Expression and purification of the Mcm2,4,6,7 complex.
During the course of overexpression of the Mcm proteins in Sf9 cells, the Mcm4 and Mcm7 proteins were produced as insoluble forms while Mcm2 and Mcm6 were recovered as soluble forms. To facilitate the isolation and purification of soluble Mcm protein complexes, the His6 tag was added to the N terminus of the Mcm4 and Mcm7 proteins and the four Mcm proteins (Mcm2, Mcm4, Mcm6, and Mcm7) were expressed simultaneously. Recombinant baculoviruses expressing the His6-Mcm4 and Mcm6 proteins, under the control of the p10 promoter and the polyhedrin promoter, respectively, were constructed. Viruses expressing the His6-Mcm7 and Mcm2 proteins were also constructed.
To express Mcm2,4,6,7 proteins, High 5 insect cells were coinfected with two recombinant baculoviruses, each carrying the Mcm4,6
genes. The recombinant Mcm2,4,6,7 proteins were recovered from the lysed cells and purified by Ni-NTA affinity column chromatography. Nearly equal amounts of the four Mcm proteins were detected in the eluate of the Ni column, which was analyzed by SDS-PAGE, and the proteins were stained with Coomassie briliant blue (data not shown). The partially purified Mcm2,4,6,7 protein complex was further purified by a histone H3/H4-Sepharose column chromatography, as described previously (20
). SDS-PAGE of proteins in the fractions eluted from the histone-Sepharose column is shown in Fig. . The peaks of the Mcm4, Mcm6, and Mcm7 proteins were detected in the histone column fractions eluted with 0.75 M NaCl, while the peak of Mcm2 protein was detected in fractions eluted with 1 M NaCl. We previously suggested that Mcm2 is the only Mcm protein that binds to histone (22
). For this reason, the Mcm proteins probably bind to the histone column as an Mcm2,4,6,7 heterotetramer, particularly through the interaction between Mcm2 and histone H3, and the Mcm4,6,7 proteins may elute from the column earlier than the Mcm2 protein. However, the possibility that the Mcm4,6,7 complex itself has a weak affinity for histone H3/H4 remains to be tested.
FIG. 1 Purification of the recombinant Mcm2, Mcm4, Mcm6, and Mcm7 proteins by histone-Sepharose column chromatography. The recombinant proteins were produced in High 5 insect cells coinfected with recombinant baculoviruses carrying the Mcm2-his-Mcm7 and his-Mcm4-Mcm6 (more ...) Characterization of DNA helicase and ATPase activities of wild-type Mcm4,6,7 complex.
The histone-Sepharose fractions that contained predominantly the Mcm4,6,7 complex were pooled and further fractionated by glycerol gradient centrifugation. The proteins in these fractions were then analyzed by SDS-PAGE and detected by staining with silver (Fig. A, top). Proteins present in these fractions were also analyzed by immunoblotting with antibodies against the Mcm6 protein (Fig. A, bottom), and the membrane was then reprobed with the antibodies against the Mcm4 protein (Fig. A, bottom). The data obtained indicated that the Mcm4, Mcm6, and Mcm7 proteins were cosedimented and peaked at approximately 350 kDa.
FIG. 2 Purified Mcm4,6,7 complex has both DNA helicase and ATPase activities. (A) The histone-Sepharose fractions that mainly contain Mcm4, Mcm6, and Mcm7 proteins were pooled and further fractionated by glycerol gradient centrifugation. Proteins in the fractions (more ...)
Although the amount of Mcm7 protein in the peak fractions appeared to exceed those of Mcm4 and Mcm6 protein in this experiment, staining of proteins in the peak fractions with Coomassie brilliant blue suggested that the amount of Mcm7 protein did not differ greatly from that of other Mcm proteins (see Fig. A). These results suggest that these proteins form a stoichiometric complex. The DNA helicase and ATPase activities present in the separated glycerol gradient fractions were examined (Fig. B and C). The DNA helicase activity was measured with a labeled 17-mer oligonucleotide annealed to a single-stranded circular M13 DNA. The results show that the DNA helicase activity cosedimented with Mcm4,6,7 (Fig. B). The peak of the helicase activity was detected at 350 kDa (fractions 4 to 6), and the activity in these fractions was almost proportional to the amount of the Mcm proteins present. The ATPase activity in the glycerol gradient fractions was measured in the presence of single-stranded DNA (Fig. C). Similar to the DNA helicase activity, the ATPase activity cosedimented with the Mcm proteins. These results suggest that the purified recombinant mouse Mcm4,6,7 protein complex possesses both DNA helicase activity and ATPase activity, which is consistent with the results obtained with the purified human Mcm4,6,7 complex (20
FIG. 4 Inhibition of Mcm4,6,7 DNA helicase activity by Mcm2. Increasing amounts of His6-tagged mouse Mcm2 proteins purified from baculovirus-infected cells were incubated with the recombinant Mcm4,6,7 complex in 50 mM Tris-HCl (pH 7.9)–20 mM 2-mercaptoethanol–5 (more ...) Mcm4,6,7 complex binds single-stranded DNA.
