Mcm Subunits Can Assemble into Two Different Active Unwinding Complexes*

The replication fork helicase in eukaryotes is a large complex that is composed of Mcm2-7, Cdc45, and GINS. The Mcm2-7 proteins form a heterohexameric ring that hydrolyzes ATP and provide the motor function for this unwinding complex. A comprehensive study of how individual Mcm subunit biochemical activities relate to unwinding function has not been accomplished. We studied the mechanism of the Mcm4-Mcm6-Mcm7 complex, a useful model system because this complex has helicase activity in vitro. We separately purified each of three Mcm subunits until they were each nuclease-free, and we then examined the biochemical properties of different combinations of Mcm subunits. We found that Mcm4 and Mcm7 form an active unwinding assembly. The addition of Mcm6 to Mcm4/Mcm7 results in the formation of an active Mcm4/Mcm6/Mcm7 helicase assembly. The Mcm4-Mcm7 complex forms a ringed-shaped hexamer that unwinds DNA with 3′ to 5′ polarity by a steric exclusion mechanism, similar to Mcm4/Mcm6/Mcm7. The Mcm4-Mcm7 complex has a high level of ATPase activity that is further stimulated by DNA. The ability of different Mcm mixtures to form rings or exhibit DNA stimulation of ATPase activity correlates with the ability of these complexes to unwind DNA. The Mcm4/Mcm7 and Mcm4/Mcm6/Mcm7 assemblies can open to load onto circular DNA to initiate unwinding. We conclude that the Mcm subunits are surprisingly flexible and dynamic in their ability to interact with one another to form active unwinding complexes.

The replication fork helicase unwinds parental duplex DNA to provide single-stranded DNA (ssDNA) 3 substrates for the replicative polymerases (1,2). Unwinding of the replication fork in eukaryotes is powered by six minichromosome maintenance proteins (Mcm2-7) acting in concert with Cdc45 and the GINS complex (3)(4)(5)(6)(7)(8). There are an estimated 30,000 Mcm mol-ecules in Saccharomyces cerevisiae, in vast excess of what is required to license origins of DNA replication (9 -13). Thus, it is widely believed that Mcm proteins participate in functions other than DNA replication. There is evidence that Mcm7 acts as a transcription factor (14), and Mcm5 is essential for Stat-1mediated transcriptional activation (15). There is also evidence that Mcm proteins are involved in chromatin remodeling (16 -21), and Mcms interact directly with histone H3 (16,22). Mcms are also important to maintain genome stability (23,24), and it has been proposed that dormant origins licensed by Mcms are required to survive replication stress (25).
Investigations of ring-shaped helicases have been primarily focused on homohexameric assemblies (1,2,38). The eukaryotic Mcms are somewhat unusual in this regard, because they form heterohexamers. It is currently not known how the different eukaryotic Mcm subunits function in coordination to unwind DNA, and a comprehensive study of how individual Mcm subunit biochemical activities relate to unwinding activity has not been accomplished.
