The Methanobacterium thermoautotrophicum MCM protein can form heptameric rings

Mini-chromosome maintenance (MCM) proteins form a conserved family found in all eukaryotes and are essential for DNA replication. They exist as heteromultimeric complexes containing as many as six different proteins. These complexes are believed to be the replicative helicases, functioning as hexameric rings at replication forks. In most archaea a single MCM protein exists. The protein from Methanobacterium thermoautotrophicum (mtMCM) has been reported to assemble into a large complex consistent with a dodecamer. We show that mtMCM can assemble into a heptameric ring. This ring contains a C-terminal helicase domain that can be fit with crystal structures of ring helicases and an N-terminal domain of unknown function. While the structure of the ring is very similar to that of hexameric replicative helicases such as bacteriophage T7 gp4, our results show that such ring structures may not be constrained to have only six subunits.     

Hexameric ring structure of human MCM10 DNA replication factor

The DNA replication factor minichromosome maintenance 10 (MCM10) is a conserved, abundant nuclear protein crucial for origin firing. During the transition from pre-replicative complexes to pre-initiation complexes, MCM10 recruitment to replication origins is required to provide a physical link between the MCM2-7 complex DNA helicase and DNA polymerases. Here, we report the molecular structure of human MCM10 as determined by electron microscopy and single-particle analysis. The MCM10 molecule is a ring-shaped hexamer with large central and smaller lateral channels and a system of inner chambers. This structure, together with biochemical data, suggests that this important protein uses its architecture to provide a docking module for assembly of the molecular machinery required for eukaryotic DNA replication.     

Nearest neighbour analysis of MCM protein complexes in Drosophila melanogaster

The MCM proteins are a group of six proteins whose action is vital for DNA replication in eukaryotes. It has been suggested that they constitute the replicative helicase, with a subset of the proteins forming the catalytic helicase (MCM4,6,7) while the others have a loading or control function. In this paper we show that all six MCM proteins are present in equivalent amounts in soluble extracts and on chromatin. We have also analysed soluble and chromatin-associated MCM protein complexes under different conditions. This suggests that all six MCM proteins are always found in a complex with each other, although the interaction between the individual MCM proteins is not equivalent as stringent salt conditions are able to break the intact complex into a number of stable subcomplexes. These data contribute to the ongoing debate about the nature of MCM complexes, supporting the hypothesis that they act as a heterohexamer rather than as a number of different subcomplexes. Finally, using protein–protein cross-linking we have shown that MCM2 interacts directly with MCM5 and MCM6; MCM5 with MCM3 and MCM2; and MCM6 with MCM2 and MCM4. This provides the first direct information about specific subunit contacts in the MCM complex.     

Structural polymorphism of Methanothermobacter thermautotrophicus MCM

The minichromosome maintenance (MCM) proteins are essential for replication initiation and elongation in eukarya and archaea. There are six MCM proteins in eukaryotes, and MCM complexes are believed to unwind DNA during chromosomal DNA replication. However, the mechanism and structure of the MCM complexes are not known. Only one MCM is found in the archaeon Methanothermobacter thermautotrophicus (mtMCM), and this provides a simpler system for study. The crystal structure of a mtMCM N-terminal fragment has been solved, but surprisingly only subtle structural changes were seen between the wild-type protein and one having a mutation corresponding to the yeast MCM5 bob1 mutation. The bob1 mutation bypasses the phosphorylation required for activation of MCM in yeast. We have used electron microscopy and three-dimensional reconstruction to examine a number of different fragments of mtMCM, and can visualize a large conformational change within the N-terminal fragment. This offers new insight into the conformational dynamics of MCM and the phosphorylation-bypass phenotype in yeast.     

Licensing of DNA replication by a multi-protein complex of MCM/P1 proteins in Xenopus eggs.