We next examined the formation of the purified Mcm4,6,7 protein complex and the single-stranded DNA binding activity of the complex (Fig. ). The Mcm proteins present in glycerol gradient fractions were electrophoresed under nondenaturing conditions to analyze the complex (Fig. A). In this native gel, a major protein band of approximately 550 kDa was detected (fractions 4 to 6), which is slightly smaller than the 600-kDa human Mcm4,6,7 complex (20
). The structure of the Mcm4,6,7 complex is estimated in Discussion.
FIG. 3 The Mcm4,6,7 protein complex can bind single-stranded DNA. (A) Proteins in the gradient fractions were electrophoresed on a 5% native polyacrylamide gel and stained with silver. As markers, thyroglobulin (669 kDa) and ferritin (440 kDa) were electrophoresed. (more ...)
The single-stranded DNA binding activity of the purified Mcm protein complex was investigated as described in Materials and Methods (Fig. B). When each glycerol gradient fraction was incubated with the labeled 37-mer single-stranded DNA, a band that migrated at the expected position of ~550 kDa was detected. The binding of the Mcm complex with the 37-mer oligonucleotide does not require ATP (data not shown). The intensity of the band appeared to be proportional to the amount of the 550-kDa Mcm4,6,7 complex present in the glycerol gradient fraction added (Fig. A). These results indicate that the recombinant Mcm4,6,7 complex can bind the 37-mer single-stranded DNA, consistent with the finding that both DNA helicase and the single-stranded DNA-dependent ATPase activities are cofractionated with the 550-kDa Mcm4,6,7 complex.
Comparison of native and recombinant Mcm4,6,7 DNA helicases.
The results described above suggest that the recombinant Mcm4,6,7 complex has DNA helicase activity similar to that of the native human Mcm4,6,7 complex. To further address this point, the DNA helicase activity of these two complexes was examined in more detail. The specific activities of the DNA helicase of the recombinant mouse and the native human Mcm4,6,7 complex were comparable; approximately 100 ng of Mcm4,6,7 complex was required to displace 5 fmol of the annealed 17-mer oligonucleotides for 30 min under standard conditions (data not shown). This means that about a 40-fold molar excess of protein, if it forms a hexamer, compared to the 17-mer oligonucleotide, was necessary to displace the 17-mer. Thus, since we added substantial amounts of Mcm4,6,7 complex compared to the 17-mer, it is difficult to conclude that this reaction is catalytic. However, this activity appears to satisfy the criteria of a DNA helicase; the DNA helicase activity of Mcm4,6,7 complex is dependent on the presence of hydrolyzable ATP, and the data suggest that the complex migrates along single-stranded DNA in the 3′-to-5′ direction (20
We reported that the incubation of Mcm4,6,7 complex with Mcm2 leads to the inhibition of DNA helicase activity, which is associated with the change from a 600-kDa Mcm4,6,7 complex to a 450-kDa Mcm2,4,6,7 complex (22
). Similary, incubation of the recombinant mouse Mcm4,6,7 complex with the Mcm2 protein resulted in inhibition of the DNA helicase activity (Fig. A and B) and in the conversion of the 550-kDa Mcm4,6,7 complex to the 450-kDa complex, which was confirmed by using anti-Mcm4 antibodies (Fig. C). The 450-kDa complex did not possess the single-stranded DNA binding activity (Fig. D). An identical effect of the Mcm2 protein on the human Mcm4,6,7 complex was observed (22
). These results indicate that the recombinant mouse Mcm4,6,7 complex has DNA helicase activity which is essentially the same as that of the native human Mcm4,6,7 complex.
Biochemical characterization of mutant Mcm4,6,7 complexes.