Study of the eukaryotic Mcms may reveal insight into how specific components of a hexameric helicase contribute to form an active unwinding assembly. The Mcm4-Mcm6-Mcm7 complex is a useful model system in this regard, because the complex purified from recombinant proteins is active as a helicase in vitro. In the past, Mcm4-Mcm6-Mcm7 was purified as a preassembled complex from partially purified subunits, because Mcm4, Mcm6, and Mcm7 subunits were not pure enough to allow for direct study with DNA (33,36,39,40). In this study, the production of highly purified, nuclease-free individual Mcm subunits is used to study how each Mcm subunit contributes to DNA unwinding. We find that the combination of individually purified Mcm4  , and a colony from the transformation was inoculated into 60 liters of LB containing 30 g/ml kanamycin. When the cells reached an A 600 of 0.6, 0.7 mM isopropyl 1-thio-␤-D-galactopyranoside was added, and the temperature was lowered to 12°C. 16 h later, cells were harvested and lysed by French press in 500 ml of solution containing 10% sucrose, 3.5 g of spermidine, 500 mM NaCl, and 50 mM Tris-HCl, pH 8.0. The lysate was applied to a 40-ml chelating Sepharose fast flow resin (GE Healthcare) precharged with nickel sulfate. The column was washed with solution containing 500 mM NaCl, 50 mM Tris-HCl, pH 8.0, and 50 mM imidazole. The Mcm4 protein was eluted in solution containing 10% glycerol, 500 mM NaCl, 50 mM Tris-HCl, pH 8.0, and 250 mM imidazole, dialyzed into Buffer H (10% glycerol, 20 mM Hepes, pH 7.5, 0.1 mM EDTA, and 1 mM DTT) containing 50 mM NaCl, and applied to a 30-ml SP-Sepharose column. The SP column was washed with Buffer H containing 100 mM NaCl, and Mcm4 was eluted in a 300-ml linear gradient from Buffer H containing 100 mM NaCl to Buffer H containing 500 mM NaCl. Peak fractions containing Mcm4 were combined and precipitated with 0.3 g/ml ammonium sulfate, and the pellet was resuspended in 22 ml of Buffer H ϩ 500 mM NaCl. 5 ml of this sample containing 22.5 mg of protein was applied to a 124-ml Superdex 200 column (GE Healthcare) that was pre-equilibrated in Buffer H ϩ 500 mM NaCl. Peak fractions containing Mcm4 were pooled and dialyzed against Buffer A (10% glycerol, 20 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, and 1 mM DTT) containing 50 mM NaCl and 10 mM magnesium chloride. The sample was applied to an ssDNA-cellulose resin, and the flow-through containing Mcm4 was flash-frozen.
Purification of Mcm6 was identical to the procedure followed previously (27), except the Mcm6 sample was further purified. The Mcm6 sample was subjected to preparative sizeexclusion chromatography with a 124-ml Superdex 200 column (GE Healthcare) that was pre-equilibrated in Buffer A ϩ 500 mM NaCl. Peak fractions containing Mcm6 were pooled and dialyzed against Buffer A containing 50 mM NaCl and 10 mM magnesium chloride. The sample was then applied to an ssDNA-cellulose resin, and the flow-through containing Mcm6 was flash-frozen.
Purification of Mcm7 was identical to the procedure followed previously (35), except the Mcm7 sample was further purified. The Mcm7 sample was subjected to preparative sizeexclusion chromatography with a 124-ml Superdex 200 column that was pre-equilibrated in Buffer A ϩ 500 mM NaCl. Peak fractions containing Mcm7 were pooled and dialyzed against Buffer A containing 50 mM NaCl and 10 mM magnesium chloride. The sample was then applied to an ssDNA-cellulose resin, and the flow-through containing Mcm7 was flash-frozen.
Radiolabeling and Annealing DNA-DNA was end-labeled with T4 polynucleotide kinase (New England Biolabs) according to the manufacturer's instructions. To anneal DNA, 500 nM radiolabeled DNA was incubated overnight at 37°C with 1 M complementary DNA in 20 mM Tris-HCl, 4% glycerol, 0.1 mM EDTA, 40 g/ml bovine serum albumin, 5 mM DTT, and 5 mM magnesium acetate in a final volume of 12 l. Following the overnight incubation, the reaction was diluted to a final concentration of 50 nM (concentration of radiolabeled DNA) with 20 mM Tris-HCl, 0.1 mM EDTA.
Helicase Assay-Each unwinding reaction contained 25.4 mM Tris-HCl, pH 7.5, 9.9 mM magnesium acetate, 23.3% glycerol, 152 M EDTA, 42.4 g/ml bovine serum albumin, 5.01 mM DTT, 4.98 mM ATP, 4.98 mM creatine phosphate, 19.9 g/ml creatine kinase, 1.2 nM 32 P-labeled DNA, and proteins as detailed in each figure legend, in a final volume of 11 l. Unless otherwise indicated, the final concentration of each Mcm subunit was 709 nM, expressed as a monomer (850 ng of Mcm4, 897 ng of Mcm6, and 750 ng of Mcm7). When UvrD was used as a control, the final concentration was 3.7 g/ml, or 45 nM monomer. All reaction samples were prepared on ice, and shifted to 37°C to initiate the reaction. At the end of the reaction, 1 l of proteinase K (10 mg/ml) was added, and the sample was incubated an additional 1 min at 37°C. 5 l of stop solution (2% SDS, 80 mM EDTA) was added, followed by 5 l of 6ϫ loading dye (15% Ficoll ϩ 0.1% xylene cyanol), and finally the reaction was flash-frozen. For DNA containing a biotin moiety, 100 nM streptavidin was preincubated with the DNA prior to adding Mcm proteins. For experiments containing T4 gp32, 26 ng (73 nM) of T4 gp32 (New England Biolabs) was preincubated with the DNA prior to adding the Mcm proteins.
