Roles of Mcm7 and Mcm4 subunits in the DNA helicase activity of the mouse Mcm4/6/7 complex.

Mcm, which is composed of six structurally related subunits (Mcm2-7), is essential for eukaryotic DNA replication. A subassembly of Mcm, the Mcm4/6/7 double-trimeric complex, possesses DNA helicase activity, and it has been proposed that Mcm may function as a replicative helicase at replication forks. We show here that conserved ATPase motifs of Mcm7 are essential for ATPase and DNA helicase activities of the Mcm4/6/7 complex. Because uncomplexed Mcm7 displayed neither ATPase nor DNA helicase activity, Mcm7 contributes to the DNA helicase activity of the Mcm complex through interaction with other subunits. In contrast, the Mcm4/6/7 complex containing a zinc finger mutant of Mcm4 with partially impaired DNA binding activity exhibited elevated DNA helicase activity. The Mcm4/6/7 complex containing this Mcm4 mutant tended to dissociate into trimeric complexes, suggesting that the zinc finger of Mcm4 is involved in subunit interactions of trimers. The Mcm4 mutants lacking the N-terminal 35 or 112 amino acids could form hexameric Mcm4/6/7 complexes, but displayed very little DNA helicase activity. In conjunction with the previously reported essential role of Mcm6 in ATP binding (You, Z., Komamura, Y., and Ishimi, Y. (1999) Mol. Cell. Biol. 19, 8003-8015), our data indicate distinct roles of Mcm4, Mcm6, and Mcm7 subunits in activation of the DNA helicase activity of the Mcm4/6/7 complex.

DNA helicase plays a central role in the replication of chromosomal DNA. It couples the free energy of ATP hydrolysis to separation of a DNA duplex into its component strands. DNA helicase is associated with a set of subactivities including DNA binding, nucleotide binding, and ATP hydrolysis, all of which cooperatively work for DNA unwinding (1,2). The Mcm family proteins (Mcm2-7) interact with one another to form a complex that plays an essential role in the initiation of DNA replication (3)(4)(5)(6)(7). A recent demonstration that the Mcm4/6/7 complex can form a hexameric complex with active helicase activity suggests that the Mcm complex may be part of the replicative DNA helicase of eukaryotes (8 -13).
All six Mcm proteins contain DNA-dependent ATPase motifs in their central domains, including Walker motifs A and B, which are widely conserved in ATPases and DNA helicases (14,15). In addition, four of the Mcm proteins (Mcm2, Mcm4, Mcm6, and Mcm7) contain a zinc finger motif, which may function in protein-protein interactions or nucleic acid binding (3,16). The ATPase and zinc finger motifs are highly conserved in Mcm proteins from yeast to mammalian cells, suggesting their crucial roles in the functions of Mcm.
We have identified DNA helicase activity in the human Mcm4/6/7 protein complex (8) and subsequently demonstrated that it is intrinsic to mammalian Mcm4/6/7 complexes (9). DNA helicase activity was also demonstrated in the Mcm4/6/7 complex of fission yeast (10,11). To elucidate the mechanism of the DNA helicase functions of the Mcm4/6/7 complex and to obtain insight into the physiological significance of this activity, biochemical studies with a series of mutants of the Mcm4/6/7 complex were undertaken. Our previous studies with mutant Mcm4/6/7 complexes in which the ATP-binding motifs of the Mcm6 protein were mutated showed that the mutation preferentially affected the high affinity binding of ATP and inactivated the DNA helicase activity, although the ATPase activity was not significantly affected. A mutation in an ATP-binding motif of the Mcm4 protein affected the ssDNA 1 binding activity of the complex and led to moderate inhibition of the DNA helicase activity (9). In the present study, the roles of ATPbinding motifs of Mcm7 and those of the zinc finger motif of Mcm4 in various activities of the Mcm4/6/7 complex were examined. Our results indicate that ATPase motifs of Mcm7 are essential for the ATP hydrolytic and DNA helicase activities of the Mcm4/6/7 complex, whereas Mcm7 alone does not possess these activities, and that zinc finger motif mutants of Mcm4 display enhanced DNA helicase activity, with altered subunit assembly and partially impaired DNA binding activity. We have shown that the N-terminal region of the Mcm4 protein is also required for DNA helicase activity. Our results indicate distinct roles of each subunit of the Mcm4/6/7 complex in the execution of its helicase activity. A possible model of the helicase action of Mcm is discussed on the basis of these findings.

