Protoplasma DOI 10.1007/s00709-015-0825-2

ORIGINAL ARTICLE

Arabidopsis thaliana MCM3 single subunit of MCM2–7 complex functions as 3′ to 5′ DNA helicase Irum Rizvi 1 & Nirupam Roy Choudhury 1 & Narendra Tuteja 1

Received: 10 February 2015 / Accepted: 27 April 2015 # Springer-Verlag Wien 2015

Abstract Minichromosome maintenance 2–7 (MCM2–7) proteins are conserved eukaryotic replicative factors essential for the DNA replication at its initiation and elongation step, and act as a licensing factor. The MCM2–7 and MCM4/6/ 7subcomplex exhibit DNA helicase activity, and are therefore regarded as the replicative helicase. The MCM proteins have not been studied in detail in plant system. Here, we present the biochemical characterization of Arabidopsis thaliana MCM3 single subunit and show that it exhibits in vitro unwinding and ATPase activities. AtMCM3 shows a greater unwinding activity with 5′ forked partial DNA duplex substrate as compared to 3′ forked and non-forked substrates. ATP and magnesium ion are indispensable for its DNA helicase activity. Specifically, ATP and dATP are the preferred nucleotides for its unwinding activity. The directionality of the AtMCM3 has been determined to be in 3′ to 5′ direction. The oligomerization status of AtMCM3 single subunit protein indicates that it is present in different multimeric forms. The unraveling of the helicase activity of AtMCM3 will provide better insights into the plant DNA replication. Keywords Arabidopsis thaliana . ATPase . DNA helicase . MCM3 . Native PAGE

Handling Editor: Pavla Binarova * Narendra Tuteja [email protected] 1

Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India

Introduction The MCM proteins were first identified as the proteins required for the maintenance of autonomously replicating sequence (ARS)-containing plasmids in Saccharomyces cerevisiae (Maine et al. 1984). In eukaryotes, the MCM proteins are conserved from yeast to mammals and include six subunits MCM2–7 that participate in DNA replication (reviewed in Forsburg 2004). The formation of prereplicative complex (pre-RC) is a requisite for the DNA replication initiation in eukaryotes. The pre-RC is composed of several replication factors which includes origin recognition complex (ORC), cell division cycle 6 (Cdc6), Cdc10dependent transcript 1 (Cdt1), and minichromosome maintenance 2–7 (MCM2–7) complex (Montagnoli et al. 2006; Labib and Diffley 2001). The eukaryotic MCMs exist as dimers, trimers, tetramers, and heterohexamers, and dimer of heterotrimers and monomers (Lee and Hurwitz 2000; Yu et al. 2004; Xu et al. 2013). The subcomplex MCM4/6/7 possess in vitro helicase activity (Ishimi et al. 1998; You et al. 1999; Lee and Hurwitz 2001; Xu et al. 2013; Stead et al. 2009). MCM2–7 complex has also been shown to exhibit unwinding activity in the presence of glutamate ions (Bochman and Schwacha 2008). In Schizosaccharomyces pombe, the dimer of MCM4/6/7 complex has the helicase activity and its interaction with either MCM2 or MCM3/5 dimer leads to disassembly of MCM4/6/7 complex and abolishment of its helicase activity (Lee and Hurwitz 2000). Similarly, the in vitro unwinding activity of mouse MCM4/6/7 complex is inhibited by the MCM2 subunit (Ishimi et al. 1998). Furthermore, Stead et al. (2009) showed the helicase activity of S. cerevisiae MCM4/6/7 complex is reduced by the ADP bound form of MCM2. The MCM4/6/7 complex forms the core catalytic complex and the remaining subunits MCM2/3/5 are the regulatory subunits (Lee and

