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.     

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.     

MCM8 is an MCM2-7-related protein that functions as a DNA helicase during replication elongation and not initiation

MCM2-7 proteins are replication factors required to initiate DNA synthesis and are currently the best candidates for replicative helicases. We show that the MCM2-7-related protein MCM8 is required to efficiently replicate chromosomal DNA in Xenopus egg extracts. MCM8 does not associate with the soluble MCM2-7 complex and binds chromatin upon initiation of DNA synthesis. MCM8 depletion does not affect replication licensing or MCM3 loading but slows down DNA synthesis and reduces chromatin recruitment of RPA34 and DNA polymerase-alpha. Recombinant MCM8 displays both DNA helicase and ATPase activities in vitro. Reconstitution experiments show that ATP binding in MCM8 is required to rescue DNA synthesis in MCM8-depleted extracts. MCM8 colocalizes with replication foci and RPA34 on chromatin. We suggest that MCM8 functions in the elongation step of DNA replication as a helicase that facilitates the recruitment of RPA34 and stimulates the processivity of DNA polymerases at replication foci.     

MCM10 mediates RECQ4 association with MCM2‐7 helicase complex during DNA replication

Mutations in RECQ4, a member of the RecQ family of DNA helicases, have been linked to the progeroid disease Rothmund–Thomson Syndrome. Attempts to understand the complex phenotypes observed in recq4‐deficient cells suggest a potential involvement in DNA repair and replication, yet the molecular basis of the function of RECQ4 in these processes remains unknown. Here, we report the identification of a highly purified chromatin‐bound RECQ4 complex from human cell extracts. We found that essential replisome factors MCM10, MCM2‐7 helicase, CDC45 and GINS are the primary interaction partner proteins of human RECQ4. Importantly, complex formation and the association of RECQ4 with the replication origin are cell‐cycle regulated. Furthermore, we show that MCM10 is essential for the integrity of the RECQ4–MCM replicative helicase complex. MCM10 interacts directly with RECQ4 and regulates its DNA unwinding activity, and that this interaction may be modulated by cyclin‐dependent kinase phosphorylation. Thus, these studies show that RECQ4 is an integral component of the MCM replicative helicase complex participating in DNA replication in human cells.     

Differences in the single-stranded DNA binding activities of MCM2-7 and MCM467: MCM2 and MCM5 define a slow ATP-dependent step

The MCM2-7 complex, a hexamer containing six distinct and essential subunits, is postulated to be the eukaryotic replicative DNA helicase. Although all six subunits function at the replication fork, only a specific subcomplex consisting of the MCM4, 6, and 7 subunits (MCM467) and not the MCM2-7 complex exhibits DNA helicase activity in vitro. To understand why MCM2-7 lacks helicase activity and to address the possible function of the MCM2, 3, and 5 subunits, we have compared the biochemical properties of the Saccharomyces cerevisiae MCM2-7 and MCM467 complexes. We demonstrate that both complexes are toroidal and possess a similar ATP-dependent single-stranded DNA (ssDNA) binding activity, indicating that the lack of helicase activity by MCM2-7 is not due to ineffective ssDNA binding. We identify two important differences between them. MCM467 binds dsDNA better than MCM2-7. In addition, we find that the rate of MCM2-7/ssDNA association is slow compared with MCM467; the association rate can be dramatically increased either by preincubation with ATP or by inclusion of mutations that ablate the MCM2/5 active site. We propose that the DNA binding differences between MCM2-7 and MCM467 correspond to a conformational change at the MCM2/5 active site with putative regulatory significance.     

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.     

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.     

Interaction of heliquinomycin with single-stranded DNA inhibits MCM4/6/7 helicase

The antibiotic heliquinomycin inhibited cellular DNA replication at IC(50) of 2.5 μM without affecting level of chromatin-bound MCM4 and without activating the DNA replication stress checkpoint system, suggesting that heliquinomycin perturbs DNA replication mainly by inhibiting the activity of replicative DNA helicase that unwinds DNA duplex at replication forks. Among the DNA helicases involved in DNA replication, DNA helicase B was inhibited by heliquinomycin at IC(50) of 4.3 μM and RECQL4 helicase at IC(50) of 14 μM; these values are higher than that of MCM4/6/7 helicase (2.5 μM). These results suggest that heliquinomycin mainly targets actions of the replicative DNA helicases. Gel-retardation experiment indicates that heliquinomycin binds to single-stranded DNA. The single-stranded DNA-binding ability of MCM4/6/7 was affected in the presence of heliquinomycin. The data suggest that heliquinomycin inhibits the DNA helicase activity of MCM4/6/7 complex by stabilizing its interaction with single-stranded DNA.     

