Influence of chromatin and single strand binding proteins on the activity of an archaeal MCM

The mini-chromosome maintenance (MCM) complex is the presumptive replicative helicase in archaea and eukaryotes. In archaea, the MCM is a homo-multimer, in eukaryotes a heterohexamer composed of six related subunits, MCM 2-7. Biochemical studies using naked DNA templates have revealed that archaeal MCMs and a sub-complex of eukaryotic MCM 4, 6 and 7 have 3' to 5' helicase activity. Here, we investigate the influence of the major chromatin proteins, Alba and Sul7d, of Sulfolobus solfataricus (Sso) on the ability of the MCM complex to melt partial duplex DNA substrates. In addition, we test the effect of Sso SSB on MCM activity. We reveal that Alba represents a formidable barrier to MCM activity and further demonstrate that acetylation of Alba alleviates repression of MCM activity.     

Archaeal MCM has separable processivity, substrate choice and helicase domains

The mini-chromosome maintenance (MCM) complex is the principal candidate for the replicative helicase of archaea and eukaryotes. Here, we describe a functional dissection of the roles of the three principal structural modules of the homomultimeric MCM of the hyperthermophilic archaeon Sulfolobus solfataricus. Our results include the first analysis of the central AAA+ domain in isolation. This domain possesses ATPase and helicase activity, defining this as the minimal helicase domain. Reconstitution experiments show that the helicase activity of the AAA+ domain can be stimulated by addition of the isolated N-terminal half in trans. Addition of the N-terminus influences both the processivity of the helicase and the choice of substrate that can be melted by the ATPase domain. The degenerate helix-turn-helix domain at the C-terminus of MCM exerts a negative effect on the helicase activity of the complex. These results provide the first evidence for extensive regulatory inter-domain communication within the MCM complex.     

Structure and regulatory role of the C-terminal winged helix domain of the archaeal minichromosome maintenance complex

The minichromosome maintenance complex (MCM) represents the replicative DNA helicase both in eukaryotes and archaea. Here, we describe the solution structure of the C-terminal domains of the archaeal MCMs of Sulfolobus solfataricus (Sso) and Methanothermobacter thermautotrophicus (Mth). Those domains consist of a structurally conserved truncated winged helix (WH) domain lacking the two typical ‘wings’ of canonical WH domains. A less conserved N-terminal extension links this WH module to the MCM AAA+ domain forming the ATPase center. In the Sso MCM this linker contains a short α-helical element. Using Sso MCM mutants, including chimeric constructs containing Mth C-terminal domain elements, we show that the ATPase and helicase activity of the Sso MCM is significantly modulated by the short α-helical linker element and by N-terminal residues of the first α-helix of the truncated WH module. Finally, based on our structural and functional data, we present a docking-derived model of the Sso MCM, which implies an allosteric control of the ATPase center by the C-terminal domain.     

The intrinsic DNA helicase activity of Methanobacterium thermoautotrophicum delta H minichromosome maintenance protein

Minichromosome maintenance proteins (MCMs) form a family of conserved molecules that are essential for initiation of DNA replication. All eukaryotes contain six orthologous MCM proteins that function as heteromultimeric complexes. The sequencing of the complete genomes of several archaebacteria has shown that MCM proteins are also present in archaea. The archaea Methanobacterium thermoautotrophicum contains a single MCM-related sequence. Here we report on the expression and purification of the recombinant M. thermoautotrophicum MCM protein (MtMCM) in both Escherichia coli and baculovirus-infected cells. We show that purified MtMCM protein assembles in large macromolecular complexes consistent in size with being double hexamers. We demonstrate that MtMCM contains helicase activity that preferentially uses dATP and DNA-dependent dATPase and ATPase activities. The intrinsic helicase activity of MtMCM is abolished when a conserved lysine in the helicase domain I/nucleotide binding site is mutated. MtMCM helicase unwinds DNA duplexes in a 3' --> 5' direction and can unwind up to 500 base pairs in vitro. The kinetics, processivity, and directionality of MtMCM support its role as a replicative helicase in M. thermoautotrophicum. This strongly suggests that this function is conserved for MCM proteins in eukaryotes where a replicative helicase has yet to be identified.     

Unwinding the structure and function of the archaeal MCM helicase

During chromosomal DNA replication, the replicative helicase unwinds the duplex DNA to provide the single-stranded DNA substrate for the polymerase. In archaea, the replicative helicase is the minichromosome maintenance (MCM) complex. The enzyme utilizes the energy of ATP hydrolysis to translocate along one strand of the duplex and unwind the complementary strand. Much progress has been made in elucidating structure and function since the first report on the biochemical properties of an archaeal MCM protein in 1999. We now know the biochemical and structural properties of the enzyme from several archaeal species and some of the mechanisms by which the enzyme is regulated. This review summarizes recent studies on the archaeal MCM protein and discusses the implications for helicase function and DNA replication in archaea.     