I. Rizvi et al.

Hurwitz 2000). However, all the subunits are required during the replication initiation and elongation stages (Labib et al. 2000; Tuteja et al. 2011). The subunits of MCM2–7 complex contribute differentially to its unwinding activity. The ATPase motifs of MCM6 and MCM7 are critical for the unwinding activity of MCM4/6/7 complex, whereas that of MCM4 is important for single-stranded DNA binding property of the MCM4/6/7 complex (You et al. 1999, 2002). The zinc finger mutant of mouse MCM4 results in enhanced helicase activity of the MCM4/6/7 complex (You et al. 2002). In MCM2–7 complex, the active sites lie at the interface of the subunits and are contributed by Walker A and Walker B of one subunit, and the arginine finger of adjacent subunit (Bochman et al. 2008). However, all the MCM subunits do not participate equally in the formation of the active site (Bochman et al. 2008). In archaea such as Methanobacterium thermoautotrophicum (Mth) and Sulfolobus solfataricus (Sso), only one homologue of eukaryotic MCMs exist which shows biochemical properties similar to that of the eukaryotic MCMs. The Mth MCM exists as both monomers and hexamers and both the forms possess helicase activity, ssDNA binding property, and DNAdependent ATPase activity (Kelman et al. 1999). Likewise, the Sso MCM forms hexamers and has helicase activity (Carpentieri et al. 2002). The EM images of Mth MCM appear it to form heptamers (Yu et al. 2002). In another study by Shechter et al. (2000), Mth MCM display DNA-dependent ATPase activity, processive 3′ to 5′ helicase activity, and exists as a double hexamer. In plants, the role of MCMs in DNA replication is evident from a study which shows that disruption of AtMCM2 leads to lethality during embryo development and its overexpression inhibits endoreduplication (Ni et al. 2009). Recently, the study of A. thaliana, MCM2–7 mutants have shown its role in the seed development (Herridge et al. 2014). In Arabidopsis, MCM5 and MCM7 exist in the nucleus in G1, S, and G2 phase, whereas in M phase, they are localized in the cytoplasm (Shultz et al. 2009). The maize homologue of MCM3 (ZmROA2) is localized in the nucleus throughout the cell cycle and appears distinct from chromatin during mitotic stages (Sabelli et al. 1999). The distribution of maize MCM3 in the root tip cells at different stages of cell cycle reinforces its role in licensing during cell cycle. In Arabidopsis, the expression of MCM3 is transcriptionally regulated by the virtue of E2F binding sites in its promoter region. One of the E2F binding sites activates its S-phase expression and the other restricts its expression in meristematic regions (Stevens et al. 2002). MCM3 is expressed in all the phases of the cell cycle to a variable extent in tobacco BY-2 cells (Dambrauskas et al. 2003). The single subunit MCM6 of pea exhibits in vitro helicase and ATPase activity (Tran et al. 2010). Overall, there is little information on the function of plant MCMs and their role in DNA replication. The

elucidation of the plant MCM2–7 complex as well as the characterization of MCM single subunits will allow us to gain further insights on plant DNA replication. In the present study, we have characterized AtMCM3 single subunit and reveal that AtMCM3 possess in vitro 3′ to 5′ DNA helicase and ATPase activities and exist in various oligomeric forms including homohexamer.

Materials and methods Cloning, expression, and purification of recombinant protein Multiple sequence alignment of the AAA+ domain of the MCM proteins from A. thaliana was performed using Clustal Omega tool. The AtMCM3 (Gene Bank Accession number: NM_123997; Locus id: AT5G46280; TAIR Accession-Sequence: 1009065989) cDNA was amplified by PCR using gene primers specific to AtMCM3 gene. The PCR amplified product was cloned in pET-28a at BamHI and SacI sites and sequenced. For the expression of AtMCM3 protein, the recombinant pET-28a-AtMCM3 construct was transformed in E. coli BL21 (DE3) cells. The protein induction was carried out overnight (12–14 h) at 18 °C with 0.25 mM isopropyl-βD-thiogalactopyranoside (IPTG). The induced protein was purified by affinity chromatography using nickel-NTA resin (Qiagen GmbH, Cologne, Germany), following the standard protocol. The eluted protein was then dialyzed against buffer containing 50 mM Tris (pH 8.0), 100 mM NaCl, 20 % glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM benzamidine. The purified protein was stored at −80 °C. ATP binding assay Different amounts of the protein were slot blotted on a nitrocellulose membrane, followed by the incubation of the membrane in blocking buffer (1× PBS+3 % BSA). After blocking, the membrane was incubated for 1 h in a binding buffer (10 mM MgCl2, 1× PBS) containing [α-32P] ATP. Next, the membrane was washed thrice using binding buffer. Finally, it was autoradiographed and scanned using typhoon scanner. ATP hydrolysis assay The ATPase assay was performed as described by Choudhury et al. (2006). Briefly, the protein concentrations were incubated with 0.2 μCi [γ-32P] ATP (6000 Ci/mmol, Perkin Elmer Life Sciences, USA) for 30 min at 37 °C in ATPase buffer [20 mM Tris-HCl (pH 8.0), 4 mM MgCl2, 50 mM KCl, 8 mM DTT, and 80 μg/ml BSA]. Subsequently, 1 μl of the reaction mixture was spotted on a polyethyleneimine thin-layer

