The PS1 Hairpin of Mcm3 Is Essential for Viability and for DNA Unwinding In Vitro

The pre-sensor 1 (PS1) hairpin is found in ring-shaped helicases of the AAA+ family (ATPases associated with a variety of cellular activities) of proteins and is implicated in DNA translocation during DNA unwinding of archaeal mini-chromosome maintenance (MCM) and superfamily 3 viral replicative helicases. To determine whether the PS1 hairpin is required for the function of the eukaryotic replicative helicase, Mcm2-7 (also comprised of AAA+ proteins), we mutated the conserved lysine residue in the putative PS1 hairpin motif in each of the Saccharomyces cerevisiae Mcm2-7 subunits to alanine. Interestingly, only the PS1 hairpin of Mcm3 was essential for viability. While mutation of the PS1 hairpin in the remaining MCM subunits resulted in minimal phenotypes, with the exception of Mcm7 which showed slow growth under all conditions examined, the viable alleles were synthetic lethal with each other. Reconstituted Mcm2-7 containing Mcm3 with the PS1 mutation (Mcm3K499A) had severely decreased helicase activity. The lack of helicase activity provides a probable explanation for the inviability of the mcm3 K499A strain. The ATPase activity of Mcm2-73K499A was similar to the wild type complex, but its interaction with single-stranded DNA in an electrophoretic mobility shift assay and its associations in cells were subtly altered. Together, these findings indicate that the PS1 hairpins in the Mcm2-7 subunits have important and distinct functions, most evident by the essential nature of the Mcm3 PS1 hairpin in DNA unwinding.


Introduction
In order for DNA replication to occur, the DNA duplex strands need to be separated by a replicative helicase [1]. Cellular replicative helicases tend to be hexameric rings that bind DNA within their central channels [2,3]. The ring shape is thought to maintain association with DNA thus enhancing the processivity of the helicase [4], and may be important for DNA unwinding by potentially excluding one strand from the central channel [5,6]. Regardless of the exact mechanism for DNA unwinding, the helicase must use nucleotide binding and hydrolysis to translocate along the bound DNA.
X-ray structures of homo-hexameric replicative helicases that are members of the AAA+ family, including the superfamily 3 (SF3) helicase from bovine papillomavirus (E1) and mini-chromosome maintenance (MCM) from archaeal species, provide insight into how DNA translocation is achieved [7][8][9][10]. Notably, a b hairpin from each subunit projects into the central channel of the helicase. The structure of the E1 hexameric helicase with singlestranded DNA in its central channel identifies residues at the tip of the hairpin that contact the sugar phosphate backbone; in particular a lysine side-chain forms a salt-bridge with the DNA backbone [11]. ATP binding and hydrolysis are thought to drive conformational changes, leading to a sweeping motion of the b hairpins that moves DNA through the central channel [9]. Later structures of archaeal MCM proteins demonstrated the existence of the b hairpins with a lysine residue near the tip [7,10]. These hairpins are referred to as the pre-sensor 1 (PS1) hairpins due to their position adjacent to the sensor 1 motif of the AAA+ domain as shown for the Sulfolobus solfataricus (Sso) MCM ( Figure 1). Mutation of the conserved lysine in archaeal MCM proteins abrogates its helicase activity, but only slightly affects DNA binding, consistent with a role in DNA translocation [12].