How to load a replicative helicase onto chromatin: A more and more complex matter during evolution

In all eukaryotes, the heterohexameric MCM2-7 complex functions as the main replicative helicase during S phase. During early G1 phase, it is recruited onto chromatin in a sequence of reactions called pre-replication complex (pre-RC) formation or DNA licensing. This process is ATP-dependent and at least two different chromatin-bound ATPase activities are required besides several others essential, but not enzymatically active, proteins. Although functionally conserved during evolution, pre-RC formation and the way the MCM2-7 helicase is loaded onto DNA are more complex in metazoans than in single-cell eukaryotes. Recently, we characterized a new essential factor for pre-RC assembly and DNA licensing, the vertebrate-specific MCM9 protein that contains not only an ATPase but also a helicase domain. MCM9 adds another layer of complexity to how vertebrates achieve and regulate the loading of the MCM2-7 helicase and DNA replication.     

Mutational analysis of conserved aspartic acid residues in the <Emphasis Type="Italic">Methanothermobacter thermautotrophicus</Emphasis> MCM helicase

Minichromosome maintenance (MCM) helicases are thought to function as the replicative helicases in archaea and eukarya, unwinding the duplex DNA in the front of the replication fork. The archaeal MCM helicase can be divided into three parts, the N-terminal, catalytic, and C-terminal regions. The N-terminal part of the protein is divided into three domains, A, B, and C, and was shown to be involved in protein multimerization and binding to single- and double-stranded DNA. Two Asp residues found in domain C are conserved among MCM proteins from different archaea. These residues are located in a loop at the interface with domain A. Mutations of these residues in the Methanothermobacter thermautotrophicus MCM protein, Asp202 and Asp203, to Asn result in a significant reduction in the ability of the enzyme to bind DNA and in lower thermal stability. However, the mutant proteins retained helicase and ATPase activities. Further investigation of the DNA binding revealed that the presence of ATP rescues the DNA binding deficiencies by these mutant proteins. Possible roles of these conserved residues in MCM function are discussed.     

Biochemical characterization of the minichromosome maintenance protein from the archaeon Thermoplasma acidophilum

Minichromosome maintenance (MCM) proteins are thought to function as the replicative helicases in archaea. Studies have shown that the MCM complex from the thermoacidophilic euryarchaeon Thermoplasma acidophilum (TaMCM) has some properties not reported in other archaeal MCM helicases. Here, the biochemical properties of the TaMCM are studied. The protein binds single-stranded DNA, has DNA-dependent ATPase activity and ATP-dependent 3′ → 5′ helicase activity. The optimal helicase conditions with regard to temperature, pH and salinity are similar to the intracellular conditions in T. acidophilum. It is also found that about 1,000 molecules of TaMCM are present per actively growing cell. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

High-temperature single-molecule kinetic analysis of thermophilic archaeal MCM helicases

The minichromosome maintenance (MCM) complex is the replicative helicase responsible for unwinding DNA during archaeal and eukaryal genome replication. To mimic long helicase events in the cell, a high-temperature single-molecule assay was designed to quantitatively measure long-range DNA unwinding of individual DNA helicases from the archaeons Methanothermobacter thermautotrophicus (Mth) and Thermococcus sp. 9°N (9°N). Mth encodes a single MCM homolog while 9°N encodes three helicases. 9°N MCM3, the proposed replicative helicase, unwinds DNA at a faster rate compared to 9°N MCM2 and to Mth MCM. However, all three MCM proteins have similar processivities. The implications of these observations for DNA replication in archaea and the differences and similarities among helicases from different microorganisms are discussed. Development of the high-temperature single-molecule assay establishes a system to comprehensively study thermophilic replisomes and evolutionary links between archaeal, eukaryal, and bacterial replication systems.     

Biochemical activities of the BOB1 mutant in Methanobacterium thermoautotrophicum MCM

Minichromosomal maintenance proteins (MCMs) are considered to be the replicative helicase. Methanobacterium thermoautotrophicum has a single MCM gene (mtMCM). The crystal structure of the mtMCM N-terminal region is a double hexamer. Structure-guided sequence alignment indicates a structural conservation of this fragment across archaeal and eukaryotic MCMs. The mtMCM structure was successfully used to analyze a Saccharomyces cerevisiae MCM5 mutant, called BOB1, which contains a single residue change from Pro to Leu and bypasses a kinase normally required for initiation of DNA replication. A domain-push model was proposed to explain the BOB1 bypass activity. Here we investigate the effects of BOB1 mutation on the biochemical activities of mtMCM. Surprisingly, the BOB1 mutation (P62L) had a major effect on the helicase activity but had no significant impact on DNA binding and ATPase activities. These results will contribute to a more detailed understanding of the BOB1 bypass activity and other aspects of DNA replication control.     

