Divergent functions of multiple eukaryote-like Orc1/Cdc6 proteins on modulating the loading of the MCM helicase onto the origins of the hyperthermophilic archaeon Sulfolobus solfataricus P2

The Cdc6 protein has been suggested as a loader for the eukaryotic MCM helicase. Archaeal replication machinery represents a core version of that in eukaryotes. In the current work, three eukaryotic Orc1/Cdc6 homologs (SsoCdc6-1, -2, and -3) from crenarchaeon Sulfolobus solfataricus were shown to have totally different effects on the interactions with SsoMCM helicase. SsoCdc6-2 stimulates the binding of the SsoMCM onto the origin DNA, but SsoCdc6-1 and SsoCdc6-3 significantly inhibit the loading activities, and these inhibitive effects can not be reversed by the stimulation of SsoCdc6-2. Using pull-down assays, we showed that three SsoCdc6 proteins interacted physically with the SsoMCM. Furthermore, the C-terminal domains of SsoCdc6 proteins were shown to physically and functionally affect the interactions with SsoMCM. This is the first report on the divergent functions of multiple eukaryote-like Orc1/Cdc6 proteins on regulating the loading of the MCM helicase onto the origins in the archaeon.     

The regulatory function of N-terminal AAA+ ATPase domain of eukaryote-like archaeal Orc1/Cdc6 protein during DNA replication initiation

Archaeal replication machinery represents a core version of this in eukaryotes. The crenarchaeon Sulfolobus solfataricus has the potential to be a powerful model system to understand the central mechanism of eukaryotic DNA replication because it contains three active origins of replication and three eukaryote-like Orc1/Cdc6 proteins (SsoCdc6-1, SsoCdc6-2, and SsoCdc6-3). In this study, we investigate the DNA-binding activities of the N-terminal AAA⁺ ATPase domains of these Orc1/Cdc6 proteins, including their functional interactions with the other SsoCdc6 proteins, on duplex DNA substrates derived from the origins of S. solfataricus. We showed that the ATPase domain of SsoCdc6-2 retained to a great extent the origin DNA-binding activity, and likewise maintained its stimulating effect on SsoCdc6-3. Second, the ATPase domain of SsoCdc6-1, which also stimulated the DNA-binding ability of SsoCdc6-3, demonstrated a significantly improved DNA-binding activity at the forked substrate, but only showed a very weak ability towards the blunt DNA. Third, the ATPase domain of SsoCdc6-3, although having lost much of its DNA-binding activity from the origin, inhibited both SsoCdc6-1 and SsoCdc6-2. These imply that the N-terminal AAA⁺ ATPase domain of archaeal Orc1/Cdc6 protein could be differentially involved in origin recognition during DNA replication initiation even if lacking conventional C-terminal winged helix DNA-binding elements. Our findings further propose that conserved AAA⁺ ATPase domains of Orc1/Cdc6 proteins determine their defined and coordinated functions not only in the archaeon species but also in eukaryotes during the early events of DNA replication.     

Three eukaryote-like Orc1/Cdc6 proteins functionally interact and mutually regulate their activities of binding to the replication origin in the hyperthermophilic archaeon Sulfolobus solfataricus P2

The crenarchaeon Sulfolobus solfataricus has the potential to be a powerful model system to understand the central mechanism of eukaryotic DNA replication because it contains three active origins of replication and three eukaryote-like Orc1/Cdc6 proteins. However, it is not known whether these SsoCdc6 proteins can functionally interact and collectively contribute to DNA replication initiation. In the current work, we found that SsoCdc6-1 stimulates DNA-binding activities of SsoCdc6-3. In contrast, SsoCdc6-3 inhibits those of both SsoCdc6-1 and SsoCdc6-2. These regulatory functions are differentially affected by the C-terminal domains of these SsoCdc6 proteins. These data, in conjunction with studies on physical interactions between these replication initiators by bacterial two-hybrid and pull-down/Western blot assays, lead us to propose the possibility that multiple SsoCdc6 proteins might coordinately regulate DNA replication in the archaeon species. This is the first report on the functional interaction among the archaeal multiple Cdc6 proteins to regulate DNA replication.     

