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The minichromosome maintenance (MCM) complex is thought to function as the replicative helicase in archaea, separating the two strands of chromosomal DNA during replication. The catalytic activity resides within the C-terminal region of the MCM protein, while the N-terminal portion plays an important role in DNA binding and protein multimerization. An alignment of MCM homologues from several archaeal species revealed a number of conserved amino acids. Here several of the conserved residues located on the surface of the helicase have been mutated and their roles in MCM functions determined. It was found that some mutations result in increased affinity for ssDNA while the affinity for dsDNA is decreased. Other mutants exhibit the opposite effect. Thus, the data suggest that these conserved surface residues may participate in MCM-DNA interactions.
The minichromosome maintenance (MCM) helicase is thought to function as the replicative helicase in eukarya and archaea. Most archaeal species contain a single MCM homologue with biochemical properties that are similar to the eukaryotic enzyme. Both archaeal and eukaryal MCM helicases exhibit ATP-dependent 3′-5′ helicase activity, can bind and translocate along single-stranded (ss) and double-stranded (ds) DNA, unwind DNA-RNA hybrids while translocating on the DNA strand, and can displace proteins from DNA (reviewed in [1–3]). The archaeal MCM protein can be divided into three parts; the N-terminal region, the AAA+ catalytic core, and a C-terminal region that may form a helix-turn-helix (HTH) domain [4–7]. The three-dimensional structure of the N-terminal portion of the Methanothermobacter thermautotrophicus and Sulfolobus solfataricus MCM proteins revealed a three domain architecture [4, 5]. Biochemical studies showed that domain A participates in regulating helicase activity, domain B participates in DNA binding, and domain C is involved in hexamer formation, DNA binding and communication between the N-terminal part and the catalytic domains (reviewed in: [1, 3]). Although the N-terminal portion of MCM is less conserved than the AAA+ region, an alignment of the N-terminal region from a number of archaeal species revealed the presence of several highly conserved residues, particularly in domain C (Figure 1(a)). Several of these residues have previously been reported to play an essential role in communicating between the N-terminal DNA binding and C-terminal catalytic activity [8, 9]. In this study, several conserved residues in domain C of the M. thermautotrophicus MCM protein were individually (except for one double mutant) mutated and the effects on MCM function were examined. All but one of the amino acids analyzed in the study are located on the surface of the helicase (Figure 1(b)), and thus they may play functional rather than structural roles. The amino acids analyzed in the study, highlighted in Figure 1(a), were Gln176, Pro210, Gly211, Asp212, Val214, and Gly218. It is shown here that while V214 or G218 to Ala substitutions do not significantly alter the MCM biochemical properties, P210 to Gly or G211 to Ala mutations promote enhanced binding to ssDNA and reduced binding to dsDNA. Q176 to Ala or D212 to Asn show better binding to dsDNA compared to ssDNA binding. The possible roles of these residues in MCM function are discussed.
ATP and [γ-32P]ATP were obtained from GE Healthcare and oligonucleotides were synthesized by the NIST/UMD DNA synthesis facility.
All M. thermautotrophicus MCM mutant proteins used in this study are derivatives of the full-length enzyme and were generated using PCR-based site-directed mutagenesis as previously described . All constructs contain a C-terminal His6-tag and were cloned into the NdeI and XhoI sites of the pET-21a vector (Novagen). The oligonucleotides used for the mutagenesis are shown in Supplementary Table 1. The wild-type and mutant proteins were overexpressed in Escherichia coli DE3 codon plus cells (Stratagene) at 37°C and purified on a Ni-column as previously described .
One hundred micrograms of wild-type or mutant MCM protein were fractionated on a superose-6 gel filtration column (HR10/30, GE Healthcare) pre-equilibrated with buffer containing 20mM Tris-HCl (pH 7.5), 150mMNaCl, and 10% glycerol. The column was run at 22°C with a flow rate of 0.2ml/min. The presence of protein was determined by ultraviolet absorbance at 280 nm.