A series of mutations that changed specific amino acids located in the conserved DNA-dependent ATPase motifs of the Mcm proteins were carried out (Fig. A) (30
). Aspartic and glutamic acid (DE) residues in motif B, which were highly conserved among various ATPases, were changed to alanine residues in the Mcm6 (DE459-AA) or Mcm4 (DE572-AA) protein. In addition, the highly conserved lysine and serine (KS) residues in motif A were converted to alanine residues in the Mcm6 protein (KS401-AA). These amino acids have been implicated in DNA binding, ATP binding, and ATP hydrolysis in other ATPases (31
FIG. 5 Mutations introduced into Mcm4 and Mcm6 proteins for the formation of various mutant Mcm4,6,7 complexes. (A) A schematic presentation of the Mcm4 and Mcm6 proteins depicts the DNA-dependent ATPase motifs A, B, C, and D in the conserved regions, and the (more ...)
The Mcm4 and/or Mcm6 protein, mutagenized at these particular sites, were coexpressed in insect cells with other wild-type Mcm proteins. The mutant Mcm complexes of Mcm2,4,6DE-AA,7, Mcm2,4DE-AA,6,7, Mcm2,4DE-AA,6DE-AA,7, and Mcm2,4,6KS-AA,7 were purified by the same procedure used for the isolation of the wild-type complex. After glycerol gradient centrifugation, a 550-kDa Mcm4,6,7 complex containing the mutated Mcm4 and/or Mcm6 protein was isolated (Fig. B), although the recovery of the Mcm4,6,7 complex varied somewhat among the mutant Mcm complexes (data not shown). These results suggest that the mutations in these conserved amino acids in the ATPase motifs of the Mcm4 and Mcm6 proteins did not significantly affect the assembly of Mcm proteins into the 550-kDa Mcm4,6,7 complex.
The mutant Mcm4,6,7 complex (Mcm4,6DE-AA,7) in which DE were converted to AA in motif B of Mcm6 protein was characterized first. The DNA helicase and ATPase activities of the mutant Mcm4,6,7 complex were compared with those of the wild type. The mutant Mcm complex did not show any DNA helicase activity even when substantially higher levels of the complex compared to the wild-type complex were added to the reaction mixtures (Fig. A). On the other hand, the levels of ATPase and single-stranded DNA binding activities detected with the Mcm4,6DE-AA,7 mutant complex were nearly comparable to those detected with the wild-type complex (Fig. B and C). Thus, the DE in motif B of the Mcm6 protein is essential for the DNA helicase activity of the Mcm4,6,7 complex, but this mutation hardly affected the ATPase and the single-stranded DNA binding activities.
FIG. 6 A defect in the DNA helicase activity of the Mcm4,6,7 complex containing mutated Mcm6. The DNA helicase (A), ATPase (B), and single-stranded DNA binding (C) activities of the mutant Mcm4,6,7 complex where DE in motif B of the Mcm6 protein was converted (more ...) ATP binding activity of the mutant Mcm complex.
Binding and hydrolysis of nucleotide triphosphates are necessary for helicase activity. It has been shown that either the Mcm4 or the Mcm6 protein, in the native human Mcm4,6,7 complex, can be affinity labeled with ATP (20
). To clarify which Mcm protein binds ATP with high affinity, the human Mcm4,6,7 complex was incubated with [α-32
P]ATP, subjected to UV irradiation, and then separated by SDS-PAGE (Fig. A). As a marker, Escherichia coli
DNA polymerase I (molecular mass, 103 kDa) was affinity labeled to provide a 100-kDa band marker on SDS-PAGE. A major band of 100 kDa was detected after incubating the human Mcm4,6,7 complex with [α-32
P]ATP. Comparison of the electrophoretic mobility of Mcm proteins and DNA polymerase I suggested that the labeled 100-kDa protein is the Mcm6 protein. Furthermore, the 100-kDa band was also detected by affinity labeling the recombinant mouse Mcm4,6,7 complex of the wild type with [α-32
P]ATP (Fig. B). The 100-kDa ATP-labeled protein band formed with the recombinant Mcm4,6,7 complex was immunodepleted with respect to the anti-Mcm4 antibodies but not to the control antibodies. This finding suggests that the Mcm6 protein in the Mcm4,6,7 complex has high affinity for ATP. However, this finding does not rule out the possibility that Mcm4 and Mcm7 proteins have a lower affinity for ATP. It is also possible that the lack of cross-linking of radiolabeled ATP to the Mcm4 and Mcm7 proteins is due to technical problems.
FIG. 7 Mutation in motif B of the Mcm6 protein results in the reduction of ATP binding activity of the Mcm4,6,7 complex. (A) Native Mcm4,6,7 complex of HeLa cells (lane 2) and E. coli DNA polymerase I (Pol. I) (lane 1) were electrophoresed in an SDS–8% (more ...)