In eukaryotic cells, the replicative helicase is comprised of six paralogous proteins of the AAA+ family, termed Mcm2-7. Each of the six subunits is essential for DNA replication in cells from yeast to mammals [13,14]. The requirement for six distinct subunits may reflect the greater need for control of DNA replication and hence cell proliferation in eukaryotic cells compared to other systems. Indeed, the Mcm2-7 subunits are differentially targeted by protein kinases for control of cell proliferation [15][16][17][18][19][20][21][22][23][24][25], and have distinct roles in the activity of the intact complex [26]. In this regard, ATP sites found within each of the Mcm subunits are formed at the interface of neighboring subunits, and contribute differently to the overall ATPase activity of the complex [26][27][28]. Not all of the ATP sites are essential for DNA unwinding, even though the ATP sites are essential for viability [28][29][30][31]. Models for DNA unwinding by the homo-hexameric helicases suggest each subunit makes an identical contribution. This is not the case for Mcm2-7 as suggested by the distinct sequences of the components and the different ATPase activity of subunit pairs [26,27]. However, the exact contribution each subunit makes to the DNA unwinding by Mcm2-7 is currently unknown. Here
To clone MCM3 (pMD562) and mcm3 K499A (pMD563) into YEplac181, a SphI-SacI fragment of pMD235 and pMD386 was ligated into the same sites of YEplac181. A fragment of SphI-SacI from pMD386 was also ligated to the same sites in YIplac211 to generate mcm3 K499A -YIplac211 (pMD561). For cloning mcm4 K658A into YIplac211, we first amplified mcm4 K658A by PCR using oligonucleotides MD85, MD274, and mcm4 K658A -YCplac111 as template. This product was cloned into YIplac211 using PstI and BamHI to give pMD438. For mcm5 K506A , a BlpI site was introduced into YIplac211 at the SmaI site. mcm5 K506A was introduced into this plasmid from mcm5 K506A -YCplac111 as a SphI-BlpI fragment (pMD439). pMD440 was constructed by inserting a SphI-BamHI fragment of mcm6 K665A -YCplac111 into YIplac211. To generate MCM3 and mcm3 K499A myc 9 N-terminally tagged expression plasmids, MCM3 and mcm3 K499A were amplified by PCR using  [7]. The Mcm proteins are members of the AAA+ family of ATPases. The ATPase active sites are formed at the interface between two subunits. The Walker A (red), Walker B (magenta), and Sensor-1 (green) motifs are contributed by one subunit; the Arginine Finger (orange) and Sensor-2 (cyan) motifs are contributed by a second subunit (reviewed in 13). The Pre-Sensor 1 motif (PS1; blue) harbors a conserved lysyl residue at the turn between the two b-strands, and is not directly involved in ATP hydrolysis; this lysyl residue is the subject of the current work. For clarity, the PS2 motif is not indicated on the 3-dimensional structure. doi:10.1371/journal.pone.0082177.g001 oligonucleotides MD84, MD556, and cloned using NotI and SacI into a derivative of YCplac111 where the DED1 promoter drives expression of a myc 9 N-terminally tagged protein [33]. The pET24a-mcm3 K499A was cloned by cutting mcm3 K499A -YCplac111 with NdeI and SacI and ligating into the same sites of pET24a.

Plasmid shuffling
Diploid heterozygous strains containing a KanMX deletion of a mcm gene were obtained from Open Biosystems. The mcm2::his3 disruption strain (MDY54) was a derivative of YMD33 [31]. Each of these was transformed with the relevant MCM-YCplac33 plasmid and sporulated to give MDY16, 17, 40, 41, 70, and 100. mcm deletion haploid strains containing their corresponding MCM-YCplac33 were transformed with a mcm KA -YCplac111 or MCM-YCplac111. The transformed strains were grown in YPD, then plated on 5-FOA-containing media to select for cells that lost the MCM-YCplac33 [34].
Imaging yeast overexpressing of MCM3 and mcm3 K499A BY4741 transformed with YCplac111-GAL10-MCM3 or YC-plac111-GAL10-mcm3 K499A was grown in minimal media lacking leucine supplemented with 2% galactose overnight. The overnight cultures were diluted to 10 6 cells/mL with minimal media lacking leucine supplemented with 2% galactose. After two hours cells were imaged under bright field using a Nikon Eclipse Ti microscope. Measurements were taken using NIS Elements Imaging Software.

Proteins
The recombinant Mcm subunits were purified from Escherichia coli and reconstituted into Mcm2-7 as described [27].

