Organization of the archaeal MCM complex on DNA and implications for the helicase mechanism

The homomultimeric archaeal mini-chromosome maintenance (MCM) complex serves as a simple model for the analogous heterohexameric eukaryotic complex. Here we investigate the organization and orientation of the MCM complex of the hyperthermophilic archaeon Sulfolobus solfataricus (Sso) on model DNA substrates. Sso MCM binds as a hexamer and slides on the end of a 3'-extended single-stranded DNA tail of a Y-shaped substrate; binding is oriented so that the motor domain of the protein faces duplex DNA. Two candidate beta-hairpin motifs within the MCM monomer have partially redundant roles in DNA binding. Notably, however, conserved basic residues within these motifs have nonequivalent roles in the helicase activity of MCM. On the basis of these findings, we propose a model for the mechanism of the helicase activity of MCM and note parallels with SV40 T antigen.     

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.     

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.     

Insights into the MCM functional mechanism: lessons learned from the archaeal MCM complex

The helicase function of the minichromosome maintenance protein (MCM) is essential for genomic DNA replication in archaea and eukaryotes. There has been rapid progress in studies of the structure and function of MCM proteins from different organisms, leading to better understanding of the MCM helicase mechanism. Because there are a number of excellent reviews on this topic, we will use this review to summarize some of the recent progress, with particular focus on the structural aspects of MCM and their implications for helicase function. Given the hexameric and double hexameric architecture observed by X-ray crystallography and electron microscopy of MCMs from archaeal and eukaryotic cells, we summarize and discuss possible unwinding modes by either a hexameric or a double hexameric helicase. Additionally, our recent crystal structure of a full length archaeal MCM has provided structural information on an intact, multi-domain MCM protein, which includes the salient features of four unusual β-hairpins from each monomer, and the side channels of a hexamer/double hexamer. These new structural data enable a closer examination of the structural basis of the unwinding mechanisms by MCM.     

ATPase cycle of an archaeal chaperonin

Recent structural data imply differences in allosteric behavior of the group I chaperonins, typified by GroEL from Escherichia coli, and the group II chaperonins, which comprise archaeal thermosome and eukaryotic TRiC/CCT. Therefore, this study addresses the mechanism of interaction of adenine nucleotides with recombinant alpha-only and native alphabeta-thermosomes from Thermoplasma acidophilum, which also enables us to analyze the role of the heterooligomeric composition of the natural thermosome. Although all subunits of the alpha-only thermosome seem to bind nucleotides tightly and independently, the native chaperonin has two different classes of ATP-binding sites. Furthermore, for the alpha-only thermosome, the steady-state ATPase rate is determined by the cleavage reaction itself, whereas, for the alphabeta-thermosome, the rate-limiting step is associated with a post-hydrolysis isomerisation into a non-covalent ADP*P(i) species prior to the release of the gamma-phosphate group. After half-saturation with ATP, a negative cooperativity in hydrolysis is observed for both thermosomes. The effect of Mg(2+) and K(+) nucleotide cycling is documented. We conclude that archaeal chaperonins have unique allosteric properties and discuss them in the light of the mechanism established for the group I chaperonins.     

Cryo-electron microscopy reveals a novel DNA-binding site on the MCM helicase

The eukaryotic MCM2-7 complex is recruited at origins of replication during the G1 phase and acts as the main helicase at the replication fork during the S phase of the cell cycle. To characterize the interplay between the MCM helicase and DNA prior to the melting of the double helix, we determined the structure of an archaeal MCM orthologue bound to a 5.6-kb double-stranded DNA segment, using cryo-electron microscopy. DNA wraps around the N-terminal face of a single hexameric ring. This interaction requires a conformational change within the outer belt of the MCM N-terminal domain, exposing a previously unrecognized helix-turn-helix DNA-binding motif. Our findings provide novel insights into the role of the MCM complex during the initiation step of DNA replication.     