Generation of Mutant Forms of Mouse Mcm7 and Mcm4 cDNAs-
Site-directed mutagenesis of the Mcm7 and Mcm4 genes was conducted with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The oligonucleotides 5Ј-GGCGTGTGCTGCATTGCTGCGT-TTGACAAGATGGCC-3Ј and 5Ј-GACCCTGGTGTGGCCGCAGCTCAG-* This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by the RIKEN Board of Directors Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
CTCCTATCTTAC-3Ј were used as primers to prepare the histidinetagged Mcm7 DE and Mcm7 KS mutants (where "DE" refers to the Asp 444 and Glu 445 residues in the Walker B motif mutated to alanine, and "KS" refers to the highly conserved Lys 386 and Ser 387 residues in the Walker A motif mutated to alanine), respectively, in pBluescript II SK (Ϫ). The  oligonucleotides 5Ј-GAGGCCTTTTTCCAAGCCCAAGTCGCTGCCCA-CACCACCCGG-3Ј and 5Ј-GCTGAGCCCTGCAGTGCTGTGCACGCCC-ACACTACCCACAGC-3Ј were used as primers to introduce Cys-to-Ala  mutations at amino acids 305 and 308 and amino acids 327 and 330 (9). The mutant Mcm4/6/7 complexes were purified by nickel column chromatography, histone-Sepharose column chromatography, and glycerol gradient centrifugation as described previously (9). The Mcm4 CC1 /6/7 or Mcm4 CC2 /6/7 complex was similarly expressed in HighFive cells after co-infection of FLAG-tagged Mcm7 with Mcm4 CC1 /6 or Mcm4 CC2 /6 and was purified by consecutive steps involving Ni 2ϩ -nitrilotriacetic acid affinity chromatography, anti-FLAG antibody M2-agarose affinity chromatography, and glycerol gradient centrifugation. On the nitrilotriacetic acid column, proteins bound to the beads were eluted three times by adding 1 bed volume of buffer containing 50 mM sodium phosphate buffer, 300 mM NaCl, and 10% glycerol supplemented with 200 mM imidazole (pH 7.5). The eluted fractions were mixed with anti-FLAG antibody M2agarose beads (Sigma) equilibrated with buffer A (50 mM sodium phosphate buffer (pH 7.5), 300 mM NaCl, and 10% glycerol) and incubated at 4°C for 1 h on a rocking platform. After washing the resin three times with 10 bed volumes of buffer A, bound proteins were eluted three times by incubation at 4°C for 15 min with 1 bed volume of buffer A containing 0.2 mg/ml FLAG peptide. The eluted fractions were pooled and further purified by glycerol gradient centrifugation at 36,000 rpm for 16 h (Beckman TLS55 rotor) on a 15-35% linear glycerol gradient as described previously (9) or on a Mono Q column in a SMART system (Amersham Biosciences) in which the peak fractions containing the zinc finger mutant complexes eluted at 0.3 M NaCl. The presence of a FLAG tag at the C terminus of Mcm7 did not affect various biochemical functions of the Mcm4/6/7 complex. The FLAG-tagged Mcm7 protein, singly expressed in insect cells, was purified by consecutive steps involving anti-FLAG antibody M2-agarose affinity chromatography, Mono Q column chromatography, a second run of anti-FLAG antibody M2-agarose affinity chromatography, and finally 15-35% glycerol gradient centrifugation at 36,000 rpm for 16 h.
The conditions for ATPase assays were the same as for DNA helicase assays, except that the substrate was omitted, and 2 Ci of [␥-32 P]ATP and 2 g of heated-denatured salmon sperm DNA or 0.75 g of 67-mer oligonucleotide were added. After incubation at 37°C for 1 h, aliquots (0.5 l) were spotted onto a polyethyleneimine-cellulose TLC plate, and the ATP and P i were separated by chromatography in 1 M formic acid and 0.5 M LiCl. The extent of ATP hydrolysis was quantified by using a Fuji BAS 2500 Bio-Image analyzer.