Mcm3 K499A purification
The mcm3 K499A pET24a plasmid was transformed into BL21 DE3 Codon+. Twelve liters of transformed cells were grown in LB media with 100 mg/L of ampicillin, and 25 mg/L of chloramphenicol to a density of A 600 = 0.6. Cells were cooled to 15uC and isopropyl b-D-1-thiogalactopyranoside added to a final concentration of 1 mM. Cells were incubated at 15uC for 20 hours prior to collecting the cell pellet. The cell pellet was resuspended in 250 mL of Buffer H (20 mM HEPES, pH 7.5, 2 mM DTT, 10% v/v glycerol, and 0.1 mM EDTA) and lysed at 15000 psi in an Emulsiflex-C3 high pressure homogenizer. Debris was pelleted by centrifugation at 15000 g for 25 minutes and the supernatant decanted. Ammonium sulfate was added to the supernatant (0.25 g/mL) with stirring at 4uC. Ammonium

Western blotting
Western blotting was performed using polyvinylidene difluoride membranes and anti-myc (Sigma-Aldrich) as described by Mutiu et al [36].

Biochemical assays
DNA unwinding and ATPase assays were performed essentially as described by Stead et al [25] with the exception that intact complex was used. ATP hydrolysis was assayed using thin-layer chromatography. Each 15-ml reaction contained 1 mM [c 32 P]ATP (20 mCi/mmole; Perkin Elmer Life Sciences), 20 mM Tris-HCl (pH 7.5), 10 mM magnesium acetate, and 2 mM DTT, and 200 nM Mcm2-7. At the indicated times, 2 mL of each reaction was removed and quenched with 2 mL of 50 mM EDTA (pH 8). One microliter was spotted onto a polyethyleneimine cellulose sheet (EM Science), developed in 0.6 M potassium phosphate (pH 3.4), dried, exposed to a PhosphorStorage screen, and scanned with a Storm 860 scanner (GE Healthcare). DNA unwinding measurements were performed with a DNA substrate containing 30 nucleotides of duplex, with 60 nucleotides of singlestranded DNA on one strand and a 59 biotin on the other strand. Each reaction (6 mL) contained 20 mM Tris-HCl (pH 7.5), 10 mM magnesium acetate, 100 mM EDTA, 5 mM DTT, 5 mM ATP, 67 nM streptavidin, 1 nM substrate with 100 nM, Table 3. Yeast strains used in this study.

DNA binding assay
The single-stranded DNA affinity chromatography was performed with a 200 mL single-stranded DNA Sepharose column (see above Mcm3 K499A purification). Five micrograms of Mcm2-7 complex were applied to the column in buffer H containing 5 mM ATP and 50 mM NaCl and eluted with buffer containing 5 mM ATP and either 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, or 500 mM NaCl. Each elution was performed twice with one column volume. 24 mL of each fraction was separated by SDS-PAGE (6%). The polyacrylamide gels were stained with colloidal blue stain, and then washed with deionized water to destain the gels for imaging. The destained gels were then silver stained according to protocol provided in Pierce Silver Stain Kit (Thermo Scientific) to detect protein in the column fractions.

Gel filtration chromatography
Proteins extracts were prepared cryogenically as described by Saleh et al [37]. Five mg of yeast extract prepared in 50 mM sodium phosphate pH 7.0, 150 mM NaCl was loaded at a flow rate of 0.3 mL/min. onto a 24 mL FPLC Superose 6HR10/30 column (Amersham Pharmacia Biotech.). Protein from 10 mL aliquots of 250 mL fractions for wild type protein and 10 mL for    [40]). Molecular graphics were generated using PyMOL (Version 1.5.0.5, Schrödinger, LLC) and electrostatic surface calculations were carried out using PDB2PQR [41] and APBS [42].