ATPase site architecture and helicase mechanism of an archaeal MCM

The subunits of the presumptive replicative helicase of archaea and eukaryotes, the MCM complex, are members of the AAA+ (ATPase-associated with various cellular activities) family of ATPases. Proteins within this family harness the chemical energy of ATP hydrolysis to perform a broad range of cellular processes. Here, we investigate the function of the AAA+ site in the mini-chromosome maintenance (MCM) complex of the archaeon Sulfolobus solfataricus (SsoMCM). We find that SsoMCM has an unusual active-site architecture, with a unique blend of features previously found only in distinct families of AAA+ proteins. We additionally describe a series of mutant doping experiments to investigate the mechanistic basis of intersubunit coordination in the generation of helicase activity. Our results indicate that MCM can tolerate catalytically inactive subunits and still function as a helicase, leading us to propose a semisequential model for helicase activity of this complex.     

DNA Induces Conformational Changes in a Recombinant Human Minichromosome Maintenance Complex

ATP-dependent DNA unwinding activity has been demonstrated for recombinant archaeal homohexameric minichromosome maintenance (MCM) complexes and their yeast heterohexameric counterparts, but in higher eukaryotes such as Drosophila, MCM-associated DNA helicase activity has been observed only in the context of a co-purified Cdc45-MCM-GINS complex. Here, we describe the production of the recombinant human MCM (hMCM) complex in Escherichia coli. This protein displays ATP hydrolysis activity and is capable of unwinding duplex DNA. Using single-particle asymmetric EM reconstruction, we demonstrate that recombinant hMCM forms a hexamer that undergoes a conformational change when bound to DNA. Recombinant hMCM produced without post-translational modifications is functional in vitro and provides an important tool for biochemical reconstitution of the human replicative helicase.     

Coupling of DNA binding and helicase activity is mediated by a conserved loop in the MCM protein

Minichromosome maintenance (MCM) helicases are the presumptive replicative helicases, thought to separate the two strands of chromosomal DNA during replication. In archaea, the catalytic activity resides within the C-terminal region of the MCM protein. In Methanothermobacter thermautotrophicus the N-terminal portion of the protein was shown to be involved in protein multimerization and binding to single and double stranded DNA. MCM homologues from many archaeal species have highly conserved predicted amino acid similarity in a loop located between β7 and β8 in the N-terminal part of the molecule. This high degree of conservation suggests a functional role for the loop. Mutational analysis and biochemical characterization of the conserved residues suggest that the loop participates in communication between the N-terminal portion of the helicase and the C-terminal catalytic domain. Since similar residues are also conserved in the eukaryotic MCM proteins, the data presented here suggest a similar coupling between the N-terminal and catalytic domain of the eukaryotic enzyme.     

Identification and characterization of a novel component of the human minichromosome maintenance complex

Minichromosome maintenance (MCM) complex replicative helicase complexes play essential roles in DNA replication in all eukaryotes. Using a tandem affinity purification-tagging approach in human cells, we discovered a form of the MCM complex that contains a previously unstudied protein, MCM binding protein (MCM-BP). MCM-BP is conserved in multicellular eukaryotes and shares limited homology with MCM proteins. MCM-BP formed a complex with MCM3 to MCM7, which excluded MCM2; and, conversely, hexameric complexes of MCM2 to MCM7 lacked MCM-BP, indicating that MCM-BP can replace MCM2 in the MCM complex. MCM-BP-containing complexes exhibited increased stability under experimental conditions relative to those containing MCM2. MCM-BP also formed a complex with the MCM4/6/7 core helicase in vitro, but, unlike MCM2, did not inhibit this helicase activity. A proportion of MCM-BP bound to cellular chromatin in a cell cycle-dependent manner typical of MCM proteins, and, like other MCM subunits, preferentially associated with a cellular origin in G(1) but not in S phase. In addition, down-regulation of MCM-BP decreased the association of MCM4 with chromatin, and the chromatin association of MCM-BP was at least partially dependent on MCM4 and cdc6. The results indicate that multicellular eukaryotes contain two types of hexameric MCM complexes with unique properties and functions.     