In the absence of ATPase activity,pre-RC formation is blocked prior to MCM2–7 hexamer dimerization

The origin recognition complex (ORC) of Saccharomyces cerevisiae binds origin DNA and cooperates with Cdc6 and Cdt1 to load the replicative helicase MCM2–7 onto DNA. Helicase loading involves two MCM2–7 hexamers that assemble into a double hexamer around double-stranded DNA. This reaction requires ORC and Cdc6 ATPase activity, but it is unknown how these proteins control MCM2–7 double hexamer formation. We demonstrate that mutations in Cdc6 sensor-2 and Walker A motifs, which are predicted to affect ATP binding, influence the ORC–Cdc6 interaction and MCM2–7 recruitment. In contrast, a Cdc6 sensor-1 mutant affects MCM2–7 loading and Cdt1 release, similar as a Cdc6 Walker B ATPase mutant. Moreover, we show that Orc1 ATP hydrolysis is not involved in helicase loading or in releasing ORC from loaded MCM2–7. To determine whether Cdc6 regulates MCM2–7 double hexamer formation, we analysed complex assembly. We discovered that inhibition of Cdc6 ATPase restricts MCM2–7 association with origin DNA to a single hexamer, while active Cdc6 ATPase promotes recruitment of two MCM2–7 hexamer to origin DNA. Our findings illustrate how conserved Cdc6 AAA+ motifs modulate MCM2–7 recruitment, show that ATPase activity is required for MCM2–7 hexamer dimerization and demonstrate that MCM2–7 hexamers are recruited to origins in a consecutive process.     

Functional differentiation and cooperative interaction between two eukaryote-like archaeal Orc1/Cdc6 proteins on the replication origin

The DNA replication apparatus of archaea represents a core version of that in eukaryotes. Archaeal Orc1/Cdc6s can be an integral component in the replication machineries cooperatively regulating DNA replication. We investigated the DNA-binding activities of two eukaryote-like Orc1/Cdc6 proteins (SsoCdc6-1 and -2) and interactions between them on the different structural duplex DNA substrates derived from oriC1 of Sulfolobus solfataricus. The results showed that two Orc1/Cdc6 proteins stimulated mutual DNA-binding activities at lower concentrations and formed bigger SsoCdc6-1/SsoCdc6-2/DNA complex at higher concentrations. Furthermore, SsoCdc6-2 stimulated the DNA-binding activity of SsoMCM and demonstrated a high affinity to the 5-forked DNA. In contrast, SsoCdc6-1 inhibited the binding of SsoMCM and demonstrated better affinity to the sequence-specific blunt DNA substrate. Finally, we found that the two proteins physically interacted with each other and with SsoMCM. Thus, the two Orc1/Cdc6 proteins were functionally different, but they may keep the coordinated interaction on the replication origin.     

Characterization of physical and functional interactions between eukaryote-like Orc1/Cdc6 proteins and Y-family DNA polymerase in the hyperthermophilic archaeon Sulfolobus solfataricus

The roles of Y-family DNA polymerases and the regulation mechanisms are not well defined in Archaea. In this study, we performed in vitro and in vivo characterization of the physical interaction between the archaeon Sulfolobus solfataricus Y-family DNA polymerase (SsoPolY) and three eukaryote-like Orc1/Cdc6 proteins (SsoCdc6-1, SsoCdc6-2, and SsoCdc6-3). The effect of SsoCdc6-2 was the strongest, and the three SsoCdc6 proteins were shown to have very different effects on the function of SsoPolY. SsoCdc6-2 inhibited both the DNA-binding activity and DNA polymerization activity of SsoPolY on the DNA substrates containing mismatched bases, while it formed a large complex with SsoPolY and stimulated DNA-binding activity on paired primer-template DNA substrates. SsoCdc6-2 and S. solfataricus PCNA (SsoPCNA) showed a cooperative effect on polymerization by SsoPolY on paired DNA templates, but SsoCdc6 reduced the stimulating effect of SsoPCNA on this polymerization on mismatched DNA substrates. Therefore, we uncovered a DNA substrate-dependent SsoCdc6/SsoPolY interaction mechanism. This is the first evidence for a physical and functional linkage between archaeal eukaryote-like Orc1/Cdc6 proteins and Y-family DNA polymerase.     