Effects of PS1 mutations in Mcm2-7 on yeast growth
Each of the Mcm subunits contains a pre-sensor 1 (PS1) hairpin adjacent to the sensor 1 motif of the AAA+ domain ( Figure 1). To determine whether the PS1 hairpin motifs are important for the function of Mcm2-7, the replicative helicase in eukaryotic cells, we mutated the conserved lysine residue to alanine in each of the Mcm subunits (Figure 2A). The mutant genes, encoded on LEU2containing centromeric plasmids, were shuffled into haploid strains bearing a deletion in the corresponding mcm and maintained by MCM on a URA3-containing centromeric plasmid. Lack of function of the mutant Mcm subunit is indicated by the absence of growth on media containing 5-fluoroorotic acid (5-FOA), which is toxic to URA3-expressing strains. Of the six subunits, only a mutation in the Mcm3 PS1 hairpin (mcm3 K499A ) resulted in a loss of viability on 5-FOA ( Figure 2B). Slow growth was noted for the mcm7 K550A strain, but it is viable. To more fully compare the relative phenotypes of the mcm3 K499A and mcm7 K550A strains, we have also incubated the strains for an extended period ( Figure S1). The presence of the mutant allele as the sole copy of the mcm KA in the viable strains was confirmed by PCR and restriction digestion.
The Mcm genes were first identified through their requirement for the maintenance of autonomously replicating chromosomes in yeast [43]. To ensure that the inviability of the mcm3 K499A strain was not due to a failure to maintain the plasmid, we integrated the mcm3 K499A mutation into the diploid yeast strain BY4743 and analyzed the viability of spore colonies after sporulation and tetrad dissection. As shown in Figure 2C viability segregates in a 2:2 manner consistent with the inability of mcm3 K499A to support growth. We also addressed whether mcm3 K499A would support growth when overexpressed on a 2-micron plasmid by plasmid shuffling ( Figure 2D). Similar to what we observed with the centromeric plasmid, no growth was detected. Further suggesting that the inability of mcm3 K499A to support viability is not the result of reduced stability of the protein, we find that myc 9 -tagged wildtype Mcm3 and Mcm3 K499A are found at a similar level ( Figure  S2). Taken together we conclude that the Mcm3 PS1 hairpin is essential for the function of the Mcm2-7 complex. To further characterize the effects of the PS1 mutations, we examined the growth of viable strains bearing the mutations at different temperatures ( Figure 3A). For these experiments the PS1 hairpin mutations were integrated into the genome. At each of the temperatures, the relative growth mcm7 K550A was reduced. The mcm2 K633A containing strain grew somewhat more slowly at 16uC. Mutations in the Mcm subunits often result in sensitivity to genotoxic agents. Therefore, we examined the growth of each of the strains with a PS1 mutation on media containing the ribonucleotide reductase inhibitor hydroxyurea, or the DNAdamaging agent methyl methanesulfonate (MMS) ( Figure 3B). Consistent with its slow growth at different temperatures, strains containing mcm7 K550A grew slowly on both agents, with a slight sensitivity to MMS noted. Similarly strains bearing the other PS1 mutations were not sensitive to hydroxyurea or MMS.
Our plasmid shuffling experiments indicate that a single mutation of the conserved PS1 lysine (K499) residue in Mcm3 results in loss of viability. In contrast, the homo-hexameric S. solfataricus Mcm (SsoMcm) accommodates several subunits with disruptions in catalytic elements and still maintains significant helicase activity [44]. Therefore, we investigated the effect of mutating two different PS1 hairpins in the Mcm2-7 complex. We mated the haploid strains containing individual PS1 hairpin mutations to produce all the possible pair-wise combinations. After sporulating the heterozygous strains, we screened the spore colonies for viable double mutants. Only spore colonies with mcm4 K658A and mcm5 K506A were viable (Table 4). These grew more slowly than wild-type or strains containing either mcm4 K658A or mcm5 K506A and were more sensitive to hydroxyurea and MMS (Figure 4).
The lysine residue on the PS1 hairpin is predicted to make an electrostatic interaction with the sugar phosphate backbone of DNA to facilitate translocation of DNA and unwinding [8]. We converted the lysine of the PS1 hairpin of Mcm3 to arginine, glutamine, or asparagine to determine whether the charge is important for function. Of the three alleles examined by plasmid shuffling, only mcm3 K499R supported viability ( Figure 5A). The strain containing this allele displayed no overt growth defects when plated on 200 mM hydroxyurea, 0.03% MMS, and 20 mM caffeine ( Figure 5B). Additionally, the rate of growth was the same as wild type at 16uC, 30uC and 37uC ( Figure 5C). This suggests that the positive charge at residue 499 of Mcm3 is essential for function.