ssDNA Binding and ATP Binding Assays-Mcm proteins were incubated with the labeled 37-mer oligonucleotide (9) at 37°C in buffer containing 10 mM sodium creatine phosphate, 5 mM ATP, 5 mM MgCl 2 , 0.3 mM DTT, 0.01% Triton X-100, and 15 mM potassium phosphate (pH 7.5) for 30 min. Glutaraldehyde (10%) was then added to the reaction mixture at a final concentration of 0.1%, and the incubation was continued for 10 min. The reaction mixture was applied to 5% native polyacrylamide gel in Tris/glycine buffer. We did not observe any effect of the presence of ATP on the gel shift patterns of the Mcm complexes under the conditions employed. Nitrocellulose filter binding assays were conducted as previously described (10). Reaction mixtures (15 l) contained 25 mM Hepes-NaOH (pH 7.5), 50 mM NaOAc, 10 mM Mg(CH 3 COO) 2 , 1 mM DTT, 0.1 mg/ml bovine serum albumin, 0.25 mM AMP-PNP, and 20 fmol of 5Ј-end-labeled 37-mer oligonucleotide. Proteins were added, and reaction mixtures were incubated at 30°C for 30 min. The reaction mixtures, diluted by addition of 85 l of wash buffer (25 mM Hepes-NaOH (pH 7.5), 50 mM NaOAc, 10 mM Mg(CH 3 COO) 2 , and 1 mM DTT), were filtered through alkaline-treated 0.45-m HA nitrocellulose filters (Millipore) (17). The filters were washed three times with wash buffer, and the radioactivity retained on the filters was subjected to liquid scintillation counting. ATP binding assays were conducted as previously described (9). Immunoprecipitation

Expression and Purification of Mouse Mutant Mcm4/6/7 Complexes Containing ATPase Motif Mutations in Mcm7-Two
mutations that changed specific amino acids located in the well conserved DNA-dependent ATPase motifs of Mcm7 were generated by site-directed mutagenesis (Fig. 1). The highly conserved lysine and serine residues (KS) in the Walker A motif and the aspartic and glutamic acid residues (DE) in the Walker B motif were changed to alanine in the Mcm7 protein (Mcm7 DE and Mcm7 KS , respectively). These amino acids have been implicated in the nucleotide hydrolytic and DNA unwinding activities of various ATPases and DNA helicases (18 -22). To express these mutant Mcm4/6/7 proteins, HighFive insect cells were co-infected with two recombinant baculoviruses, Mcm2/ His 6 -Mcm7 (DE or KS) and His 6 -Mcm4/6. The mutant Mcm4/ 6/7 complexes were purified to near homogeneity by Ni 2ϩagarose chromatography, histone H3/H4-Sepharose column chromatography (23), and glycerol gradient centrifugation (9) as described previously ( Fig. 2A).
Glycerol gradient sedimentation indicated that the mutant complexes sedimented at ϳ550 kDa, a value almost identical to that of the wild-type complex (data not shown). Examination of the purified proteins by SDS-PAGE revealed that the three subunits largely formed stoichiometric complexes in both mutants ( Fig. 2A). The levels of contaminating Mcm2 protein in the purified Mcm4/6/7 complexes were examined by Western blotting (Fig. 2B) (Fig. 3A). The levels of ATPase activities of the mutant complexes were also significantly lower than that of the wild-type Mcm4/6/7 complex (Fig. 3B). However, they retained ssDNA and ATP binding activities that were nearly comparable to those detected with the wild-type complex (Fig. 3, C  Procedures") was further fractionated by glycerol gradient centrifugation. The proteins in these fractions were then analyzed by SDS-PAGE (Fig. 4A). The major peak of the Mcm7 protein sedimented at fractions close to catalase (230 kDa), indicating that Mcm7 may form a trimeric complex. The ATPase and DNA helicase activities were then measured using this purified protein fraction. As shown in Fig. 4 (B and C) (Fig. 1). These mutant complexes, carrying a histidine tag and a FLAG tag at the N terminus of Mcm4 and at the C terminus of Mcm7, respectively, were purified by Ni 2ϩ -nitrilotriacetic acid column chromatography, FLAG affinity column chromatography, and glycerol gradient centrifugation (Fig. 5A).
The wild-type Mcm4/6/7 complex sedimented at 500 -600 kDa, whereas the Mcm4 CC2 /6/7 complex peaked at fractions slightly larger than catalase (232 kDa) (Fig. 5A, upper panels). Examination of each fraction on a nondenaturing acrylamide gel revealed that the mutant complex contained a significant amount of 280-kDa trimers, whereas the wild-type complex was almost exclusively composed of 550-kDa hexameric complexes (Fig. 5A, lower panels). Similar results were obtained with the Mcm4 CC1 /6/7 complex (data not shown). These results indicate that mutations in the zinc finger result in disassembly of the hexameric complex into trimers. Therefore, the zinc finger motif of Mcm4 is likely to be involved in the stable assembly of the hexameric structure.