Cdc6 ATPase activity regulates ORC x Cdc6 stability and the selection of specific DNA sequences as origins of DNA replication

DNA replication, as with all macromolecular synthesis steps, is controlled in part at the level of initiation. Although the origin recognition complex (ORC) binds to origins of DNA replication, it does not solely determine their location. To initiate DNA replication ORC requires Cdc6 to target initiation to specific DNA sequences in chromosomes and with Cdt1 loads the ring-shaped mini-chromosome maintenance (MCM) 2-7 DNA helicase component onto DNA. ORC and Cdc6 combine to form a ring-shaped complex that contains six AAA+ subunits. ORC and Cdc6 ATPase mutants are defective in MCM loading, and ORC ATPase mutants have reduced activity in ORC x Cdc6 x DNA complex formation. Here we analyzed the role of the Cdc6 ATPase on ORC x Cdc6 complex stability in the presence or absence of specific DNA sequences. Cdc6 ATPase is activated by ORC, regulates ORC x Cdc6 complex stability, and is suppressed by origin DNA. Mutations in the conserved origin A element, and to a lesser extent mutations in the B1 and B2 elements, induce Cdc6 ATPase activity and prevent stable ORC x Cdc6 formation. By analyzing ORC x Cdc6 complex stability on various DNAs, we demonstrated that specific DNA sequences control the rate of Cdc6 ATPase, which in turn controls the rate of Cdc6 dissociation from the ORC x Cdc6 x DNA complex. We propose a mechanism explaining how Cdc6 ATPase activity promotes origin DNA sequence specificity; on DNA that lacks origin activity, Cdc6 ATPase promotes dissociation of Cdc6, whereas origin DNA down-regulates Cdc6 ATPase resulting in a stable ORC x Cdc6 x DNA complex, which can then promote MCM loading. This model has relevance for origin specificity in higher eukaryotes.     

Physical and functional interaction between the mini-chromosome maintenance-like DNA helicase and the single-stranded DNA binding protein from the crenarchaeon Sulfolobus solfataricus

Mini-chromosome Maintenance (MCM) proteins play an essential role in both initiation and elongation phases of DNA replication in Eukarya. Genes encoding MCM homologs are present also in the genomic sequence of Archaea and the MCM-like protein from the euryarchaeon Methanobacterium thermoautotrophicum (Mth MCM) was shown to possess a robust ATP-dependent 3'-5' DNA helicase activity in vitro. Herein, we report the first biochemical characterization of a MCM homolog from a crenarchaeon, the thermoacidophile Sulfolobus solfataricus (Sso MCM). Gel filtration and glycerol gradient centrifugation experiments indicate that the Sso MCM forms single hexamers (470 kDa) in solution, whereas the Mth MCM assembles into double hexamers. The Sso MCM has NTPase and DNA helicase activity, which preferentially acts on DNA duplexes containing a 5'-tail and is stimulated by the single-stranded DNA binding protein from S. solfataricus (Sso SSB). In support of this functional interaction, we demonstrated by immunological methods that the Sso MCM and SSB form protein.protein complexes. These findings provide the first in vitro biochemical evidence of a physical/functional interaction between a MCM complex and another replication factor and suggest that the two proteins may function together in vivo in important DNA metabolic pathways.     

Interactions between the archaeal Cdc6 and MCM proteins modulate their biochemical properties

The origin recognition complex, Cdc6 and the minichromosome maintenance (MCM) complex play essential roles in the initiation of eukaryotic DNA replication. Homologs of these proteins may play similar roles in archaeal replication initiation. While the interactions among the eukaryotic initiation proteins are well documented, the protein–protein interactions between the archaeal proteins have not yet been determined. Here, an extensive structural and functional analysis of the interactions between the Methanothermobacter thermautotrophicus MCM and the two Cdc6 proteins (Cdc6-1 and -2) identified in the organism is described. The main contact between Cdc6 and MCM occurs via the N-terminal portion of the MCM protein. It was found that Cdc6–MCM interaction, but not Cdc6–DNA binding, plays the predominant role in regulating MCM helicase activity. In addition, the data showed that the interactions with MCM modulate the autophosphorylation of Cdc6-1 and -2. The results also suggest that MCM and DNA may compete for Cdc6-1 protein binding. The implications of these observations for the initiation of archaeal DNA replication are discussed.     

Stimulation of MCM helicase activity by a Cdc6 protein in the archaeon Thermoplasma acidophilum

Replicative DNA helicases are ring-shaped hexamers that play an essential role in chromosomal DNA replication. They unwind the two strands of the duplex DNA and provide the single-stranded (ss) DNA substrate for the polymerase. The minichromosome maintenance (MCM) proteins are thought to function as the replicative helicases in eukarya and archaea. The proteins of only a few archaeal organisms have been studied and revealed that although all have similar amino acid sequences and overall structures they differ in their biochemical properties. In this report the biochemical properties of the MCM protein from the archaeon Thermoplasma acidophilum is described. The enzyme has weak helicase activity on a substrate containing only a 3′-ssDNA overhang region and the protein requires a forked DNA structure for efficient helicase activity. It was also found that the helicase activity is stimulated by one of the two T.acidophilum Cdc6 homologues. This is an interesting observation as it is in sharp contrast to observations made with MCM and Cdc6 homologues from other archaea in which the helicase activity is inhibited when bound to Cdc6.     