To begin to investigate how Mcm3 K499A disrupts function, we addressed whether its overexpression would have a dominant negative effect. A plasmid expressing mcm3 K499A or MCM3 from a GAL10 promoter was transformed into BY4741 (MCM3), and serial dilutions plated onto media containing glucose, raffinose, or galactose. In the presence of glucose or raffinose, where the GAL10 promoter is transcriptionally repressed or not induced respectively, there was no effect on growth, whereas in galactose-containing media induction of mcm3 K499A expression resulted in a slow growth phenotype ( Figure 6A). In addition, there was an increase of approximately three-fold in cell diameter for the GAL10-mcm3 K499A transformed strain compared to GAL10-MCM3 transformed strain when grown in galactose-containing media ( Figure 6B and 6C). Based on these observations, overexpression of mcm3 K499A in the context of a wild type background leads to a dominant negative effect that is likely associated with a cell cycle defect.  expressed as a recombinant protein in E. coli, and checked for the absence of contaminating nuclease or ATPase activity. Individual Mcm subunits were mixed in equal molar ratios to reconstitute the hexameric complex, the final step of the reconstitution being elution from a gel filtration column. Mcm2-7 3K499A eluted at a volume corresponding to the MCM hexamer, similar to wild-type Mcm2-7 (,600 kDa; Figure 7A). We examined the DNA unwinding of wild type and mutant Mcm complexes using a radiolabeled synthetic fork substrate where DNA unwinding is measured as the amount of single stranded DNA liberated from the duplex substrate. At a concentration of 200 nM the wild-type complex converted 1.5 fmoles of substrate to single-stranded DNA in 10 minutes (Figures 7B and 7C). By contrast, the 200 nM concentration of Mcm2-7 3K499A unwound 0.1 fmol of the fork substrate. Therefore Mcm2-7 3K499A has a ,15-fold reduction in helicase activity, indicating that the Mcm3 PS1 hairpin is critical for DNA unwinding.
The loss of helicase activity in Mcm2-7 3K499A may be due to a role for the Mcm3 PS1 hairpin in the ATPase activity of the complex. Interestingly, of the isolated dimer pairs, the pair of Mcm3 and Mcm7 has the highest ATPase activity, approaching that of the intact Mcm2-7 hexamer [25]. ATP hydrolysis was measured for intact wild-type Mcm2-7 and Mcm2-7 3K499A complexes. As shown in Figure 8A, the ATP hydrolysis rate for Mcm2-7 3K499A was not significantly different from the wild-type Mcm2-7. We next addressed whether Mcm2-7 3K499A is capable of single-stranded DNA binding. Mutant and wild-type complexes were chromatographed on a single-stranded Sepharose affinity  column in the presence of ATP, and eluted with increasing salt concentration. As shown in Figure 8B, wild type Mcm2-7 eluted from this column primarily in the 200 and 300 mM NaCl wash fractions (upper panel). The elution profile for Mcm2-7 3K499A closely resembled that of the wild type complex (middle panel) indicating that the Mcm2-7 3K499A is capable of binding single stranded DNA. Lack of binding by the peptidyl prolyl isomerase Pin-1 (lower panel), a relatively basic protein with a pI of 9.4, indicated that the binding by the Mcm complexes was specific for DNA and not simply due to charge interactions. We next used an electrophoretic mobility shift assay in an attempt to detect more subtle differences in DNA binding. A 59 radiolabeled oligonucleotide of 90 bases was used as the substrate. As shown in Figure 8C (lanes 3-6) increasing concentrations of wild-type Mcm2-7 depleted the substrate band and resulted in the appearance of a discrete band of reduced mobility. To confirm that the band of slower mobility was