To address the molecular basis of the instability of the  (Fig. 5B, lanes 3 and 4) 7 complexes containing the zinc finger mutants contained the three subunits at a stoichiometry identical to that of the wild-type complex, as judged by SDS-PAGE analyses (Fig. 5C). The two zinc finger mutant Mcm4/6/7 complexes retained the DNA-dependent ATPase activities at a level comparable to that of the wild-type complex in the presence of ssDNA (Fig. 5D) (data not shown). In gel shift assays, the complex formation was significantly reduced in the Mcm4 CC1 /6/7 complex and reduced by ϳ50% in Mcm4 CC2 /6/7 (Fig. 5E). A similar reduction of DNA binding was observed also in filter binding assays with the mutant complexes (Fig. 5F). These results indicate that the zinc finger mutations reduce the DNA binding activity of the Mcm4/6/7 complex.
In contrast, these mutant complexes exhibited increased DNA helicase activities. The specific activities of both zinc finger mutants were 2-fold higher than that of the wild-type complex (Fig. 5G). Possible mechanisms of how reduced stability of hexameric structures and DNA binding activity may contribute to enhanced DNA helicase activity will be discussed later.
Attenuation  (Fig. 6B). The N-terminal deletion of Mcm4 did not significantly affect the assembly of the mutant Mcm4 proteins into the Mcm4/6/7 hexameric complex (Fig. 6B) (data not shown). The two truncated Mcm4 mutants were poorly phosphorylated by Cdk2/cyclin A kinase in vitro, consistent with the notion that the major cyclin-dependent kinase phosphorylation sites of Mcm4 lie in the N-terminal region of the protein (28, 29) (data not shown).
These two mutants displayed significantly reduced DNA helicase activity (Fig. 6C). In contrast, the ATPase and ATP binding activities of the Mcm4/6/7 complex were not affected by the N-terminal truncations of Mcm4 (Fig. 6D) (data not shown). Mutagenesis studies with various helicases and other nucleotide-binding proteins have shown that the Walker A and B motifs are critical for nucleotide hydrolysis (18 -22).
In contrast, two mutant complexes showed the wild-type level of ATP and DNA binding activities (Table I)  were mixed as indicated, and immunoprecipitation was carried out with anti-Mcm4 antibody. Input (15% of total) and bound proteins were run on 8% SDS-polyacrylamide gel (37.5:1 acrylamide/bisacrylamide), and proteins were detected by autoradiography. C, the final preparations of the wild-type and zinc finger mutants of Mcm4/6/7 (150 ng each), used in the various assays described for D-G, were prepared by concentrating glycerol gradient peak fractions (wild-type complex, pool of fractions 3-5; and zinc finger mutant complex, pool of fractions 6 -8) and subjected to 10% SDS-PAGE, followed by silver staining. D, ATPase assays were conducted under the standard conditions with various amounts of a zinc finger mutant (Mcm4 CC2 / 6/7) or the wild-type Mcm4/6/7 complex as indicated. The reactions contained a 67mer oligonucleotide DNA at 50 ng/l. The released phosphate (P i in pmol; the background in the absence of added protein was taken as 0) was quantified, and the values are presented at the bottom of each lane. E, the ssDNA binding activities of the wildtype and zinc finger mutant Mcm4/6/7 complexes were measured as described under "Experimental Procedures." The gray arrow indicates the position of the Mcm4/6/7 hexamer (550 kDa) in complex with the 37-mer DNA. The positions of protein molecular mass markers (Thy, thyroglobulin; Fer, ferritin) upon glycerol gradient and native gel electrophoresis are indicated by black arrows. The protein-free oligonucleotides have run off the gel. The intensities of the shifted bands were quantified, and the relative extent of binding, with the intensity of the shifted DNA with 50 ng of the wild-type protein being taken as 100, is presented below each lane. F, nitrocellulose filter binding assays of the wild-type and mutant Mcm4/6/7 complexes were conducted with 20 fmol of labeled 37-mer oligonucleotide. The amounts of DNA-protein complexes generated are plotted against the concentration of Mcm proteins. The values are the averages of three independent experiments, and error bars are shown. G, the DNA helicase activities of the zinc finger mutant Mcm4/6/7 complexes, as indicated, were measured along with the wild-type complex as described under "Experimental Procedures." The displaced oligonucleotides (%) were quantified and are plotted.