For experiments with oligonucleotide substrates, samples were analyzed by an 8% native polyacrylamide gel containing 1ϫ TBE (90 mM Tris-HCl borate, 2 mM EDTA) at 225 V. The gel was dried for 2 h at 60°C and exposed to a phosphorimaging screen overnight. For experiments with M13 circular ssDNA substrates, samples were analyzed by a 1% native agarose gel containing 1ϫ TBE at 100 V. The gel was dried for 2 h at 60°C and exposed to a phosphorimaging screen overnight. . The samples were applied to a 24-ml Superose 6 column (GE Healthcare) pre-equilibrated in Buffer A ϩ 150 mM NaCl. 250-l fractions from the column were analyzed by SDS-PAGE followed by staining with Coomassie Blue. The elution peaks of molecular weight standards were determined under the same condi-tions. 5 l of each fraction of the elution was further analyzed for helicase activity.
Electron Microscopy-Samples for electron microscopy contained 20 mM Tris-HCl, pH 7.5, 200 M DTT, 5 mM magnesium acetate, 5 mM ATP, and Mcm proteins, each at a concentration of 709 nM (monomer), which is 77.3 g/ml Mcm4, 81.5 g/ml Mcm6, and 67.4 g/ml Mcm7. The sample was allowed to incubate at 37°C for 16 min. For samples containing glutaraldehyde, 0.04% glutaraldehyde was added after the initial incubation, and the sample was incubated at 37°C for an additional 30 s as described previously (41). The reaction was quenched by the addition of 50 mM EDTA. The reactions were kept on ice until they could be fixed by negative staining. After 100-fold dilution into helicase reaction buffer, 20 l of sample was applied to a copper mesh 300 grid for 30 s and wicked away, and 20 l of 1% uranyl acetate was applied to the grid for 30 s and then wicked away. Images were taken with a Philips CM-12 transmission electron microscope at a voltage of 80 kV with a magnification of ϫ140,000. Scion Image software was used to create an average of selected images. Scion Image was also used to measure the outer distance of the observed ring particles.
ATP Hydrolysis Assay-The ATP hydrolysis assay in a total volume of 11 l contained 25.4 mM Tris-HCl, pH 7.5, 9.9 mM magnesium acetate, 23.

Mcm4 and Mcm7
Form an Active Helicase Assembly-Previously, the Mcm4-Mcm6-Mcm7 complex was studied by purification of the intact hexameric assembly (29,35,40,42,43). To study how the individual subunits assemble to form a functional helicase, we improved the production and purification of individual Mcm subunits, and SDS-PAGE analysis of the purified subunits is shown (Fig. 1A). The new preparations of Mcm subunits were incubated with one another and examined for helicase activity (Fig. 1, B and C). The DNA substrate used in this unwinding assay is duplex DNA bearing a single strand extension at a 3Ј end and a biotin-streptavidin group attached to the 5Ј end. The 3Ј-single strand extension serves as a loading strand for the helicase, whereas the biotin-streptavidin is a bulky group that blocks helicase translocation along dsDNA.
Previously   (Fig. 2, A and C). The proteins eluted as a complex containing all three Mcm subunits ( Fig. 2A). The elution volume of the assembly exhibited an activity peak that matched that of thyroglobulin (Fig. 2C), suggesting that mixing the three Mcm subunits results in a single hexameric helicase assembly that contains all three subunits.