Arabidopsis MCM3 functions as DNA helicase

chromatography (TLC) plate (Sigma-Aldrich, St. Louis, MO, USA) and air dried. The separation of products by thin-layer chromatography was done in a running solvent, a mix of 0.5 M lithium chloride (LiCl) and 1 M formic acid (HCOOH). The TLC paper was allowed to air dry and then autoradiographed. The relative intensities of the released Pi were estimated by densitometric scanning using a Typhoon scanner. Helicase assay The strand displacement assay was done as previously described by Choudhury et al. (2006). For ds DNA substrate preparation, approx. 2 pmols of oligonucleotide was labeled at 5′ end using [γ-32P] ATP and T4 polynucleotide kinase. Then, the annealing of radiolabeled DNA fragment and M13mp18 DNA (5 pmols) was done in annealing buffer by incubating the reaction mix at 99 °C and then gradually bringing it down to room temperature. For the preparation of 23and 31 mer non-forked substrate, oligonucleotides designated as oligo 1 (CCCAGTCACGACGTTGTAAAACG) and oligo 2 (GACTCTAGAGGATCCCCGGGTACCGAGCTCG) were used, respectively. Similarly, the oligonucleotides designated as oligo 3 (CCAAAACCCAGTCACGACGTTGTA AAACG) and oligo 4 (CCCAGTCACGACGTTGTAAAAC GTGCCGG) were used for forked substrate preparation containing an overhang of 6 nt at the 5′ and 3′ ends, respectively. The helicase assay was performed by incubating desired quantities of protein with the ds DNA substrate in a buffer composed of 20 mM Tris-HCl (pH 8.0), 4 mM MgCl2, 50 mM KCl, 8 mM DTT, 4 mM ATP, and 80 μg/ml BSA at 37 °C for half an hour. The reaction was terminated by the addition of dye (0.1 % SDS, 15 mM EDTA, 5 % glycerol) and the products of reaction were electrophoresed on 15 % polyacrylamide gel. The gel was autoradiographed and analyzed using Typhoon scanner. For determination of directionality of MCM3 unwinding activity, two different substrates were prepared. In one, unlabeled oligo 2 was annealed to M13mp18 and its 3′ end extended using Klenow polymerase along with dATP, dTTP, and [α-32P] dCTP. The extended product was restriction digested by SmaI to generate a 3′ labeled ds substrate with 19-mer duplex as shown in Fig. 5. Another substrate was prepared by annealing 5′ labeled oligo 2 to M13mp18, followed by its restriction digestion with SmaI to produce a 5′ labeled 17-mer duplex. Non-denaturing polyacrylamide gel electrophoresis (PAGE) Proteins were electrophoresed in a native 6.5 % polyacrylamide gel using Tris-glycine buffer [25 mM Tris and 250 mM glycine (pH 8.3)]. The electrophoresed proteins were visualized by staining with Coomassie blue.