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.     

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.     

Localization of MCM2-7, Cdc45, and GINS to the site of DNA unwinding during eukaryotic DNA replication

Little is known about the architecture and biochemical composition of the eukaryotic DNA replication fork. To study this problem, we used biotin-streptavidin-modified plasmids to induce sequence-specific replication fork pausing in Xenopus egg extracts. Chromatin immunoprecipitation was employed to identify factors associated with the paused fork. This approach identifies DNA pol alpha, DNA pol delta, DNA pol varepsilon, MCM2-7, Cdc45, GINS, and Mcm10 as components of the vertebrate replisome. In the presence of the DNA polymerase inhibitor aphidicolin, which causes uncoupling of a highly processive DNA helicase from the stalled replisome, only Cdc45, GINS, and MCM2-7 are enriched at the pause site. The data suggest the existence of a large molecular machine, the "unwindosome," which separates DNA strands at the replication fork and contains Cdc45, GINS, and the MCM2-7 holocomplex.     

Phosphorylation of MCM4 at sites inactivating DNA helicase activity of the MCM4-MCM6-MCM7 complex during Epstein-Barr virus productive replication

Induction of Epstein-Barr virus (EBV) lytic replication blocks chromosomal DNA replication notwithstanding an S-phase-like cellular environment with high cyclin-dependent kinase (CDK) activity. We report here that the phosphorylated form of MCM4, a subunit of the MCM complex essential for chromosomal DNA replication, increases with progression of lytic replication, Thr-19 and Thr-110 being CDK2/CDK1 targets whose phosphorylation inactivates MCM4-MCM6-MCM7 (MCM4-6-7) complex-associated DNA helicase. Expression of EBV-encoded protein kinase (EBV-PK) in HeLa cells caused phosphorylation of these sites on MCM4, leading to cell growth arrest. In vitro, the sites of MCM4 of the MCM4-6-7 hexamer were confirmed to be phosphorylated with EBV-PK, with the same loss of helicase activity as with CDK2/cyclin A. Introducing mutations in the N-terminal six Ser and Thr residues of MCM4 reduced the inhibition by CDK2/cyclin A, while EBV-PK inhibited the helicase activities of both wild-type and mutant MCM4-6-7 hexamers, probably since EBV-PK can phosphorylate MCM6 and another site(s) of MCM4 in addition to the N-terminal residues. Therefore, phosphorylation of the MCM complex by redundant actions of CDK and EBV-PK during lytic replication might provide one mechanism to block chromosomal DNA replication in the infected cells through inactivation of DNA unwinding by the MCM4-6-7 complex.     

Visualization of the MCM DNA helicase at replication factories before the onset of DNA synthesis

In mammalian cells, DNA synthesis takes place at defined nuclear structures termed “replication foci” (RF) that follow the same order of activation in each cell cycle. Intriguingly, immunofluorescence studies have failed to visualize the DNA helicase minichromosome maintenance (MCM) at RF, raising doubts about its physical presence at the sites of DNA synthesis. We have revisited this paradox by pulse-labeling RF during the S phase and analyzing the localization of MCM at labeled DNA in the following cell cycle. Using high-throughput confocal microscopy, we provide direct evidence that MCM proteins concentrate in G1 at the chromosome structures bound to become RF in the S phase. Upon initiation of DNA synthesis, an active “MCM eviction” mechanism contributes to reduce the excess of DNA helicases at RF. Most MCM complexes are released from chromatin, except for a small but detectable fraction that remains at the forks during the S phase, as expected for a replicative helicase.     

The ORC/Cdc6/MCM2-7 complex facilitates MCM2-7 dimerization during prereplicative complex formation

The replicative mini-chromosome-maintenance 2–7 (MCM2-7) helicase is loaded in Saccharomyces cerevisiae and other eukaryotes as a head-to-head double-hexamer around origin DNA. At first, ORC/Cdc6 recruits with the help of Cdt1 a single MCM2-7 hexamer to form an ‘initial’ ORC/Cdc6/Cdt1/MCM2-7 complex. Then, on ATP hydrolysis and Cdt1 release, the ‘initial’ complex is transformed into an ORC/Cdc6/MCM2-7 (OCM) complex. However, it remains unclear how the OCM is subsequently converted into a MCM2-7 double-hexamer. Through analysis of MCM2-7 hexamer-interface mutants we discovered a complex competent for MCM2-7 dimerization. We demonstrate that these MCM2-7 mutants arrest during prereplicative complex (pre-RC) assembly after OCM formation, but before MCM2-7 double-hexamer assembly. Remarkably, only the OCM complex, but not the ‘initial’ ORC/Cdc6/Cdt1/MCM2-7 complex, is competent for MCM2-7 dimerization. The MCM2-7 dimer, in contrast to the MCM2-7 double-hexamer, interacts with ORC/Cdc6 and is salt-sensitive, classifying the arrested complex as a helicase-loading intermediate. Accordingly, we found that overexpression of the mutants cause cell-cycle arrest and dominant lethality. Our work identifies the OCM complex as competent for MCM2-7 dimerization, reveals MCM2-7 dimerization as a limiting step during pre-RC formation and defines critical mechanisms that explain how origins are licensed.     