To study how rapidly the Mcm4 and Mcm7 proteins interact with one another to form an active helicase complex, we next studied how preincubating the Mcm subunits with one another affects the unwinding rate (Fig. 2, D and E). In this experiment, Mcm4 was preincubated with Mcm7 for varying times to allow the formation of an active complex. The Mcm4-Mcm7 complex was then incubated with radiolabeled DNA substrate, and the fraction of unwound DNA was determined by native gel electrophoresis. As the preincubation time varied from 0 to 90 min, the fraction of unwound DNA product did not change (Fig. 2, D 16 min, and DNA unwinding was monitored. The data indicate that the fraction of unwound DNA did not vary as a function of preincubation time (Fig. 2F). Furthermore, at every time point, the unwinding percentage was significantly greater than in a control experiment, in which buffer was added instead of   (Fig. 3A, filled squares,  and Fig. 1). However, Mcm4/Mcm7 did not unwind DNA with a reverse arrangement, because no activity was observed with DNA bearing a 5Ј-ssDNA extension and biotinstreptavidin positioned at the 3Ј end (Fig. 3A, filled triangles)  substrate shown in Fig. 3B. In this tandem substrate, the bottom strand is continuous, whereas the top strand bears a nick to create two duplexes in tandem. The duplex on the left bears a 3Ј-single strand extension for Mcm loading and no bulky group attached to the 5Ј end. The duplex on the right bears a long 5Ј-single strand extension to promote steric exclusion of this strand. When Mcm4/Mcm6/Mcm7 is incubated with this tan-dem duplex DNA, the protein complexes load on the 3Ј-single strand extension and then move in the 3Ј to 5Ј direction toward the duplex (Fig. 3B) (Fig. 3, C and  E). The only difference between the substrates in Fig. 3, C and E, is the position of the radioactive label, and the rate of tandem duplex unwinding is plotted (Fig. 3, D and F). Mcm4/Mcm7 did not unwind the left duplex of the tandem duplex substrate (Fig.  3, C and E, lanes 3-6, and Fig. 3, D and F, open diamonds), but it did unwind the right duplex (Fig. 3, C and E, lanes 3-6, and  Fig. 3, D and F, filled diamonds). This result contrasts with that of UvrD, an SF1 helicase that efficiently unwound both DNA strands of this tandem duplex (Fig. 3 (35,40), and we next examined if Mcm4/Mcm7 requires the 3Ј-single strand extension to load onto the tandem substrate. When the 3Ј-single strand extension of the tandem substrate was removed, the Mcm4/Mcm7 unwinding rate of the right duplex decreased by roughly one-half (Fig. 3, D and F, compare filled diamonds with filled squares), whereas the left duplex was still not unwound (Fig. 3, D and F, open squares) (Fig. 4A). The Mcm4/Mcm7 rings were sparsely populated on the microscopy grid, and we postulated that upon dilution and deposition on the grid, many of the Mcm4/Mcm7 rings may disassemble. Thus, we cross-linked the Mcm4/Mcm7 sample with glutaraldehyde and then imaged the assembly with negative stain electron microscopy (Fig. 4A).  3-6). The left duplex bears a 3Ј-60(dT) ssDNA extension, and the right duplex bears a 5Ј-60(dT) ssDNA extension. UvrD was used as a SF1 helicase family control (lane 2), and a no protein control is also shown (lane 1). D, data from experiments similar to C were quantified and plotted as a function of time. Data are shown for the tandem duplex substrate bearing a 3Ј-ssDNA extension (diamonds) and for a similar substrate that lacks the 3Ј-ssDNA extension (squares). For each DNA product, the time point data were fit to a linear equation. E, this experiment is similar to C, except a different strand is radiolabeled. F, data from experiments similar to E were quantified and plotted as a function of time for each DNA product. This technique has been used in the past to identify ring-shaped particles of T7 gp4 helicase (41). Using this approach, a dense array of particles appeared that are very similar to those of Mcm4/Mcm6/Mcm7 (Fig. 4A)  Mcm4/Mcm7 particles that appeared to be ring-shaped were selected for averaging, and the averaged image appears to be that of a single hexamer (Fig. 4B). We also measured the outer diameter of 100 of these ring-shaped particles, and we found that the mean diameter, 12.4 nm, is