Results Sequence analysis of AtMCMs and purification of A. thaliana MCM3 protein The multiple sequence alignment of amino acids in the AAA+ region of AtMCM2–7 showed 57–67 % conservation. In the AAA+ domain, the motifs such as Walker A, Walker B, and arginine finger are present (Fig. 1a, shown within rectangle box). The amino acid residues which are identical in all the MCMs are represented by an asterisk below the alignment. The AtMCM3 gene was amplified and cloned for expression. An induced polypeptide of 86 kDa was observed by Coomassie staining of the denaturing PAGE gel (Fig. 1b, lane 2), which was not seen in the uninduced fraction (Fig. 1b, lane 1). The recombinant AtMCM3 was affinity purified using nickel-NTA chromatography (Fig. 1c, lanes 1 and 2). The Western blot using anti-His antibody detected a protein band of 86 kDa which accords with the predicted size of AtMCM3 protein (Fig. 1d, lanes 1 and 2). A. thaliana MCM3 forms multimeric complexes We investigated the oligomerization status of AtMCM3 protein by native PAGE electrophoresis. On the basis of standard protein markers (Fig. 1e, lanes 1 to 4), the Coomassie-stained non-denaturing PAGE gel indicated the presence of multimers of various orders (Fig. 1e, lanes 5 and 6). The denatured and heated AtMCM3 protein sample distinctly showed the dissociation of higher forms into lower order oligomers (Fig. 1e, lane 7). A. thaliana MCM3 binds ATP and possess ATPase and ATP-dependent helicase activities Since MCM3 belongs to AAA+ family of proteins, its ability to bind ATP was examined. As evident from the autoradiogram, the homogeneous-purified AtMCM3 protein binds ATP in a concentration-dependent manner (Fig. 2a). Next, AtMCM3 was checked for the presence of ATPase activity. The AtMCM3 protein hydrolysed [γ- 32 P] ATP in a concentration-dependent manner (Fig. 2b). However, the ATPase activity was independent of the presence of DNA. MCM3 is a component of a larger complex MCM2–7 which can unwind double-stranded DNA duplex. Therefore, we investigated the helicase activity of AtMCM3 single subunit using strand displacement assay. The AtMCM3 has the ability to unwind partial duplex DNA substrate (Fig. 2c–e). The single subunit AtMCM3 could unwind both non-forked (Fig. 2c) and forked substrates (Fig. 2d, e). The unwinding activity was 67 % (Fig. 2e, lane 4) with 5′ forked substrate as compared to 43 % (Fig. 2d, lane 4) and 51 % (Fig. 2c, lane 4) with 3′ forked substrate and non-forked substrate, respectively. Thus, 5′

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Walker A

--------------------------------NMMMVGDPSVAKSQLLRAIMNIAPLAIS --------------------------------NILLVGDPGTSKSQLLQYIHKLSPRGIY ------------------HRQLKDGMKIRGDVHICLMGDPGVAKSQLLKHIINVAPRGVY YGHEDIKTALALAMFGGQEKNIKGKHRLRGDINVLLLGDPGTAKSQFLKYVEKTGQRAVY --------------------------------NVLLLGDPSTAKSQFLKFVEKTAPIAVY --------------------------------NVCIVGDPSCAKSQFLKYTAGIVPRSVY .: ::***. :***:*: .:

AtMCM3 AtMCM4 AtMCM7 AtMCM2 AtMCM5 AtMCM6

TTGRGSSGVGLTAAVTSDQETGERRLEAGAMVLADKGIVCIDEFDKMNDQDRVAIHEVME TSGRGSSAVGLTAYVAKDPETGETVLESGALVLSDRGICCIDEFDKMSDSARSMLHEVME TTGKGSSGVGLTAAVMRDQVTNEMVLEGGALVLADMGICAIDEFDKMDESDRTAIHEVME TTGKGASAVGLTAAVHKDPVTREWTLEGGALVLADRGICLIDEFDKMNDQDRVSIHEAME TSGKGSSAAGLTASVIRDSSTREFYLEGGAMVLADGGVVCIDEFDKMRPEDRVAIHEAME TSGKSSSAAGLTATVAKEPETGEFCIEAGALMLADNGICCIDEFDKMDIKDQVAIHEAME *:*:.:*..**** * : * * :*.**::*:* *: ******* . : :**.**

AtMCM3 AtMCM4 AtMCM7 AtMCM2 AtMCM5 AtMCM6

QQTVTIAKAGIHASLNARCSVVAAANPIYGTYDRSLTPTKNIGLPDSLLSRFDLLFIV QQTVSIAKAGIIASLNARTSVLACANPSGSRYNPRLSVIENIHLPPTLLSRFDLI--QQTVSIAKAGITTSLNARTAVLAAANPAWGRYDLRRTPAENINLPPALLSRFDL---QQSISISKAGIVTSLQARCSVIAAANPVGGRYDSSKSFAQNVELTDPILSRFDI---QQTISIAKAGITTVLNSRTSVLAAANPPSGRYDDLKTAQDNIDLQTTILSRF-----QQTISITKAGIQATLNARTSILAAANPVGGRYDKSKPLKYNVNLPPAILSRF-----**:::*:**** : *::* :::*.*** . *: *: * :****