The ATPase activity of MCM2-7 is dispensable for pre-RC assembly but is required for DNA unwinding

Eukaryotes have six minichromosome maintenance (MCM) proteins that are essential for DNA replication. The contribution of ATPase activity of MCM complexes to their function in replication is poorly understood. We have established a cell-free system competent for replication in which all MCM proteins are supplied by purified recombinant Xenopus MCM complexes. Recombinant MCM2-7 complex was able to assemble onto chromatin, load Cdc45 onto chromatin, and restore DNA replication in MCM-depleted extracts. Using mutational analysis in the Walker A motif of MCM6 and MCM7 of MCM2-7, we show that ATP binding and/or hydrolysis by MCM proteins is dispensable for chromatin loading and pre-replicative complex (pre-RC) assembly, but is required for origin unwinding during DNA replication. Moreover, this ATPase-deficient mutant complex did not support DNA replication in MCM-depleted extracts. Altogether, these results both demonstrate the ability of recombinant MCM proteins to perform all replication roles of MCM complexes, and further support the model that MCM2-7 is the replicative helicase. These data establish that mutations affecting the ATPase activity of the MCM complex uncouple its role in pre-RC assembly from DNA replication.     

Regulation of the functional interactions between archaeal eukaryote-like Cdc6/Orc1 proteins on the replication origin by two different mechanisms

The crenarchaeon Sulfolobus solfataricus contains three active origins of replication and three eukaryote-like Cdc6/Orc1 proteins known as SsoCdc6 proteins. It has the potential to become a powerful model system in understanding the central mechanism of the eukaryotic DNA replication. In this research, we designed a group of duplex DNA substrates containing specific origin recognition boxes (ORBs) of the archaeon and identified the DNA-binding activities of different SsoCdc6 proteins. Furthermore, we showed that the DNA-protein interaction between the DNA substrate and the SsoCdc6-1 or SsoCdc6-3 strikingly regulated their DNA-binding activities of each other on the origin. On the other hand, the protein-protein interactions between SsoCdc6-1 and SsoCdc6-2 were observed to mutually modulate the stimulating or inhibitive effects on the DNA-binding activities of each other. Thus, two different mechanisms were demonstrated to be involved in the regulations of the functions of the SsoCdc6 proteins on the replication origins. The results of this study imply that the interactions between multiple SsoCdc6 proteins and origin DNA collectively contribute to the positive or negative regulation of DNA replication initiation in the archaeon species.     

Functions of sensor 1 and sensor 2 regions of Saccharomyces cerevisiae Cdc6p in vivo and in vitro

Cdc6p is a key regulator of the cell cycle in eukaryotes and is a member of the AAA(+) (ATPases associated with a variety of cellular activities) family of proteins. In this family of proteins, the sensor 1 and sensor 2 regions are important for their function and ATPase activity. Here, site-directed mutagenesis has been used to examine the role of these regions of Saccharomyces cerevisiae Cdc6p in controlling the cell cycle progression and initiation of DNA replication. Two important amino acid residues (Asn(263) in sensor 1 and Arg(332) in sensor 2) were identified as key residues for Cdc6p function in vivo. Cells expressing mutant Cdc6p (N263A or R332E) grew slowly and accumulated in the S phase. In cells expressing mutant Cdc6p, loading of the minichromosome maintenance (MCM) complex of proteins was decreased, suggesting that the slow progression of S phase in these cells was due to inefficient MCM loading on chromatin. Purified wild type Cdc6p but not mutant Cdc6p (N263A and R332E) caused the structural modification of origin recognition complex proteins. These results are consistent with the idea that Cdc6p uses its ATPase activity to change the conformation of origin recognition complex, and then together they recruit the MCM complex.     

Thermoplasma acidophilum Cdc6 protein stimulates MCM helicase activity by regulating its ATPase activity

The minichromosome maintenance (MCM) proteins are thought to function as the replicative helicases in archaea. In most archaeal species studied, the interaction between MCM and the initiator protein, Cdc6, inhibits helicase activity. To date, the only exception is the helicase and Cdc6 proteins from the archaeon Thermoplasma acidophilum. It was previously shown that when the Cdc6 protein interacts with MCM it substantially stimulates helicase activity. It is shown here that the mechanism by which the Cdc6 protein stimulates helicase activity is by stimulating the ATPase activity of MCM. Also, through the use of site-specific substitutions, and truncated and chimeric proteins, it was shown that an intact Cdc6 protein is required for this stimulation. ATP binding and hydrolysis by the Cdc6 protein is not needed for the stimulation. The data suggest that binding of Cdc6 protein to MCM protein changes the structure of the helicase, enhancing the catalytic hydrolysis of ATP and helicase activity.     