similar to that reported previously for Mcm4/Mcm6/Mcm7 (Fig. 4C)  ssDNA Stimulates Mcm4/Mcm7 Hydrolysis of ATP-One hallmark of helicase activity is DNA-dependent stimulation of ATP hydrolysis, because helicases use the energy derived from ATP binding and hydrolysis to power unwinding. We first mixed individual Mcm subunits and measured the rate of ATP hydrolysis in the absence of DNA (Fig. 5, A and B). We found that combining Mcm4 with Mcm7 resulted in a dramatic increase in ATP hydrolysis (Fig. 5, A and B), similar to a previous report (27). However, we also found low levels of ATP hydrolysis for other binary combinations of Mcms (Fig. 5B,  inset).  (Fig. 5, A and B). In contrast, the effect of DNA on ATP hydrolysis of other Mcm mixtures was modest (Fig. 5B, inset) oligonucleotides that are annealed to one another provided the duplex bears a 3Ј-single strand extension and a bulky group positioned at the 5Ј end (Fig. 1). Furthermore, these Mcm complexes are ring-shaped, and they may unwind DNA by a steric exclusion mechanism. There are two likely modes whereby ring-shaped particles can assemble onto the 3Ј-single strand extension of these DNA duplexes (50). In one model, the ring-shaped particle remains intact, and the single-stranded DNA is inserted into the helicase ring, much like the threading of a needle. In a second model, the helicase ring opens or disassembles, and the subunits then reassemble into a ring to surround the DNA strand.
To determine which of these two models is more likely for Mcm4/Mcm7, the complex was incubated with circular ssDNA with an annealed radiolabeled oligonucleotide, and unwinding was monitored (Fig. 6, A, B, and C). The annealed oligonucleotide contains a 5Ј-single strand extension to prevent the helicase from encircling two DNA strands. Mcm4/Mcm7 rapidly unwound the radiolabeled oligonucleotide, suggesting that the complex efficiently loads onto circular ssDNA (Fig. 6, B and C).
To determine whether ssDNA binding is required for the helicase activity observed, the DNA was preincubated with gp32 from T4 phage, a protein that binds tightly to ssDNA. Preincubation with T4-gp32 markedly inhibited the unwinding activity of the Mcm4-Mcm7 complex, suggesting that the Mcm4- Mcm7 complex requires loading onto ssDNA to unwind the annealed oligonucleotide.
To determine whether Mcm4/Mcm7 loading onto the 5Ј-single strand extension was required for unwinding of the annealed oligonucleotide, a biotin-streptavidin group was substituted for the 5Ј-single strand extension (Fig. 6D). Mcm4/ Mcm7 unwound this oligonucleotide as well, and this unwinding was largely blocked by preincubation with T4 gp32. These data suggest that the Mcm4/Mcm7 ring likely can open or disassemble and then reform on circular ssDNA. Similar results were found with the Mcm4-Mcm6-Mcm7 complex (Fig. 6E). Thus, one feature of these Mcm helicase complexes is the ability of these ring-shaped particles to assemble around circular ssDNA. What Makes an Active Mcm Helicase?-One feature that Mcm helicases have in common is that they form ring-shaped structures. Other replication fork helicases are ring-shaped, including eukaryotic viral helicases such as SV40 T antigen, papillomavirus E1 helicase, the bacterial DnaB helicase, and T7 and T4 phage helicases (51)(52)(53)(54)(55)(56)(57)(58). The ability of these protein complexes to form rings may be critical for the capacity of these complexes to function as helicases. In our study of Mcm4, Mcm6, and Mcm7, the ability of different mixtures to form rings correlated with their ability to unwind DNA. The ringshaped nature of these complexes may be important for unwinding DNA by steric exclusion, as has been previously proposed (59). Additionally, the ring-shaped nature may be important for these helicases because they act through a cyclic mechanism to translocate along DNA. Different models for DNA translocation have been proposed for the ring-shaped helicases, including the "bucket brigade" mechanism for T7 gp4 (55) and the "concerted escort" model for papilloma E1 (56). In both of these models, the ring-shaped architecture enables the helicases to bind and hydrolyze ATP in a cyclic manner, and this activity is critical for DNA translocation and hence DNA unwinding.