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Fig. 1 Sequence analysis, cloning, and purification of AtMCM3 protein. a Multiple sequence alignment of AAA+ domain of MCM2–7 of Arabidopsis thaliana. The identical amino acid residues are shown by asterisk below the alignment. Likewise, the colon and dot below the alignment show amino acids with properties highly and weakly similar, respectively. b Coomassie stained denatured PAGE showing uninduced (lane 1) and induced (lane 2) cell lysate. M protein marker. c SDS-PAGE

showing purified fractions of His-tagged AtMCM3 (lanes 1 and 2). d Immunoblot using anti-His antibody to detect 86 kDa AtMCM3-purified protein (lanes 1 and 2). e A 6.5 % native PAGE showing multimeric forms of AtMCM3. Lanes 1 to 4 are protein markers as mentioned in the figure, lanes 5 and 6 are two different concentrations of purified AtMCM3 protein samples, and lane 7 is partially denatured AtMCM3 protein

forked substrate was preferred by AtMCM3 over the 3′ tailed and non-forked substrate. Hence, further biochemical assays were performed using the 5′ forked substrate and 0.6 μg of MCM3 protein. In each of the scenario, the helicase activity increased with the higher concentration of the enzyme (Fig. 2c–e). The helicase activity of the protein was ATP and Mg2+ ion dependent as the absence of either of them abolished the in vitro helicase activity (Fig. 3a, b, right panels). The optimum concentration of ATP required by AtMCM3 to unwind the duplex was 4 mM (Fig. 3a, lane 8). ATP concentration of 8 mM inhibited its unwinding activity (Fig. 3a, lane 10). In case of the Mg2+ ion, the optimal concentration was determined to be 4 mM (Fig. 3b, lane 7) and concentrations

higher than 6 mM reduced the unwinding activity of AtMCM3 protein (Fig. 3b lanes 9 and 10). Similarly, the optimal salt (KCl) concentration was observed to be 50 mM (Fig. 3c, lane 7). Salt concentrations of 150 mM and 200 mM were completely inhibitory to the helicase activity (Fig. 3c lanes 9 and 10). The AtMCM3 unwinding activity increased with time up to 90 min (Fig. 4a, b). AtMCM3 preferentially utilize ATP and dATP The helicases exhibit nucleotide-dependent unwinding activity. We studied the ability of AtMCM3 to utilize various nucleotides for the strand separation. The unwinding by AtMCM3 was

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Fig. 2 Functional characterization of AtMCM3 enzyme. a Autoradiogram showing ATP binding activity of AtMCM3 using [α-P32] ATP by slot blot analysis. Lane 1–3: 0.05, 0.1 and 0.2 μg MCM3 protein, respectively. b ATPase assay using different concentrations ranging from 0.6 to1.8 μg (lanes 2–6) of AtMCM3 protein. Lane 1: no protein. c Helicase activity of AtMCM3 with non-forked substrate. Lane 1: heated substrate; lane 2: no protein; lanes 3 and 4: 0.4 and 0.6 μg of AtMCM3 protein, respectively. d

Helicase activity of AtMCM3 with 3′ forked substrate. Lane 1: no protein; lane 2: heated substrate; lanes 3 and 4: 0.4 and 0.6 μg of AtMCM3 protein, respectively. e Helicase activity of AtMCM3 with 5′ forked substrate. Lane 1: no protein; lane 2: heated substrate; lanes 3 and 4: 0.4 and 0.6 μg of AtMCM3 protein, respectively. The percentage unwinding is mentioned below the autoradiograms

exhibited only in the presence of nucleotides ATP and dATP (Fig. 4c, lanes 3 and 4). Other nucleotides such as CTP, GTP, UTP, dCTP, dGTP, and dTTP did not support DNA unwinding by AtMCM3 (Fig. 4c, d). Thus, the AtMCM3 specifically utilizes only ATP or dATP for the in vitro helicase activity.

and are therefore replicative helicase (Ishimi et al. 1998; You et al. 1999; Xu et al. 2013; Stead et al. 2009; Bochmann and Schwacha 2008). The MCMs have been classified into core catalytic subunits MCM4/6/7, and regulatory subunits MCM2/3/5 (Lee and Hurwitz 2000; Ishimi et al. 1998; Stead et al. 2009). Although each of the eukaryotic MCM subunits is essential for survival, these subunits are known to contribute differentially to the overall helicase activity (Labib et al. 2000; Bochman et al. 2008). In plants, MCMs are known to influence various processes such as cell cycle (Shultz et al. 2009; Sabelli et al. 1999; Stevens et al. 2002; Dambrauskus et al. 2003), early embryonic development (Ni et al. 2009), and salinity stress tolerance (Tran et al. 2010). The pea MCM6 single subunit is reported to possess strand displacement activity (Tran et al. 2010). The study of the MCM complex and its subunits will shed light on their functions. In the present study, we have investigated the biochemical properties of MCM3 subunit from A. thaliana. We have shown it to be an ATP-dependent helicase and also to exhibit ATPase activity.