Molecular determinants of origin discrimination by Orc1 initiators in archaea

Unlike bacteria, many eukaryotes initiate DNA replication from genomic sites that lack apparent sequence conservation. These loci are identified and bound by the origin recognition complex (ORC), and subsequently activated by a cascade of events that includes recruitment of an additional factor, Cdc6. Archaeal organisms generally possess one or more Orc1/Cdc6 homologs, belonging to the Initiator clade of ATPases associated with various cellular activities (AAA(+)) superfamily; however, these proteins recognize specific sequences within replication origins. Atomic resolution studies have shown that archaeal Orc1 proteins contact double-stranded DNA through an N-terminal AAA(+) domain and a C-terminal winged-helix domain (WHD), but use remarkably few base-specific contacts. To investigate the biochemical effects of these associations, we mutated the DNA-interacting elements of the Orc1-1 and Orc1-3 paralogs from the archaeon Sulfolobus solfataricus, and tested their effect on origin binding and deformation. We find that the AAA(+) domain has an unpredicted role in controlling the sequence selectivity of DNA binding, despite an absence of base-specific contacts to this region. Our results show that both the WHD and ATPase region influence origin recognition by Orc1/Cdc6, and suggest that not only DNA sequence, but also local DNA structure help define archaeal initiator binding sites.     

Cyclin A-dependent kinase activity affects chromatin binding of ORC, Cdc6, and MCM in egg extracts of Xenopus laevis.

The initiation of DNA replication in eukaryotes requires the loading of the origin recognition complex (ORC), Cdc6, and minichromosome maintenance (MCM) proteins onto chromatin to form the preinitiation complex. In Xenopus egg extract, the proteins Orc1, Orc2, Cdc6, and Mcm4 are underphosphorylated in interphase and hyperphosphorylated in metaphase extract. We find that chromatin binding of ORC, Cdc6, and MCM proteins does not require cyclin-dependent kinase activities. High cyclin A-dependent kinase activity inhibits the binding and promotes the release of Xenopus ORC, Cdc6, and MCM from sperm chromatin, but has no effect on chromatin binding of control proteins. Cyclin A together with ORC, Cdc6 and MCM proteins is bound to sperm chromatin in DNA replicating pseudonuclei. In contrast, high cyclin E/cdk2 was not detected on chromatin, but was found soluble in the nucleoplasm. High cyclin E kinase activity allows the binding of Xenopus ORC and Cdc6, but not MCM, to sperm chromatin, even though the kinase does not phosphorylate MCM directly. We conclude that chromatin-bound cyclin A kinase controls DNA replication by protein phosphorylation and chromatin release of Cdc6 and MCM, whereas soluble cyclin E kinase prevents rereplication during the cell cycle by the inhibition of premature MCM chromatin association.     

Biochemical characterization of two Cdc6/ORC1-like proteins from the crenarchaeon Sulfolobus solfataricus

The biological role of archaeal proteins, homologous to the eukaryal replication initiation factors of cell division control (Cdc6) and origin recognition complex (ORC1), has not yet been clearly established. The hyperthermophilic crenarchaeon Sulfolobus solfataricus (referred to as Sso) possesses three Cdc6/ORC1-like factors, which are named Sso Cdc6-1, Cdc6-2 and Cdc6-3. This study is a report on the biochemical characterization of Sso Cdc6-1 and Cdc6-3. It has been found that either Sso Cdc6-1 or Cdc6-3 behave as monomers in solutions by gel filtration analyses. Both factors are able to bind to various single-stranded and double-stranded DNA ligands, but Sso Cdc6-3 shows a higher DNA-binding affinity. It has also been observed that either Sso Cdc6-1 or Cdc6-3 inhibit the DNA unwinding activity of the S. solfataricus homo-hexameric mini-chromosome maintenance (MCM)-like DNA helicase (Sso MCM); although they strongly stimulate the interaction of the Sso MCM with bubble-containing synthetic oligonucleotides. The study has also showed, with surface plasmon resonance measurements, that Sso Cdc6-2 physically interacts with either Sso Cdc6-1 or Sso Cdc6-3. These findings may provide important clues needed to understand the biological role that is played by each of these three Cdc6 factors during the DNA replication initiation process in the S. solfataricus cells.     