Since the mutant Mcm complex (Mcm6DE-AA) showed no DNA helicase activity, we investigated the ATP binding activity of this complex. The ATP binding activity of the mutant complex was markedly reduced compared to that of the wild-type complex (Fig. C). These results suggest that the DE mutation in motif B of the Mcm6 protein affects the DNA helicase activity of the Mcm4,6,7 complex by lowering the affinity of the complex for ATP. However, it is also possible that the Mcm6DE-AA mutation affects DNA-unwinding activity by altering the Mcm6 protein structure and hence the interaction of Mcm6 with Mcm4 and Mcm7.
Characteristics of the other mutants of the Mcm4,6,7 complex.
The results shown in Fig. and indicated that the DE-to-AA changes in motif B of the Mcm6 protein of the Mcm4,6,7 complex affected both the DNA helicase and ATP binding activities but did not affect the ATPase or the single-stranded DNA binding activities of the complex. We next asked whether other amino acid changes in the conserved ATPase domain of the other proteins would affect the activities of the Mcm4,6,7 complex. These include the changes DE to AA in motif B of the Mcm4 protein, KS to AA in motif A of the Mcm6 protein, and the double mutant in which the changes DE to AA were made in motif B of both the Mcm4 and Mcm6 proteins. These Mcm4,6,7 mutant complexes were purified, and the influence of these changes on the DNA helicase, DNA-dependent ATPase, single-stranded DNA binding, and ATP binding activities were examined. The DNA helicase activities of these mutants are shown in Fig. A and Table . The Mcm4,6,7 complexes containing the mutated Mcm4DE or Mcm6KS retained DNA helicase activity, but the specific activity was lower than that of the wild-type Mcm4,6,7 complex. No DNA helicase activity was detected in the complex containing mutations in both Mcm6DE and Mcm4DE.
FIG. 8 Characterization of various mutant forms of Mcm4,6,7 complex. The activities of DNA helicase (A), ATPase (B), single-stranded DNA binding (C), and ATP binding (D) were investigated. The designations of the mutant Mcm4,6,7 complexes are described in the (more ...)
TABLE 1 Summary of the biochemical properties of mutants of MCM4,6,7complexesa
The ATPase activity of the three Mcm4,6,7 mutants was also examined (Fig. B and Table ). The Mcm4,6,7 complexes containing the mutated Mcm4DE or the mutated Mcm6DE exhibited wild-type levels of ATPase activity. A slightly reduced ATPase activity was detected in the Mcm4,6,7 complex in which Mcm6KS was mutated. The ATPase activity of the double mutant, Mcm4DE and Mcm6DE, was significantly reduced.
We next compared the single-stranded DNA binding activities of the various mutants (Fig. C and Table ). The two mutants which showed either a decrease in helicase activity (Mcm4DE) or no helicase activity (Mcm4DE6DE) also showed a reduced DNA binding activity compared to the wild-type Mcm4,6,7 complex. The mutant complex containing Mcm6KS possessed single-stranded DNA binding activity comparable to that of the wild-type complex, and the other mutant containing Mcm6DE showed a level of activity slightly higher than that of the wild-type complex. Thus, the DE residues of motif B of Mcm4 but not of Mcm6 may play a significant role in the single-stranded DNA binding activity of the complex. However, it is also possible that the DNA binding defect in Mcm4DE is due to a coincidental alteration of Mcm4 protein structure and/or interaction of Mcm4 with Mcm6 and 7 proteins.
The ATP binding activity of the mutated Mcm4,6,7 complexes was investigated (Fig. D and Table ). The activity of the Mcm4DE mutant was similar to that of the wild-type Mcm4,6,7 complex, whereas the other Mcm6 mutants with mutation in either motif A or motif B exhibited almost no ATP binding activity. The Mcm4,6,7 complex mutated in both the DE of Mcm4 and the DE of Mcm6 also exhibited no ATP binding activity. These results suggest that Mcm6 plays an important role in ATP binding, consistent with the observation that the Mcm6 protein has a high affinity for ATP (Fig. ).
Based on the biochemical activities observed with the mutated Mcm complexes, the following conclusion can be drawn. The loss of ATP binding activity observed with the Mcm6DE or KS mutant complex leads to the inactivation of the DNA helicase activity. In addition, the results with the two Mcm6 mutants indicated that the mutations can affect the ATP binding, ATPase, and single-stranded DNA binding activities differently. Similarly, the mutations in the Mcm4 protein uncoupled the single-stranded DNA binding activity from the ATPase and ATP binding activities. Finally, the results suggest that defects in the ATP binding or the single-stranded DNA binding activities lead to loss of the DNA helicase activity of the Mcm4,6,7 complex.