Inhibition of unwinding and ATPase activities of pea MCM6 DNA helicase by actinomycin and nogalamycin

Pea mini-chromosome maintenance 6 (MCM6) single subunit (93 kDa) forms homohexamer (560 kDa) and contains an ATP-dependent and replication fork stimulated 3′ to 5′ DNA unwinding activity along with intrinsic DNA-dependent ATPase and ATP-binding activities¹ (Plant Mol Biol 2010; DOI: 10.1007/s11103-010-9675-7). Here, we have determined the effect of various DNA-binding agents, such as actinomycin, nogalamycin, daunorubicin, doxorubicin, distamycin, camptothecin, cyclophosphamide, ellipticine, VP-16, novobiocin, netropsin, cisplatin, mitoxantrone and genistein on the DNA unwinding and ATPase activities of the pea MCM6 DNA helicase. The results show that actinomycin and nogalamycin inhibited the DNA helicase (apparent Ki values of 10 and 1 µM, respectively) and ATPase (apparent Ki values of 100 and 17 µM, respectively) activities. Although, daunorubicin and doxorubicin also inhibited the DNA helicase activity of pea MCM6, but with less efficiency; however, these could not inhibit the ATPase activity. These results suggest that the intercalation of the inhibitors into duplex DNA generates a complex that impedes translocation of MCM6, resulting in the inhibitions of the activities. This study could be useful in our better understanding of the mechanism of plant nuclear DNA helicase unwinding.Key words: ATPase, actinomycin, DNA-binding agents, DNA helicase, nogalamycin, pea MCM6     

Polymorphism and double hexamer structure in the archaeal minichromosome maintenance (MCM) helicase from Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum minichromosome maintenance complex (mtMCM), a cellular replicative helicase, is a useful model for the more complex eukaryotic MCMs. Biochemical and crystallographic evidence indicates that mtMCM assembles as a double hexamer (dHex), but previous electron microscopy studies reported only the presence of single heptamers or single hexamers (Pape, T., Meka, H., Chen, S., Vicentini, G., Van Heel, M., and Onesti, S. (2003) EMBO Rep. 4, 1079-1083; Yu, X., VanLoock, M. S., Poplawski, A., Kelman, Z., Xiang, T., Tye, B. K., and Egelman, E. H. (2002) EMBO Rep. 3, 792-797). Here we present the first three-dimensional electron microscopy reconstruction of the full-length mtMCM dHex in which two hexamers contact each other via the structurally well defined N-terminal domains. The dHex has obvious side openings that resemble the side channels of LTag (large T antigen). 6-fold and 7-fold rings were observed in the same mtMCM preparation, but we determined that assembly as a double ring favors 6-fold structures. Additionally, open rings were also detected, which suggests a direct mtMCM loading mechanism onto DNA.     

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.     

Conformational rearrangements of an archaeal chaperonin upon ATPase cycling

Chaperonins are double-ring protein assemblies with a central cavity that provides a sequestered environment for in vivo protein folding. Their reaction cycle is thought to consist of a nucleotide-regulated alternation between an open substrate-acceptor state and a closed folding-active state. The cavity of ATP-charged group I chaperonins, typified by Escherichia coli GroEL [1], is sealed off by a co-chaperonin, whereas group II chaperonins--the archaeal thermosome and eukaryotic TRiC/CCT [2]--possess a built-in lid [3-5]. The mechanism of the lid's rearrangements requires clarification, as even in the absence of nucleotides, thermosomes of Thermoplama acidophilum appear open in vitrified ice [6] and closed in crystals [4]. Here we analyze the conformation of the thermosome at each step of the ATPase cycle by small-angle neutron scattering. The apo-chaperonin is open in solution, and ATP binding induces its further expansion. Closure seems to occur during ATP hydrolysis and before phosphate release, and represents the rate-limiting step of the cycle. The same closure can be triggered by the crystallization buffer. Thus, the allosteric regulation of group II chaperonins appears different from that of their group I counterparts.     