a Mcm2-7-DNA complex, Mcm7 antibody was pre-incubated with Mcm2-7 prior to addition of radiolabeled oligonucleotide ( Figure 8C lanes 7-10). In the presence of the antibody the band of slower mobility was diminished, indicating that it contained Mcm2-7. When the DNA binding activity of Mcm2-7 3K499A was assayed (lanes [11][12][13][14], the amount of Mcm2-7 3K499A -DNA complex was reduced suggesting that Mcm2-7 3K499A is less able to bind the 90 base single-stranded DNA or that the binding is unstable under the electrophoresis conditions. The loss of helicase activity is the most pronounced functional effect of the Mcm3 hairpin mutation, and may explain the inviability of the mcm3 K499A strain. To investigate the effects of the Mcm3 hairpin mutation in cells, we analyzed myc 9 -Mcm3 K499A expressed in yeast to determine if its ability to associate with other components required for replication differs from the wild type protein. Whole cell extracts containing myc 9 -tagged Mcm3 or Mcm3 K499A were prepared from cells grown to mid-log phase in YPD media and analyzed by gel filtration chromatography on a Superose 6 column. As shown in Figure 9, wild type Mcm3 elutes from the Superose 6 column in two peaks. The first peak is broad and corresponds to complexes with a molecular mass greater than 2 MDa. We suspect that this may represent the Mcm2-7 complex associated with chromatin. The second peak elutes in the molecular mass range from 150 to 350 kDa. This likely represents Mcm3 in association with other molecules, and perhaps an equilibrium between subcomplexes of Mcms. The elution profile of myc 9 -Mcm3 K499A resembled that of the wild type in that it eluted as two peaks, but with significant differences for the highand low-molecular weight complexes. In the high molecular weight complex, Mcm3 appears as a single band that migrates with a mass of 135 kDa, while in the mcm3 K499A strain, Mcm3 appears as a 135 kDa band but there are also two prominent bands at approximately 150 kDa and 175 kDa. These were not detected in the wild-type Mcm3 cells, even after prolonged exposure of the film. The reduced mobility forms of Mcm3 are likely the result of protein modification, but the nature of this modification is unclear. The second difference between wild-type and mcm3 K499A cells is that the smaller complex from wild-type cells elutes with a peak at 200 kDa (fraction 34 on the profile, Figure 9), which was shifted to approximately 260 kDa in the mcm3 K499A strain (fraction 32). Since the purified Mcm2-7 3K499A hexamer assembles and behaves similarly to the wild-type complex in vitro, these results suggest that in cells the altered activities of the Mcm2-7 3K499A complex result in changes in its molecular associations.
Mcm3 has a unique role in the initiation process in that it is able to recruit its neighboring subunits, Mcm5 and Mcm7, to the origin recognition complex (ORC) independent of Cdt1 [44]. A winged helix domain only present at the C-terminal of Mcm3 interacts with the Cdc6/ORC complex to stimulate the ATP hydrolysis required for time-dependent stable double hexamer loading [45,46]. It may be that these processes are compromised by the poor helicase activity of Mcm3 K499A , leading to the differences we observe in Mcm3 K499A -containing complexes.
The  although the degree to which it obstructs the central channel will depend on its structure, which has not been modeled. In addition to the funnel shape, the surface charge of the central channel exhibits a systematic change: the large opening formed by the Cterminal domains carries a negative surface charge, while the surface formed by the more constricted N-terminal domains is positively charged ( Figure 10B).