In eukaryotes, chromosomal DNA is licensed for a single round of replication in each cell cycle. Xenopus MCM3 protein has been implicated in the licensing of replication in egg extract. We have cloned cDNAs encoding five immunologically distinct proteins associated with Xenopus MCM3 as members of the MCM/P1 family. Six Xenopus MCM proteins formed a physical complex in the egg extract, bound to unreplicated chromatin before the formation of nuclei, and apparently displaced from replicated chromatin. The requirement of six XMCM proteins for the replication activity of the egg extract before nuclear formation suggests that their re-association with replicated chromatin at the end of the mitotic cell cycle is a key step for the licensing of replication.     

The 1.8-Å Crystal Structure of the N-Terminal Domain of an Archaeal MCM as a Right-Handed Filament

Mini-chromosome maintenance (MCM) proteins are the replicative helicase necessary for DNA replication in both eukarya and archaea. Most of archaea only have one MCM gene. Here, we report a 1.8-Å crystal structure of the N-terminal MCM from the archaeon Thermoplasma acidophilum (tapMCM). In the structure, the MCM N-terminus forms a right-handed filament that contains six subunits in each turn, with a diameter of 25 Å of the central channel opening. The inner surface is highly positively charged, indicating DNA binding. This filament structure with six subunits per turn may also suggests a potential role for an open-ring structure for hexameric MCM and dynamic conformational changes in initiation and elongation stages of DNA replication.     

Functional conservation of beta-hairpin DNA binding domains in the Mcm protein of Methanobacterium thermoautotrophicum and the Mcm5 protein of Saccharomyces cerevisiae

Mcm proteins are an important family of evolutionarily conserved helicases required for DNA replication in eukaryotes. The eukaryotic Mcm complex consists of six paralogs that form a heterohexameric ring. Because the intact Mcm2-7 hexamer is inactive in vitro, it has been difficult to determine the precise function of the different subunits. The solved atomic structure of an archaeal minichromosome maintenance (MCM) homolog provides insight into the function of eukaryotic Mcm proteins. The N-terminal positively charged central channel in the archaeal molecule consists of beta-hairpin domains essential for DNA binding in vitro. Eukaryotic Mcm proteins also have beta-hairpin domains, but their function is unknown. With the archaeal atomic structure as a guide, yeast molecular genetics was used to query the function of the beta-hairpin domains in vivo. A yeast mcm5 mutant with beta-hairpin mutations displays defects in the G1/S transition of the cell cycle, the initiation phase of DNA replication, and in the binding of the entire Mcm2-7 complex to replication origins. A similar mcm4 mutation is synthetically lethal with the mcm5 mutation. Therefore, in addition to its known regulatory role, Mcm5 protein has a positive role in origin binding, which requires coordination by all six Mcm2-7 subunits in the hexamer.     

Plant MCM proteins: role in DNA replication and beyond

Mini-chromosome maintenance (MCM) proteins form heterohexameric complex (MCM2–7) to serve as licensing factor for DNA replication to make sure that genomic DNA is replicated completely and accurately once during S phase in a single cell cycle. MCMs were initially identified in yeast for their role in plasmid replication or cell cycle progression. Each of six MCM contains highly conserved sequence called “MCM box”, which contains two ATPase consensus Walker A and Walker B motifs. Studies on MCM proteins showed that (a) the replication origins are licensed by stable binding of MCM2–7 to form pre-RC (pre-replicative complex) during G1 phase of the cell cycle, (b) the activation of MCM proteins by CDKs (cyclin-dependent kinases) and DDKs (Dbf4-dependent kinases) and their helicase activity are important for pre-RC to initiate the DNA replication, and (c) the release of MCMs from chromatin renders the origins “unlicensed”. DNA replication licensing in plant is, in general, less characterized. The MCMs have been reported from Arabidopsis, maize, tobacco, pea and rice, where they are found to be highly expressed in dividing tissues such as shoot apex and root tips, localized in nucleus and cytosol and play important role in DNA replication, megagametophyte and embryo development. The identification of six MCM coding genes from pea and Arabidopsis suggest six distinct classes of MCM protein in higher plant, and the conserved function right across the eukaryotes. This overview of MCMs contains an emphasis on MCMs from plants and the novel role of MCM6 in abiotic stress tolerance.     