Dissection of the functional domains of an archaeal Holliday junction helicase

Helicases and nucleases form complexes that play very important roles in DNA repair pathways some of which interact with each other at Holliday junctions. In this study, we present in vitro and in vivo analysis of Hjm and its interaction with Hjc in Sulfolobus. In vitro studies employed Hjm from the hyperthermophilic archaeon Sulfolobus tokodaii (StoHjm) and its truncated derivatives, and characterization of the StoHjm proteins revealed that the N-terminal module (residues 1-431) alone was capable of ATP hydrolysis and DNA binding, while the C-terminal one (residues 415-704) was responsible for regulating the helicase activity. The region involved in StoHjm-StoHjc (Hjc from S. tokodaii) interaction was identified as part of domain II, domain III (Winged Helix motif), and domain IV (residues 366-645) for StoHjm. We present evidence supporting that StoHjc regulates the helicase activity of StoHjm by inducing conformation change of the enzyme. Furthermore, StoHjm is able to prevent the formation of Hjc/HJ high complex, suggesting a regulation mechanism of Hjm to the activity of Hjc. We show that Hjm is essential for cell viability using recently developed genetic system and mutant propagation assay, suggesting that Hjm/Hjc mediated resolution of stalled replication forks is of crucial importance in archaea. A tentative pathway with which Hjm/Hjc interaction could have occurred at stalled replication forks is discussed.     

Molecular architecture of the recombinant human MCM2-7 helicase in complex with nucleotides and DNA

DNA replication is a key biological process that involves different protein complexes whose assembly is rigorously regulated in a successive order. One of these complexes is a replicative hexameric helicase, the MCM complex, which is essential for the initiation and elongation phases of replication. After the assembly of a double heterohexameric MCM2-7 complex at replication origins in G1, the 2 heterohexamers separate from each other and associate with Cdc45 and GINS proteins in a CMG complex that is capable of unwinding dsDNA during S phase. Here, we have reconstituted and characterized the purified human MCM2-7 (hMCM2-7) hexameric complex by co-expression of its 6 different subunits in insect cells. The conformational variability of the complex has been analyzed by single particle electron microscopy in the presence of different nucleotide analogs and DNA. The interaction with nucleotide stabilizes the complex while DNA introduces conformational changes in the hexamer inducing a cylindrical shape. Our studies suggest that the assembly of GINS and Cdc45 to the hMCM2-7 hexamer would favor conformational changes on the hexamer bound to ssDNA shifting the cylindrical shape of the complex into a right-handed spiral conformation as observed in the CMG complex bound to DNA.     

The ATPase cycle mechanism of the DEAD-box rRNA helicase, DbpA

DEAD-box proteins are ATPase enzymes that destabilize and unwind duplex RNA. Quantitative knowledge of the ATPase cycle parameters is critical for developing models of helicase activity. However, limited information regarding the rate and equilibrium constants defining the ATPase cycle of RNA helicases is available, including the distribution of populated biochemical intermediates, the catalytic step(s) that limits the enzymatic reaction cycle, and how ATP utilization and RNA interactions are linked. We present a quantitative kinetic and equilibrium characterization of the ribosomal RNA (rRNA)-activated ATPase cycle mechanism of DbpA, a DEAD-box rRNA helicase implicated in ribosome biogenesis. rRNA activates the ATPase activity of DbpA by promoting a conformational change after ATP binding that is associated with hydrolysis. Chemical cleavage of bound ATP is reversible and occurs via a γ-phosphate attack mechanism. ADP-P_i and RNA binding display strong thermodynamic coupling, which causes DbpA-ADP-P_i to bind rRNA with > 10-fold higher affinity than with bound ATP, ADP or in the absence of nucleotide. The rRNA-activated steady-state ATPase cycle of DbpA is limited both by ATP hydrolysis and by P_i release, which occur with comparable rates. Consequently, the predominantly populated biochemical states during steady-state cycling are the ATP- and ADP-P_i-bound intermediates. Thermodynamic linkage analysis of the ATPase cycle transitions favors a model in which rRNA duplex destabilization is linked to strong rRNA and nucleotide binding. The presented analysis of the DbpA ATPase cycle reaction mechanism provides a rigorous kinetic and thermodynamic foundation for developing testable hypotheses regarding the functions and molecular mechanisms of DEAD-box helicases.     