The GINS complex from the thermophilic archaeon, <Emphasis Type="Italic">Thermoplasma acidophilum</Emphasis> may function as a homotetramer in DNA replication

The eukaryotic GINS heterotetramer, consisting of Sld5, Psf1, Psf2, and Psf3, participates in “CMG complex” formation with mini-chromosome maintenance (MCM) and Cdc45 as a key component of a replicative helicase. There are only two homologs of the GINS proteins in Archaea, and these proteins, Gins51 and Gins23, form a heterotetrameric GINS with a 2:2 molar ratio. The Pyrococcus furiosus GINS stimulates the ATPase and helicase activities of its cognate MCM, whereas the Sulfolobus solfataricus GINS does not affect those activities of its cognate MCM, although the proteins bind each other. Intriguingly, Thermoplasma acidophilum, as well as many euryarchaea, have only one gene encoding the sequence homologous to that of archaeal Gins protein (Gins51) on the genome. In this study, we investigated the biochemical properties of the gene product (TaGins51). A gel filtration and electron microscopy revealed that TaGins51 forms a homotetramer. A physical interaction between TaGins51 and TaMcm was detected by a surface plasmon resonance analysis. Unexpectedly, TaGins51 inhibited the ATPase activity, but did not affect the helicase activity of its cognate MCM. These results suggest that another factor is required to form a stable helicase complex with MCM and GINS at the replication fork in T. acidophilum cells.     

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.     

Control of DNA replication: regulation and activation of eukaryotic replicative helicase, MCM

DNA replication is a key event of cell proliferation and the final target of signal transduction induced by growth factor stimulation. It is also strictly regulated during the ongoing cell cycle so that it occurs only once during S phase and that all the genetic materials are faithfully duplicated. DNA replication may be arrested or temporally inhibited due to a varieties of internal and external causes. Cells have developed intricate mechanisms to cope with the arrested replication forks to minimize the adversary effect on the stable maintenance of genetic materials. Helicases play a central role in DNA replication. In eukaryotes, MCM (minichromosome maintenance) protein complex plays essential roles as a replicative helicase. MCM4-6-7 complex possesses intrinsic DNA helicase activity which translocates on single-stranded DNA form 3' to 5'. Mammalian MCM4-6-7 helicase and ATPase activities are specifically stimulated by the presence of thymine-rich single-stranded DNA sequences onto which it is loaded. The activation appears to depend on the thymine content of this single-strand, and sequences derived from human replication origins can serve as potent activators of the MCM helicase. MCM is a prime target of Cdc7 kinase, known to be essential for activation of replication origins. We will discuss how the MCM may be activated at the replication origins by template DNA, phosphorylation, and interaction with other replicative proteins, and will present a model of how activation of MCM helicase by specific sequences may contribute to selection of replication initiation sites in higher eukaryotes.     

Modular organization of the Sulfolobus solfataricus mini-chromosome maintenance protein

Mini-chromosome maintenance (MCM) proteins form ring-like hexameric complexes that are commonly believed to act as the replicative DNA helicase at the eukaryotic/archaeal DNA replication fork. Because of their simplified composition with respect to the eukaryotic counterparts, the archaeal MCM complexes represent a good model system to use in analyzing the structural/functional relationships of these important replication factors. In this study the domain organization of the MCM-like protein from Sulfolobus solfataricus (Sso MCM) has been dissected by trypsin partial proteolysis. Three truncated derivatives of Sso MCM corresponding to protease-resistant domains were produced as soluble recombinant proteins and purified: the N-terminal domain (N-ter, residues 1-268); a fragment comprising the AAA+ and C-terminal domains (AAA+-C-ter, residues 269-686); and the C-terminal domain (C-ter, residues 504-686). All of the purified recombinant proteins behaved as monomers in solution as determined by analytical gel filtration chromatography, suggesting that the polypeptide chain integrity is required for stable oligomerization of Sso MCM. However, the AAA+-C-ter derivative, which includes the AAA+ motor domain and retains ATPase activity, was able to form dimers in solution when ATP was present, as analyzed by size exclusion chromatography and glycerol gradient sedimentation analyses. Interestingly, the AAA+-C-ter protein could displace oligonucleotides annealed to M13 single-stranded DNA although with a reduced efficiency in comparison with the full-sized Sso MCM. The implications of these findings for understanding the DNA helicase mechanism of the MCM complex are discussed.     

The interplay of DNA binding, ATP hydrolysis and helicase activities of the archaeal MCM helicase

The MCM (minichromosome maintenance) proteins of archaea are widely believed to be the replicative DNA helicase of these organisms. Most archaea possess a single MCM orthologue that forms homo-multimeric assemblies with a single hexamer believed to be the active form. In the present study we characterize the roles of highly conserved residues in the ATPase domain of the MCM of the hyperthermophilic archaeon Sulfolobus solfataricus. Our results identify a potential conduit for communicating DNA-binding information to the ATPase active site.