ATPase activity was measured in reaction mixtures (15μl) containing 25mM Hepes-NaOH (pH 7.5), 1mM dithiothreitol, 5mM MgCl2, 100μg/ml bovine serum albumin (BSA), and 1500 pmol of [γ-32P]ATP (3000Ci/mmol; GE healthcare) and 10 or 30nM MCM protein (as monomer) in the presence or absence of 50ng ssDNA (5′-GCAGATAACAGTTGTCCTGGAGAACGACCTGGTTGACACCCTCACACCC-3′). After incubation at 60°C for 60min, samples were placed on ice, then an aliquot (1μl) was spotted onto a polyethyleneimine cellulose thin layer plate, and ATP and P i were separated by chromatography in 1M formic acid and 0.5M lithium chloride. The extent of ATP hydrolysis was quantitated by phosphorimager analysis. All ATPase assays were repeated three times, and their averages with standard deviations are shown in the figure.
Single stranded and dsDNA substrates for filter binding assays were prepared by labeling the oligonucleotide using [γ-32P]ATP and T4 polynucleotide kinase. Unincorporated [γ-32P]ATP was removed from the DNA substrate by extraction from polyacrylamide gel as previously described . For ssDNA binding a 49-mer oligonucleotide with the sequence 5′-GCAGATAACAGTTGTCCTGGAGAACGACCTGGTTGACACCCTCACACCC-3′ was used. For dsDNA the same oligonucleotide was hybridized to its complementary sequence.
Filter binding assays were carried out in a reaction mixture (20μl) containing 25mM Hepes-NaOH (pH 7.5), 2mM DTT, 10mM MgCl2, 100μg/ml BSA, 50fmol of32P-labeled ss or dsDNA substrates and 10, 30, or 90nM of MCM protein (as monomer). After incubation at 60°C for 10min the mixture was filtered through an alkaline-washed nitrocellulose filter (Millipore, HA 0.45μm)  which was subsequently washed with 20mM Tris-HCl (pH 7.5). The radioactivity adsorbed to the filter was measured by liquid scintillation counting. All DNA binding experiments were repeated three times, and their averages with standard deviations are shown in the figure.
Substrates for helicase assays were made as previously described  using the following oligonucleotides. For forked substrate the two oligonucleotides were 5′-GGGACGCGTCGGCCTGGCACGTCGGCCGCTGCGGCCAGGCACCCGATGGCGTTTGTTTGTTTGTTTGTTTGTTT-3′ and 5′-TTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGCCGACGTGCCAGGCCGACGCGTCCC-3′ and for the substrate with only a 3′-overhang region (flat substrate) the oligonucleotides used were 5′-CCGACGTGCCAGGCCGACGCGTCCC-3′ and 5′-GGGACGCGTCGGCCTGGCACGTCGGCCGCTGCGGCCAGGCACCCGATGGC-3′.
DNA helicase activity assays were measured in reaction mixtures (15μl) containing 20mM Tris-HCl (pH 8.5), 2mM DTT, 10mM MgCl2, 100μg/ml BSA, 3.33mM ATP, 10fmol (0.66nM) of 32P-labeled substrate and 10, 30 or 90nM MCM protein (as monomer) for flat substrate or 3, 9, or 27nM MCM protein (as monomer) for forked substrate. Following incubation at 60°C for 1hr the reactions were stopped by adding 5μl of loading buffer containing 1% SDS, 100mM EDTA, 0.1% xylene cyanol, 0.1% bromophenol blue, and 50% glycerol and chilling on ice. Aliquots (10μl) were loaded onto an 8% native polyacrylamide gel in 0.5X TBE and electrophoresed for 40min at 180V. Gels were visualized and quantitated by phosphorimaging. All helicase experiments were repeated three times, and their averages with standard deviations are shown in the figure.
DSC was used to determine the denaturation temperature of the wild-type and mutant proteins as an indicator of the thermostability of the enzymes. The purified proteins were dialyzed at room temperature against buffer containing 20mM potassium phosphate (pH 7.5), 100mM NaCl and 10% glycerol. The protein concentrations were determined using an absorbance at 280 nm and a calculated extinction coefficient of A280 of 28730cm−1·M−1. The solution outside the dialysis bag was retained and used as reference for the experiments.