Direction of unwinding of A. thaliana MCM3 The experimental strategy used to determine the directionality of AtMCM3 is represented in Fig. 5a. The AtMCM3 could displace only the 5′ labeled oligonucleotide (Fig. 5c) and was unable to displace the 3′ labeled oligo (Fig. 5b). Hence, it was confirmed that AtMCM3 translocates in 3′ to 5′ direction and is therefore a 3′ to 5′ helicase.

Discussion It is known from the previous studies that MCM2–7 complex as well as MCM4/6/7 entity possess in vitro helicase activity

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Fig. 3 Biochemical characterization of AtMCM3 DNA helicase activity by using 5′ forked partial DNA duplex substrate. a Effect of increasing concentrations of ATP on the DNA unwinding activity of AtMCM3. Lane 1: heated substrate; lane 2: no protein; lane 3: no ATP; lanes 4 to 10: 0.25 mM to 8 mM of ATP concentration. b Effect of increasing concentrations of Mg2+ on the unwinding activity of AtMCM3. Lane 1: no protein; lane 2: heated substrate; lane 3: no Mg2+; lanes 4 to 10: 0.5 mM

to 10 mM of Mg2+ ion concentration. c Effect of increasing concentrations of KCl on the unwinding activity of AtMCM3. Lane 1: heated substrate; lane 2: no protein; lane 3: no KCl; lanes 4 to 10: 5 mM to 200 mM of KCl concentration. The left panel is the autoradiogram of gel and right panel is the graphical representation (quantitation) of the corresponding autoradiogram. S annealed substrate, UD unwound DNA

The multiple sequence alignment of AAA+ region of the MCM2–7 of A. thaliana reveals its 57–67 % conserved nature. The individual characteristics of MCMs may arise from the variations in the amino acid sequence or their unique interactions with cellular factors. The purified recombinant 86 kDa AtMCM3 protein was used for various biochemical assays. As MCM proteins are known to form hetero- or homooligomers (Lee and Hurwitz 2000; Xu et al. 2013; Yu et al. 2004; Ma et al. 2010), we investigated the oligomeric status of AtMCM3. The native PAGE indicated that AtMCM3 exists in different oligomeric forms including hexamer. The replicative helicases usually form a hexameric complex to unwind the DNA. Earlier, the MCM6 single subunit from pea has been

shown to form homohexamer (Tran et al. 2010). In archaea such as M. thermoautotrophicum and S. solfataricus, the MCM homologue exists as homohexamers (Kelman et al. 1999; Carpentieri et al. 2002). Using [α-32P] ATP, we have shown that AtMCM3 has the ability to bind ATP. The ATP binding property of AtMCM3 protein is similar to that of MCM4/6/7 (You et al. 1999, 2002) and Mth MCM (Poplawski et al. 2001). Since MCM3 protein belongs to AAA+ domain containing family of proteins, its ATPase and helicase activities were investigated. Similar to the S. cerevisiae MCM dimers (MCM3/7, MCM4/7) and MCM2–7 (Davey et al. 2003; Schwacha and Bell 2001), the AtMCM3 single subunit

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activity in any system. Our studies have focused on MCM3 subunit of A. thaliana and our results show that AtMCM3 single subunit possess in vitro DNA helicase activity. There are several reports which have demonstrated in vitro DNA

exhibited ATPase activity independent of the presence of ssDNA. The Sso MCM also has an ATPase activity which is not affected by DNA (Carpentieri et al. 2002). The MCM3 single subunit has not been reported to possess DNA helicase

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Fig. 5 Direction of DNA unwinding by AtMCM3 protein. a Schematic representation of the experimental strategy followed to determine the directionality of AtMCM3. The star represents the incorporated radioactive nucleotide. b Autoradiogram showing the absence of helicase activity in 5′ to 3′ direction. Lane 1: heated substrate; lane 2: empty lane; lane 3: no protein; lanes 4 to 7: 0.2 μg to 0.8 μg of AtMCM3. c Autoradiogram showing AtMCM3 unwinds in 3′ to 5′ direction. Lane 1: heated substrate; lane 2: empty lane; lane 3: no protein; lanes 4 to 7: 0.2 μg to 0.8 μg of AtMCM3