Ancient diversification of eukaryotic MCM DNA replication proteins

Background  

Yeast and animal cells require six mini-chromosome maintenance proteins (Mcm2-7) for pre-replication complex formation, DNA replication initiation and DNA synthesis. These six individual MCM proteins form distinct heterogeneous subunits within a hexamer which is believed to form the replicative helicase and which associates with the essential but non-homologous Mcm10 protein during DNA replication. In contrast Archaea generally only possess one MCM homologue which forms a homohexameric MCM helicase. In some eukaryotes Mcm8 and Mcm9 paralogues also appear to be involved in DNA replication although their exact roles are unclear.     

Schizosaccharomyces pombe Mcm3p, an essential nuclear protein, associates tightly with Nda4p (Mcm5p).

MCM proteins are required for the proper regulation of DNA replication. There are six MCM proteins in all eukaryotes which interact to form a large complex. We report the cloning of fission yeast mcm3 +. mcm3 + is essential and spores carrying a Delta mcm3 disruption arrest with an apparently replicated DNA content. The protein is found constitutively in the nucleus and levels remain constant throughout the cell cycle. Mcm3p binds particularly tightly to Nda4p (Mcm5p), but is loosely associated with the other Schizosaccharomyces pombe MCM proteins. Thus, Mcm3p is a peripheral MCM subunit.     

Structural insight into how the human helicase subunit MCM2 may act as a histone chaperone together with ASF1 at the replication fork

MCM2 is a subunit of the replicative helicase machinery shown to interact with histones H3 and H4 during the replication process through its N-terminal domain. During replication, this interaction has been proposed to assist disassembly and assembly of nucleosomes on DNA. However, how this interaction participates in crosstalk with histone chaperones at the replication fork remains to be elucidated. Here, we solved the crystal structure of the ternary complex between the histone-binding domain of Mcm2 and the histones H3-H4 at 2.9 Å resolution. Histones H3 and H4 assemble as a tetramer in the crystal structure, but MCM2 interacts only with a single molecule of H3-H4. The latter interaction exploits binding surfaces that contact either DNA or H2B when H3-H4 dimers are incorporated in the nucleosome core particle. Upon binding of the ternary complex with the histone chaperone ASF1, the histone tetramer dissociates and both MCM2 and ASF1 interact simultaneously with the histones forming a 1:1:1:1 heteromeric complex. Thermodynamic analysis of the quaternary complex together with structural modeling support that ASF1 and MCM2 could form a chaperoning module for histones H3 and H4 protecting them from promiscuous interactions. This suggests an additional function for MCM2 outside its helicase function as a proper histone chaperone connected to the replication pathway.     

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.     

Transient association of MCM complex proteins with the nuclear matrix during initiation of mammalian DNA replication

The minichromosome maintenance complex (MCM2-7) is the putative DNA helicase in eukaryotes, and essential for DNA replication. By applying serial extractions to mammalian cells synchronized by release from quiescence, we reveal dynamic changes to the sub-nuclear compartmentalization of MCM2 as cells pass through late G1 and early S phase, identifying a brief window when MCM2 becomes transiently attached to the nuclear-matrix. The data distinguish 3 states that correspond to loose association with chromatin prior to DNA replication, transient highly stable binding to the nuclear-matrix coincident with initiation, and a post-initiation phase when MCM2 remains tightly associated with chromatin but not the nuclear-matrix. The data suggests that functional MCM complex loading takes place at the nuclear-matrix.     

Transient association of MCM complex proteins with the nuclear matrix during initiation of mammalian DNA replication

The minichromosome maintenance complex (MCM2-7) is the putative DNA helicase in eukaryotes, and essential for DNA replication. By applying serial extractions to mammalian cells synchronized by release from quiescence, we reveal dynamic changes to the sub-nuclear compartmentalization of MCM2 as cells pass through late G1 and early S phase, identifying a brief window when MCM2 becomes transiently attached to the nuclear-matrix. The data distinguish 3 states that correspond to loose association with chromatin prior to DNA replication, transient highly stable binding to the nuclear-matrix coincident with initiation, and a post-initiation phase when MCM2 remains tightly associated with chromatin but not the nuclear-matrix. The data suggests that functional MCM complex loading takes place at the nuclear-matrix.     