Pumps, paradoxes and ploughshares: mechanism of the MCM2-7 DNA helicase

In eukaryotes, numerous lines of evidence have coalesced into a convincing case that the MCM2-7 complex - a heterohexameric ATPase - is the replicative DNA helicase. However, almost nothing is known about how this enzyme functions in a cellular context. Some models for the mechanism of the MCM2-7 helicase envision that it translocates along single-stranded DNA (ssDNA), whereas, more recently, it is has been suggested that it pumps double-stranded DNA (dsDNA) through its central channel. In particular, one model in which a double hexamer of MCM2-7 pumps dsDNA towards the hexamer interface and extrudes ssDNA laterally as a result of torsional strain is gaining popularity. Here, we discuss existing models and propose a new variation in which a single hexamer is the functional unit of the helicase. Duplex DNA is pumped into MCM2-7 and, as it emerges from the complex, a rigid protein that we term the 'ploughshare' splits the duplex.     

DNA binding and helicase actions of mouse MCM4/6/7 helicase

Helicases play central roles in initiation and elongation of DNA replication. We previously reported that helicase and ATPase activities of the mammalian Mcm4/6/7 complex are activated specifically by thymine-rich single-stranded DNA. Here, we examined its substrate preference and helicase actions using various synthetic DNAs. On a bubble substrate, Mcm4/6/7 makes symmetric dual contacts with the 5′-proximal 25 nt single-stranded segments adjacent to the branch points, presumably generating double hexamers. Loss of thymine residues from one single-strand results in significant decrease of unwinding efficacy, suggesting that concurrent bidirectional unwinding by a single double hexameric Mcm4/6/7 may play a role in efficient unwinding of the bubble. Mcm4/6/7 binds and unwinds various fork and extension structures carrying a single-stranded 3′-tail DNA. The extent of helicase activation depends on the sequence context of the 3′-tail, and the maximum level is achieved by DNA with 50% or more thymine content. Strand displacement by Mcm4/6/7 is inhibited, as the GC content of the duplex region increases. Replacement of cytosine–guanine pairs with cytosine–inosine pairs in the duplex restored unwinding, suggesting that mammalian Mcm4/6/7 helicase has difficulties in unwinding stably base-paired duplex. Taken together, these findings reveal important features on activation and substrate preference of the eukaryotic replicative helicase.     

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.     

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.     

Multi-scale architecture of archaeal chromosomes

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Hexameric structures of the archaeal secretion ATPase GspE and implications for a universal secretion mechanism

The secretion superfamily ATPases are conserved motors in key microbial membrane transport and filament assembly machineries, including bacterial type II and IV secretion, type IV pilus assembly, natural competence, and archaeal flagellae assembly. We report here crystal structures and small angle X-ray scattering (SAXS) solution analyses of the Archaeoglobus fulgidus secretion superfamily ATPase, afGspE. AfGspE structures in complex with ATP analogue AMP-PNP and Mg(2+) reveal for the first time, alternating open and closed subunit conformations within a hexameric ring. The closed-form active site with bound Mg(2+) evidently reveals the catalytically active conformation. Furthermore, nucleotide binding results and SAXS analyses of ADP, ATPgammaS, ADP-Vi, and AMP-PNP-bound states in solution showed that asymmetric assembly involves ADP binding, but clamped closed conformations depend on both ATP gamma-phosphate and Mg(2+) plus the conserved motifs, arginine fingers, and subdomains of the secretion ATPase superfamily. Moreover, protruding N-terminal domain shifts caused by the closed conformation suggest a unified piston-like, push-pull mechanism for ATP hydrolysis-dependent conformational changes, suitable to drive diverse microbial secretion and assembly processes by a universal mechanism.