DSC measurements were performed using a VP-DSC Microcalorimeter from Microcal Inc. (Northampton, MA). The volumes of the solution and the reference vessel were 0.511ml and the scan was either at slow rate (15Khr−1) or medium rate (60Khr−1), with the temperature ranging from 25–85°C. Since the scans of protein samples were irreversible, the second scan for each sample was used as the baseline. After subtraction of the baseline from the protein scan, the resulting raw data of differential powers as a function of time were divided by the scan rate to convert into a heat capacity as a function of temperature. Utilizing the EXAM program , a two state, AB, transition model was then fitted to the heat capacity as a function of temperature scan to determine the van't Hoff enthalpy (ΔH VH) for the scan from the shape of the transition peak; a transition temperature (T m), and a calorimetric transition enthalpy (ΔH cal) was calculated from the area under the transition peak (mJ) divided by the moles of protein in the sample cell (concentration of protein × 0.511ml). DSC scans on the samples were repeated twice.
In order to gain useful information from a mutant enzyme it is necessary to show that the mutation did not affect the overall structure of the molecule. All residues studied except G218 are located on the surface of the N-terminal part of MCM protein (Figure 1(b)) and thus are not expected to substantially alter the overall structure of the molecule. As shown in Figure 2 this indeed is the case. The size of all mutant proteins is similar to that of the wild-type enzyme (Figure 2(a)) although some mutant proteins elute earlier suggesting that they may have a greater tendency to form filaments. It was previously shown that the filaments are in equilibrium with the dodecameric and hexameric forms in a concentration-dependent manner . The mutation may change the equilibrium between the different forms. According to the CD analysis, all mutant proteins retain similar overall secondary structure suggesting that the mutations introduced do not likely impact the overall structure of the proteins (Figure 2(b)). In addition, DSC analysis shows that all mutant proteins have similar thermal stability as the wild-type enzyme although the heat capacity change varies (Table 1). It is not clear what contributes to the large deviations and differences in the heat capacity change. Nevertheless, taken together, these results suggest that the mutations do not substantially alter the structures of the proteins.
After establishing that the mutant proteins retain their overall structure, the effect of each mutation on MCM helicase activity was determined. As shown in Figure 3(a), most mutant proteins show wild-type enzyme-like activity on a forked DNA substrate. The exception is the D212N mutant enzyme, which decreases in activity as the concentration of the enzyme increases. It has been previously shown that MCM proteins are more active on forked substrate in comparison to substrate that contains only a 3′-overhang ssDNA region (flat substrate) (e.g., see: ). This difference in activity was previously used to analyze the effect of mild mutation on MCM helicase activity (e.g., ). Thus, helicase assays were also performed with flat substrate. As shown in Figure 3(b), the P210G, G211A, and PG210,211GA mutant enzymes have reduced helicase activity, particularly at low enzyme concentration. The D212N mutant protein also exhibits reduced helicase activity on a flat substrate but with no inhibition of helicase activity at high protein concentration (Figure 3(b)).
The data presented in Figure 3 show that several mutant enzymes have reduced helicase activity. This could be due to reduced DNA binding or reduced ATP hydrolysis as both are needed for DNA unwinding. To test this, DNA binding and ATPase assays were performed with the wild-type and mutant enzymes. All mutant proteins showed lower binding to dsDNA in comparison to the wild-type protein (Figure 4(a)). In particular, G218A showed ~25%, and Q176A, P210G, and G211A showed ~50% reduction compared to the duplex binding of the wild-type enzyme. When the ability of the mutant proteins to bind ssDNA it was found that the P210G, G211A or PG210,211GA mutant enzymes showed better binding than the wild-type protein while the D212N and Q176A exhibit reduced ability to bind ssDNA (Figure 4(b)). However, regardless their ability to bind DNA all proteins possess helicase activity (Figure 3), in particular the Q176A mutant enzyme which has helicase activity indistinguishable from the wild-type MCM on forked and flat substrate (Figure 3) but exhibits reduced ability to bind DNA (Figures 4(a) and 4(b)). It was previously shown that ATP enhances ssDNA binding by M. thermautotrophicus MCM [17, 18] and therefore the effect of ATP on ssDNA binding by the mutant enzymes were evaluated (Figure 4(c)). In the presence of ATP all proteins exhibit similar DNA binding activity. The ability of the mutant proteins to bind ssDNA in the presence of ATP may explain why some mutant proteins had wild-type helicase activity while no measurable ssDNA binding could be detected in the absence of ATP.