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.     

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.     

Minichromosome maintenance proteins interact with checkpoint and recombination proteins to promote s-phase genome stability

The minichromosome maintenance (MCM) complex plays essential, conserved roles throughout DNA synthesis: first, as a component of the prereplication complex at origins and, then, as a helicase associated with replication forks. Here we use fission yeast (Schizosaccharomyces pombe) as a model to demonstrate a role for the MCM complex in protecting replication fork structure and promoting recovery from replication arrest. Loss of MCM function generates lethal double-strand breaks at sites of DNA synthesis during replication elongation, suggesting replication fork collapse. MCM function also maintains the stability of forks stalled by hydroxyurea that activate the replication checkpoint. In cells where the checkpoint is activated, Mcm4 binds the Cds1 kinase and undergoes Cds1-dependent phosphorylation. MCM proteins also interact with proteins involved in homologous recombination, which promotes recovery from arrest by ensuring normal mitosis. We suggest that the MCM complex links replication fork stabilization with checkpoint arrest and recovery through direct interactions with checkpoint and recombination proteins and that this role in S-phase genome stability is conserved from yeast to human cells.     

Thermococcus kodakarensis encodes three MCM homologs but only one is essential

The minichromosome maintenance (MCM) complex is thought to function as the replicative helicase in archaea and eukaryotes. In eukaryotes, this complex is an assembly of six different but related polypeptides (MCM2-7) but, in most archaea, one MCM protein assembles to form a homohexameric complex. Atypically, the Thermococcus kodakarensis genome encodes three archaeal MCM homologs, here designated MCM1-3, although MCM1 and MCM2 are unusual in having long and unique N-terminal extensions. The results reported establish that MCM2 and MCM3 assemble into homohexamers and exhibit DNA binding, helicase and ATPase activities in vitro typical of archaeal MCMs. In contrast, MCM1 does not form homohexamers and although MCM1 binds DNA and has ATPase activity, it has only minimal helicase activity in vitro. Removal of the N-terminal extension had no detectable effects on MCM1 but increased the helicase activity of MCM2. A T. kodakarensis strain with the genes TK0096 (MCM1) and TK1361 (MCM2) deleted has been constructed that exhibits no detectable defects in growth or viability, but all attempts to delete TK1620 (MCM3) have been unsuccessful arguing that that MCM3 is essential and is likely the replicative helicase in T. kodakarensis. The origins and possible function(s) of the three MCM proteins are discussed.     

Pairwise interactions of the six human MCM protein subunits

The eukaryotic minichromosome maintenance (MCM) proteins have six subunits, Mcm2 to 7p. Together they play essential roles in the initiation and elongation of DNA replication, and the human MCM proteins present attractive targets for potential anticancer drugs. The six MCM subunits interact and form a ring-shaped heterohexameric complex containing one of each subunit in a variety of eukaryotes, and subcomplexes have also been observed. However, the architecture of the human MCM heterohexameric complex is still unknown. We systematically studied pairwise interactions of individual human MCM subunits by using the yeast two-hybrid system and in vivo protein-protein crosslinking with a non-cleavable crosslinker in human cells followed by co-immunoprecipitation. In the yeast two-hybrid assays, we revealed multiple binary interactions among the six human MCM proteins, and a subset of these interactions was also detected as direct interactions in human cells. Based on our results, we propose a model for the architecture of the human MCM protein heterohexameric complex. We also propose models for the structures of subcomplexes. Thus, this study may serve as a foundation for understanding the overall architecture and function of eukaryotic MCM protein complexes and as clues for developing anticancer drugs targeted to the human MCM proteins.