations, mcm10-1 (P269L), cdc23-M36 (D232C), and cdc23-1E2 (C239Y) have been characterized to genetically effect the interaction of MCM10 with members of Mcm2-7 group. The positions of these mutant sites overlap with the minimal region required for the direct physical interaction between the Mcm4,6,7 subcomplex (30), and lie within the middle region of Mcm10p that is the most highly conserved between species (34). In addition, an in vivo complementation assay of the cdc23-M36ts strain was performed with DNAs encoding full-length and truncated Mcm10p derivatives. MCM10 and its truncated derivatives were subcloned into the S. pombe expression vector pREP1 containing the thiamine-repressible nmt promoter, and the complementation phenotype was determined by cell growth in minimal medium lacking thiamine at the non-permissive temperature (30). Plasmids expressing full-length Mcm10p and mutants Mcm10p-(96–593) and Mcm10p-(1–497) supported growth and rescued the position 232 point mutant, whereas those expressing Mcm10p-(295–593), Mcm10p-(416–593), Mcm10p-(1–303), and Mcm10p-(1–230) did not. These data indicate that the C-terminal truncated derivatives that stimulated pol activity did not restore the ts growth defects of cdc23-M36. Mcm10p is likely to have a number of different functional domains required in vivo, and it is likely that spMcm10p amino acid position 232 resides in a domain important for interaction with Mcm2-7 complex.
A motif present in all species of Mcm10p identified is the zinc-finger domain located between amino acids 298 and 324 in spMcm10p (Fig. 8). A mutant allele of the S. cerevisiae MCM10 has been isolated within a conserved Cys residue (C320Y) of this motif, indicating the importance of the zinc-finger in vivo. In this study, we found that the region encoding the zinc-finger domain overlaps with a portion of Mcm10p that, in part, is required for both ssDNA binding as well as pol -primase interaction. Zinc-finger domains have been implicated in numerous functions, including hetero- and homo-protein-protein interactions, ssDNA binding, sequence-specific DNA recognition, and stabilization of protein folding (39). The exact function of the zinc-finger within the Mcm10p, however, is presently unclear.
The in vitro studies reported here with highly purified proteins indicate that Mcm10p interacts directly with the pol -primase complex and helps to recruit the polymerase to primed DNA templates, suggesting that Mcm10p may play an indirect role in lagging strand synthesis during the elongation step. Genetic studies suggest that Mcm10 interacts with the other replicative polymerases, pol and pol , raising the possibility that Mcm10p may activate these polymerases as well. The role of the Mcm10p-pol -primase interaction in the recruitment of pol -primase to the pre-initiation complex is presently unclear. Recent studies in Xenopus and in budding yeast suggest that the recruitment of pol -primase to the pre-initiation complex requires a large number of protein factors that include the SLD5-psf1,2,3 complex (40), and Dpb11, which in conjunction with Cdc45p, are required to activate the Mcm2-7 helicase. Thus, the association of pol -primase with the activated pre-initiation complex is likely to involve multiple proteins. The precise role played by Mcm10p in this complicated process remains to be explored further.
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FOOTNOTES |
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** To whom correspondence should be addressed. E-mail: j-hurwitz{at}ski.mskcc.org.
1 The abbreviations used are: pre-RC, pre-replication complex; pol, polymerase; BSA, bovine serum albumin; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; Ni-NTA, nickel-nitrilotriacetic acid; ds, double-stranded; ss, single-stranded; SSB, single-stranded DNA-binding protein; RPA, replication protein A; sp, S. pombe.
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