MCM proteins, including M. thermautotrophicus MCM, utilize ATP hydrolysis to translocate along DNA. The ATPase activity of M. thermautotrophicus MCM is stimulated in the presence of DNA [19–21]. The helicase and DNA binding experiments described above suggest that ATPase activity may be impaired in some of the mutants. As shown in Figure 5, the P210G, G211A, and PG210,211GA mutant proteins exhibit much greater (>10 fold) basal ATPase activity (in the absence of DNA) compared to the wild-type enzyme. Stimulation by DNA cannot be assessed for these proteins as maximal ATPase activity levels are presence in the absence of DNA. It is interesting to note, however, that those three mutant proteins (P210G, G211A, and PG210,211GA) also exhibit better ssDNA binding in comparison to the wild-type enzyme (Figure 4(b)).
The residues studied could also play a role in protein-protein interactions. The archaeal Cdc6 proteins were shown to interact with MCM and to inhibit helicase activity [11, 22, 23]. The M. thermautotrophicus genome contains two Cdc6 homologues, Cdc6-1 and Cdc6-2, and both proteins were shown to inhibit MCM helicase activity . It is possible that the surface residues mutated in the current study interact with the Cdc6 protein. To address this possibility the effect of Cdc6 proteins on the helicase activity by the mutant proteins was evaluated. As shown in supplementary Figure 1 Cdc6 proteins inhibit helicase activity of all mutant proteins to a similar extent suggesting that the mutated residues are not needed for the interaction with the Cdc6 protein.
The role of several conserved residues in the M. thermautotrophicus MCM protein was examined using mutant enzymes. Mutations P210G, G211A and PG210,211GA exhibit elevated DNA binding (Figure 4) and ATPase activity (Figure 5). The P210 and G211 are located on the surface of domain C where they face the AAA+ catalytic domains in close proximity to helix 2 [3, 7]. Deletion of part of helix 2, known as the helix 2 insertion (H2i), resulted in an enzyme with no helicase activity and increased affinity for DNA . It is possible that mutations of P210 and G211 affect the local structure of helix 2 and/or H2i. These local alterations are likely to be mild, as helicase activity could be detected with all three mutant enzymes (P210G, G211A, and PG210,211GA).
While the P210G, G211A, and PG210,211GA mutant enzymes demonstrated increased ssDNA binding they exhibit reduction in binding to dsDNA (Figure 4). The Q176A and D212N mutant proteins, on the other hand, exhibited the opposite effect, showing better binding to dsDNA than to ssDNA (Figure 4). The MCM protein may adopt a particular conformation when it binds ssDNA and a different one when it binds dsDNA. The P210G, G211A, and PG210,211GA mutations may cause the MCM proteins to adopt a conformation that favors binding to ssDNA while the Q176A and D212N may stabilize the other conformation.
The reduction in ssDNA binding observed with the Q176A mutant protein could be restored in the presence of ATP (Figure 4(c)). Furthermore, in size exclusion chromatography, the Q176A mutant protein showed a relatively broad elution profile (Figure 2(a)), suggesting that the mutation may cause destabilization of the dodecameric ring. Q176 is located at the end of the β-sheet 7 (β7), which runs across domains B and C. Domain B was implicated in both DNA binding via the Zinc finger [10, 18] and dodecamer formation [16, 25]. It is possible that mutating Q176A may affect the structure and/or orientation of domain B in relation to the rest of the molecule thus affecting both DNA binding and the stability of the dodecamer.
It is well established that the positively charged residues inside the central cavity of the hexameric MCM are responsible for both ss and dsDNA binding [4, 26]. It has also been reported that the helicase interacts with DNA via residues that are located on the surface of the MCM [27, 28]. Although it is not yet clear how, or where, the MCM protein binds to ss or dsDNA outside of the central cavity, the data presented here may suggest that the interactions, at least in part, are via P210, G211, and Q176. The data also suggest that this interaction on the surface of MCM may play a regulatory role in switching between ss and dsDNA binding. These interactions on the surface of the molecule, however, are not essential for helicase activity in vitro. In the future, when the methods are developed, it will be of interest to study the effect of these mutations in vivo.
Supplementary Table 1 shows the oligonucleotides used to generate the mutant proteins using PCR-mediated mutagenesis.
Supplementary Figure 1 shows the effect of the two M. thermautotrophicus Cdc6 proteins on the helicase activity of the wild-type and mutant MCM proteins.
The authors would like to thank Dr. Lori Kelman for her comments on the paper. This work was supported by a Grant from the NSF (MCB-0815646) awarded to Z. Kelman.