Mcm2/3/4/5/6/7 hexameric complex is independent of DNA (32). 2 Thus, the mechanisms of ATP hydrolysis by both complexes may be intrinsically different from each other. It was shown that T7 phage-encoded DNA helicase (T7gp4) in hexamer form possesses two classes of dTTP-binding sites and catalyzes the sequential hydrolysis of two nucleotides (33). The helicase action of Mcm4/6/7 may be similar to that of T7gp4 in that two Mcm7 molecules in the Mcm4/6/7 hexamer could be responsible for sequential ATP hydrolysis, and the "two-site sequential NTPase model" (1) (24 -26). Our results indicate that mutations in the zinc finger motif of the Mcm4 protein destabilize the hexameric structures, which are the functional forms for binding to ssDNA. This may be the reason why these mutants show reduced binding to ssDNA. The zinc finger regions of SV40 and polyoma virus T antigens were also reported to contribute to protein-protein interactions required for hexamer and double-hexamer formation at the origins (35,36  ϩϩϩϩϩϩ ϩϩϩϩ ϩϩϩ ND a ϩϩϩϩϩϩ, 140 -160% of the wild-type level; ϩϩϩϩ, wild-type level of activity; ϩϩϩ, 60-80% of the wild-type level; ϩϩ, 40-60% of the wild-type level; ϩ, 20-40% of the wild-type level; Ϫ, not detectable; ND, not done. The upper half is a summary of our previous study (9), and the lower half is a summary of this study. DNA binding activities (39). It should be noted that the Nterminal region of Mcm4 contains multiple serine/threonine residues, some of which may be phosphorylation sites of cyclindependent kinase and other kinases. It is an intriguing possibility that the interaction of Mcm with DNA and its helicase activity are regulated by phosphorylation of the N-terminal region of the Mcm4 protein. However, at the moment, we cannot rule out the possibility that the truncations caused alterations of the overall structure of the protein complex.
Both a Walker A mutation described previously (Table I) (9) and a zinc finger mutation described in this study resulted in reduced DNA binding activity, but showed differential effects on DNA helicase activity. This indicates that these segments are involved in interaction with DNA in distinct manners. The zinc finger mutation may alter the overall structure of the complex, affecting subunit assembly as well as DNA binding activity, which turned out to be stimulatory for DNA helicase. On the other hand, the Walker A mutant may be deficient in coupling of ATP hydrolysis to DNA unwinding, as shown in T7gp4 helicase (19).
Models of the DNA Helicase Actions of the Mcm4/6/7 Complex-Two models, termed "inchworm" and "active rolling," have been proposed for the actions of DNA helicases (2,40). The processive functions of the Mcm4/6/7 helicase, involving three heterologous subunits, may require complex and coordinated roles for each subunit to permit the highly regulated actions of this putative eukaryotic replicative helicase. The DnaB homohexameric helicase forms mainly trimers in the absence of Mg 2ϩ , and addition of Mg 2ϩ converts the trimers to hexamers (41). Mcm4/6/7 also forms stable trimers, which are converted to hexamers, and this conversion involves the zinc finger motif of Mcm4. Our results suggest the presence of at least two separate protein-protein interfaces on Mcm4, one for trimer formation and the other for multimerization of trimers. Electron microscopic studies of the Mcm4/6/7 hexamer demonstrated a doughnut shape with a 2-3-nm diameter hole in the center (42). An active Mcm4/6/7 helicase of fission yeast appears to consist of two hexamers at a fork (11). The mammalian Mcm4/6/7 complex also binds to a fork structure as a dimer of hexamers. 2 Our earlier studies established that Mcm4 and Mcm6 play a major role in DNA binding and ATP binding, respectively (9). Now we have determined that Mcm7 contributes to DNA helicase activity through facilitating hydrolysis of ATP. Based on these findings, we propose a model of sequential DNA unwinding by the Mcm4/6/7 hexamer. The Mcm4/6/7 complex binds to DNA through the actions of Mcm4, and then ATP binds to Mcm6. After formation of a transition-state structure facilitating ATP hydrolysis, nucleotide hydrolysis is carried out by the Mcm7 protein. Efficient helicase action is likely to be provided by close communication of the three subunits to achieve coor-dinated DNA translocation and duplex DNA unwinding. Further structural information is needed to understand the helicase actions of Mcm more precisely.