DISCUSSION
Although formation of a ring-shaped particle correlates nicely with unwinding activity in this study, the hydrolysis of ATP is more complex. For example, we found that Mcm6 with Mcm7 hydrolyzes ATP at a greater rate than the sum of both   (3,4,7,8,60). However, there is a 100-fold excess of Mcm proteins relative to replication origins in the cell, raising the possibility that Mcm proteins are involved in functions aside from DNA replication. Furthermore, it has been demonstrated that Mcm proteins function in activities other than DNA replication such as transcription (14,15), chromatin remodeling (16,17,20,21), and genome stability (23,24). Intriguingly, a hypomorphic mutation in Mcm4 causes chromosome instability and mammary adenocarcinomas in mice (24), and deregulated minichromosomal maintenance protein MCM7 contributes to oncogene-driven tumorigenesis (23). These data suggest that Mcm4 and Mcm7 proteins may have roles in genome maintenance aside from their role as part of the replicative helicase. Thus, it is possible that the Mcm4-Mcm7 complex may function as a helicase in a process in the cell that maintains genome stability, such as DNA repair. Moreover, the Mcm4-Mcm6-Mcm7 complex is an active helicase in vitro using proteins from mouse, Drosophila, Xenopus, S. pombe, and S. cerevisiae (3,29,33,35,37). The observation that Mcm4/ Mcm6/Mcm7 activity is conserved across species suggests that the complex may act as a DNA helicase in an important cellular process such as genome maintenance. A radiolabeled DNA oligonucleotide is annealed to M13 circular ssDNA. The radiolabeled oligonucleotide bears a 5Ј-single strand extension to promote steric exclusion, and unwinding of the radiolabeled strand is monitored by native gel electrophoresis. The experiment is also performed in the presence of T4 gp32 to coat the free ssDNA with a nonspecific protein. B, equimolar mixture of Mcm4/ Mcm7 was incubated with DNA substrate for the time points incubated at 37°C in the absence (lanes 3-6) and presence (lanes 7-10) of T4 gp32. The position of free ssDNA and DNA annealed to M13 were determined by standards analyzed on the same gel (lanes 11 and 12). UvrD was also tested (lane 2), as well as a no protein control (lane 1). C, data from experiments similar to B were quantified and plotted as a function of time in the absence (filled squares) and presence (open squares) of T4 gp32. The data in the absence of T4 gp32 were fit to a logarithmic equation, and the data in the presence of T4 gp32 were fit to a linear equation. D, experiment is similar to that shown in C, except the radiolabeled oligonucleotide bears a 5Ј-biotinstreptavidin in the place of the ssDNA extension. Unwinding rates were determined in the absence (

Potential Role of Eukaryotic Mcm Subunit Specialization in
DNA Unwinding-Most replication fork helicases that have been studied are homohexameric. Because each subunit of the homohexamer is identical in its covalent structure, some models for activity attribute equivalent functional roles for each of the six subunits. For example, in both the bucket brigade model for T7 gp4 and the "coordinated escort" model for papilloma E1 virus, each subunit of the hexamer binds and hydrolyzes ATP in a sequential manner, and each subunit binds and later releases DNA in a sequential manner (55,56). Thus, each subunit is functionally equivalent. The Mcm helicase system is different from these homohexameric systems, in that each of the six subunits of the Mcm2-7 complex has a unique amino acid sequence. The high degree of sequence conservation across species for Mcm subunits suggests that each subunit has evolved for a particular function. There have been numerous reports ascribing specific biological functions to particular Mcm subunits. However, an important question that arises is whether different subunits within the Mcm2-7 complex function equivalently in unwinding DNA, consistent with the bucket brigade or coordinated escort models, or whether specific subunits within the Mcm complex are designated to perform particular functions as part of an unwinding machine.
In this study, we find that different Mcm subunits have distinct biochemical properties, in particular with regard to the ability of various mixtures to unwind DNA, form ring-shaped particles, and hydrolyze ATP. These observations extend the current literature that demonstrates specific functions for Mcm subunits. However, we also find that