Different phenotypes in vivo are associated with ATPase motif mutations in Schizosaccharomyces pombe minichromosome maintenance proteins

The six conserved MCM proteins are essential for normal DNA replication. They share a central core of homology that contains sequences related to DNA-dependent and AAA(+) ATPases. It has been suggested that the MCMs form a replicative helicase because a hexameric subcomplex formed by MCM4, -6, and -7 proteins has in vitro DNA helicase activity. To test whether ATPase and helicase activities are required for MCM protein function in vivo, we mutated conserved residues in the Walker A and Walker B motifs of MCM4, -6, and -7 and determined that equivalent mutations in these three proteins have different in vivo effects in fission yeast. Some mutations reported to abolish the in vitro helicase activity of the mouse MCM4/6/7 subcomplex do not affect the in vivo function of fission yeast MCM complex. Mutations of consensus CDK sites in Mcm4p and Mcm7p also have no phenotypic consequences. Co-immunoprecipitation analyses and in situ chromatin-binding experiments were used to study the ability of the mutant Mcm4ps to associate with the other MCMs, localize to the nucleus, and bind to chromatin. We conclude that the role of ATP binding and hydrolysis is different for different MCM subunits.     

Identification of Mcm2 phosphorylation sites by S-phase-regulating kinases

Minichromosome maintenance 2-7 proteins play a pivotal role in replication of the genome in eukaryotic organisms. Upon entry into S-phase several subunits of the MCM hexameric complex are phosphorylated. It is thought that phosphorylation activates the intrinsic MCM DNA helicase activity, thus allowing formation of active replication forks. Cdc7, Cdk2, and ataxia telangiectasia and Rad3-related kinases regulate S-phase entry and S-phase progression and are known to phosphorylate the Mcm2 subunit. In this work, by in vitro kinase reactions and mass spectrometry analysis of the products, we have mapped phosphorylation sites in the N terminus of Mcm2 by Cdc7, Cdk2, Cdk1, and CK2. We found that Cdc7 phosphorylates Mcm2 in at least three different sites, one of which corresponds to a site also reported to be phosphorylated by ataxia telangiectasia and Rad3-related. Three serine/proline sites were identified for Cdk2 and Cdk1, and a unique site was phosphorylated by CK2. We raised specific anti-phosphopeptide antibodies and found that all the sites identified in vitro are also phosphorylated in cells. Importantly, although all the Cdc7-dependent Mcm2 phosphosites fluctuate during the cell cycle with kinetics similar to Cdc7 kinase activity and Cdc7 protein levels, phosphorylation of Mcm2 in the putative cyclin-dependent kinase (Cdk) consensus sites is constant during the cell cycle. Furthermore, our analysis indicates that the majority of the Mcm2 isoforms phosphorylated by Cdc7 are not stably associated with chromatin. This study forms the basis for understanding how MCM functions are regulated by multiple kinases within the cell cycle and in response to external perturbations.     

A single subunit MCM6 from pea forms homohexamer and functions as DNA helicase

The initiation of DNA replication starts from origins and is controlled by a multiprotein complex, which involves about twenty protein factors. One of the important factors is hetrohexameric minichromosome maintenance (MCM2-7) protein complex which is evolutionary conserved and functions as essential replicative helicase for DNA replication. Here we report the isolation and characterization of a single subunit of pea MCM protein complex, the MCM6. The deduced amino acid (827) sequence contains all the known canonical MCM motifs including zinc finger, MCM specific Walker A and Walker B and arginine finger. The purified recombinant protein contains ATP-dependent 3′–5′ DNA helicase, ATP-binding and ATPase activities. The helicase activity was stimulated by replication fork like substrate and anti-MCM6 antibodies curtail all the enzyme activities of MCM6 protein. In vitro it self-interacts and forms a homohexamer which is active for DNA helicase and ATPase activities. The complete protein is required for self-interaction as the truncated MCM6 proteins were unable to self-interact. Western blot analysis and in vivo immunostaining followed by confocal microscopy showed the localization of MCM6 both in the nucleus and cytosol. These findings provide first direct evidence that single subunit MCM6 contains DNA helicase activity which is unique to plant MCM6 protein, as this activity was only reported for heteromultimers of MCM proteins in animal system. This discovery should make an important contribution to a better understanding of DNA replication in plants.