The second important observation from the Mcm2-7 model is that the PS1 hairpins of all Mcm subunits are somewhat recessed and do not project into the interior of the central channel. Together with the reduced stability of DNA binding by Mcm2-7 3K499A , these observations lead us to propose that the PS1 hairpin may be a component of an exit channel that directs one strand of incoming duplex DNA through the side of the Mcm2-7 hexamer. A structural model of the SsoMCM hexamer suggests that side channels are formed at the interface of subunits and run from the central channel to the outside of the ring [7]; the side channels are wide enough to accommodate single-stranded DNA. Similar channels are seen in electron micrographs of eukaryotic Mcm2-7 [47] and are present in our Mcm2-7 model ( Figure 10C). The extrusion of single-stranded DNA through a side channel of the Mcm2-7 complex is a possible explanation for reduced helicase activity upon mutation of the Mcm3 PS1 hairpin. Of note, the channel incorporating the Mcm3 PS1 hairpin is formed at the interface between Mcm3 and Mcm7 ( Figure 10C), which is critically important for Mcm2-7 function. For example, when the various Mcm subunits are expressed independently to generate dimeric species, it is the isolated Mcm3/7 dimer that has the highest ATPase activity, almost as high as the ATPase of the intact Mcm2-7 hexamer [26,27]. Furthermore, expression of Mcm7 with mutations in its Walker A or Walker B motif, or expression of Mcm3 with an R542A mutation in its ''arginine finger'' lead to a strong dominant-lethal phenotype [26]. Taken together, the shape and charge features of the Mcm2-7 ''funnel'', along with the functional importance of catalytic components found in the Mcm3/7 interface, are consistent with either double-stranded DNA or single-stranded DNA entering Mcm2-7 at the larger Cterminal end. If double-stranded DNA enters the channel, it may be destabilized due to the negative surface charge of the channel interior: one of the separated strands would be actively extruded through the Mcm3/7 interface, while the other strand could exit through the positively charged N-terminal end of the Mcm2-7 hexamer, or through another side channel. In a second possible scenario, single-stranded DNA would enter the Mcm2-7 channel and would be actively extruded through the Mcm3/7 interface, while the other strand would be sterically excluded from entering the channel.
The importance of the PS1 hairpins in Mcm function is most apparent from the loss of viability when PS1 of Mcm3 was mutated. We demonstrate that the Mcm3 PS1 hairpin participates in DNA unwinding by Mcm2-7, and based upon our in vitro experiments suggest that it may do so by altering the interaction of the complex with single-stranded DNA. This result is similar to findings with SsoMCM and the SF3 viral replicative helicases where the PS1 hairpin is essential for helicase activity [8,9,48]. We also note that the interactions of Mcm3 K499A are altered in cells as demonstrated by changes in its elution from a Superose 6 column. Whether these changes are due to or the cause of the defects in cellular function of the protein is unclear.
A key finding of our study is that of the six PS1 hairpins in the heterohexameric Mcm2-7 complex, only the PS1 hairpin of Mcm3 is essential. This strongly suggests that it has a unique role in Mcm2-7 function. The finding that the PS1 hairpin of Mcm3 is essential for viability is somewhat surprising since Mcm3 has been proposed to act principally in the regulation of the other Mcm2-7 subunits rather than have a direct role in DNA unwinding [49,50].  The PS1 Hairpins in  The finding that the mcm4 K658A mcm5 K506A double mutation strain was viable, in contrast to the lack of viability of other pairwise combinations is also another clear indication that each subunit contributes differently to the function of Mcm2-7. Figure S1 Growth of mcm3 K499A and mcm7 K550A plasmid shuffled yeast strains. Haploid yeast strains deleted for MCM3 or MCM7 and bearing MCM3 or MCM7 on a URA3-CEN plasmid were transformed with LEU2-CEN plasmids containing either MCM3, mcm3 K499A , MCM7, mcm7 K550A or the empty LEU2-CEN plasmid (Vector). The transformed yeast were grown overnight at 30uC in YPD media, serially diluted, and then spotted onto a YPD plate or a plate containing 5-FOA. The plates were incubated at 30uC for the number of days indicated. (TIF) Figure S2 Expression levels of Mcm 2 and 3. Yeast strains MDY70 (MCM2), MDY71 (mcm2 K633A ), MDY405 (DED1-myc 9 -MCM3) and MDY406 (DED1-myc 9 -mcm3 K499A ) were grown to midlog phase; yeast extracts were prepared by grinding with glass beads, and 10, 20 or 40 mg of total protein separated by SDS-PAGE. Blots of these gels were probed with anti-Mcm2, (Santa Cruz Biotech) or anti-myc (Sigma-Aldrich) antibody to assess the level of Mcm subunit. We note that for Mcm3 detection the plasmids were transformed into BY4741 and thus contain wildtype Mcm3. (TIF)