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Fig. 4 Time kinetic study and influence of nucleotides on the unwinding activity of AtMCM3. a DNA helicase activity of AtMCM3 at various time points. Lane 1: no protein; lane 2: heated substrate; lanes 3 to 10: time points from 0 min to 120 min. b Graphical depiction of the time kinetics of AtMCM3 strand displacement activity. c Effect of various nucleotides on the unwinding activity of AtMCM3. Lane 1: no protein; lane 2: no nucleotide; lane 3 to 10: nucleotides ATP, dATP, GTP, dGTP, CTP, dCTP, UTP, and dTTP, respectively. d Graphical depiction of the NTPs/dNTPs requirement by AtMCM3 for strand displacement activity. S annealed substrate, UD unwound DNA

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helicase activity of MCM2–7 complex and MCM4/6/7 subcomplex (Bochman and Schwacha 2008; Ishimi et al. 1998; You et al. 1999; Xu et al. 2013; Stead et al. 2009). The pea MCM6 single subunit possess DNA unwinding activity and is involved in imparting salinity stress tolerance to plant (Tran et al. 2010; Dang et al. 2011). It has been suggested that plants might utilize the unwinding of the MCM6 which is upregulated under salt stress condition (Dang et al. 2011). The ability to unwind dsDNA by single subunit MCMs such as pea MCM6 (Tran et al. 2010) and AtMCM3 (this study) could be unique to plant MCMs because none of the MCM single subunit from other eukaryotic systems have been reported so far to possess such characteristic. The AtMCM3 single subunit protein showed a higher percentage of unwinding with 5′ forked substrate as compared to 3′ forked and nonforked partial DNA duplex substrate. The PsMCM6 single subunit was shown to prefer forked substrate for maximum DNA unwinding activity (Tran et al. 2010). AtMCM3 possessed optimal DNA helicase activity at 4 mM Mg2+, 4 mM ATP, and 50 mM KCl. In case of pea MCM6, the optimal helicase activity is obtained at 2 mM ATP, 2 mM Mg2+, and 50 mM of KCl. We demonstrated AtMCM3 as an ATP- and Mg2+-dependent helicase as it failed to unwind DNA in the absence of ATP and Mg2+. This observation correlates with the requirement of nucleoside triphosphate by the MCM complexes (MCM2–7 and MCM4/6/7) and MCM6 single subunit for the unwinding activity (Bochman et al. 2008; Lee and Hurwitz 2000; Ishimi 1997; Tran et al. 2010). Helicases, in general, are termed as the motor proteins which hydrolyze nucleoside triphosphate to generate the energy required for strand separation. However, not all the nucleotides may be utilized equally by a particular motor protein. AtMCM3 could utilize only ATP or dATP for the DNA helicase activity and other NTPs/dNTPs did not support DNA unwinding by AtMCM3. Similar to AtMCM3, the Mth MCM, S. pombe MCM4/6/7 and MCM4/6/7 from HeLa cells also utilize only ATP and dATP for unwinding (Kelman et al. 1999; Lee and Hurwitz 2000; Ishimi 1997). The Sso MCM mainly utilize only ATP for the helicase activity (Carpentieri et al. 2002). However, pea MCM6 can utilize various nucleotides for DNA unwinding (Tran et al. 2010). AtMCM3 was shown to have 3′ to 5′ helicase activity which is similar to the unwinding directionality exhibited by PsMCM6 single subunit (Tran et al. 2010). This result also corroborates well with the fact that eukaryotic Cdc45\MCM2–7\GINS and MCM4\6\7 complex possess 3′ to 5′ unwinding activity (Moyer et al. 2006; Lee and Hurwitz 2000; Ishimi 1997). The DNA unwinding activity by single subunit AtMCM3 could be possibly involved in the DNA replication in stress or other specific cellular conditions presently unknown. Our study establishes that the single subunit AtMCM3 possess DNA unwinding activity and will provide better understanding of DNA replication in plants.

Acknowledgment The work on helicases in NT’s laboratory is partially supported by the Department of Science and Technology (DST) and the Department of Biotechnology (DBT), Government of India. IR is thankful to the Department of Biotechnology (DBT), India, for funding her scholarship. We also thank Dr. Meerambika Mishra for her help in correction of the manuscript. Conflict of interest The authors declare that they have no conflict of interest.

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