Stepwise regulated chromatin assembly of MCM2-7 proteins

Acquisition of the competence to replicate requires the assembly of the MCM2-7 (minichromosome maintenance) protein complex onto pre-replicative chromatin, a step of the licensing reaction. This step is thought to occur through binding of a heterohexameric MCM complex containing the six related MCM subunits. Here we show that assembly of the MCM complex onto pre-replicative chromatin occurs through sequential stabilization of specific MCM subunits. Inhibition of licensing with 6-dimethylaminopurine results in chromatin containing specifically bound MCM4 and MCM6. A similar result was obtained by interference of the assembly reaction with an MCM3 antibody. The presence of chromatin-bound MCM intermediates was confirmed by reconstitution experiments in vitro with purified proteins and by the observation of an ordered association of MCM subunits with chromatin. These results indicate that the assembly of the MCM complex onto pre-replicative chromatin is regulated at the level of distinct subunits, suggesting an additional regulatory step in the formation of pre-replication complexes.     

Mechanochemistry of t7 DNA helicase

The bacteriophage T7 helicase is a ring-shaped hexameric motor protein that unwinds double-stranded DNA during DNA replication and recombination. To accomplish this it couples energy from the nucleotide hydrolysis cycle to translocate along one of the DNA strands. Here, we combine computational biology with new biochemical measurements to infer the following properties of the T7 helicase: (1) all hexameric subunits are catalytic; (2) the mechanical movement along the DNA strand is driven by the binding transition of nucleotide into the catalytic site; (3) hydrolysis is coordinated between adjacent subunits that bind DNA; (4) the hydrolysis step changes the affinity of a subunit for DNA allowing passage of DNA from one subunit to the next. We construct a numerical optimization scheme to analyze transient and steady-state biochemical measurements to determine the rate constants for the hydrolysis cycle and determine the flux distribution through the reaction network. We find that, under physiological and experimental conditions, there is no dominant pathway; rather there is a distribution of pathways that varies with the ambient conditions. Our analysis methods provide a systematic procedure to study kinetic pathways of multi-subunit, multi-state cooperative enzymes.     

Activation of the MCM helicase from the thermophilic archaeon,Thermoplasma acidophilum by interactions with GINS and Cdc6-2

In DNA replication studies, the mechanism for regulation of the various steps from initiation to elongation is a crucial subject to understand cell cycle control. The eukaryotic minichromosome maintenance (MCM) protein complex is recruited to the replication origin by Cdc6 and Cdt1 to form the pre-replication complex, and participates in forming the CMG complex formation with Cdc45 and GINS to work as the active helicase. Intriguingly, Thermoplasma acidophilum, as well as many other archaea, has only one Gins protein homolog, contrary to the heterotetramer of the eukaryotic GINS made of four different proteins. The Gins51 protein reportedly forms a homotetramer (TaGINS) and physically interacts with TaMCM. In addition, TaCdc6-2, one of the two Cdc6/Orc1 homologs in T. acidophilum reportedly stimulates the ATPase and helicase activities of TaMCM in vitro. Here, we found a reaction condition, in which TaGINS stimulated the ATPase and helicase activities of TaMCM in a concentration dependent manner. Furthermore, the stimulation of the TaMCM helicase activity by TaGINS was enhanced by the addition of TaCdc6-2. A gel retardation assay revealed that TaMCM, TaGINS, and TaCdc6-2 form a complex on ssDNA. However, glutaraldehyde-crosslinking was necessary to detect the shifted band, indicating that the ternary complex of TaMCM–TaGINS–TaCdc6-2 is not stable in vitro. Immunoprecipitation experiment supported a weak interaction of these three proteins in vivo. Activation of the replicative helicase by a mechanism including a Cdc6-like protein suggests the divergent evolution after the division into Archaea and Eukarya.