Unwinding 20 Years of the Archaeal Minichromosome Maintenance Helicase

Replicative DNA helicases are essential cellular enzymes that unwind duplex DNA in front of the replication fork during chromosomal DNA replication. Replicative helicases were discovered, beginning in the 1970s, in bacteria, bacteriophages, viruses, and eukarya, and, in the mid-1990s, in archaea. This year marks the 20th anniversary of the first report on the archaeal replicative helicase, the minichromosome maintenance (MCM) protein.

ABSTRACT

Replicative DNA helicases are essential cellular enzymes that unwind duplex DNA in front of the replication fork during chromosomal DNA replication. Replicative helicases were discovered, beginning in the 1970s, in bacteria, bacteriophages, viruses, and eukarya, and, in the mid-1990s, in archaea. This year marks the 20th anniversary of the first report on the archaeal replicative helicase, the minichromosome maintenance (MCM) protein. This minireview summarizes 2 decades of work on the archaeal MCM.

INTRODUCTION

In 1996, the complete genome of the first archaeon, Methanocaldococcus jannaschii (named Methanococcus jannaschii at the time) was published (1). Since then, many aspects of archaeal biology and physiology have been studied. Because many archaeal species are extremophiles, some of these studies focused on the biotechnological applications of archaea and archaeal enzymes (e.g., PCR, molecular cloning, and environmental remediation), while others concentrated on exploring the similarities and differences between Archaea and the other two domains, Bacteria and Eukarya, with respect to physiology and cellular processes. Figure 1 summarizes the timeline of research on the archaeal minichromosome maintenance (MCM) helicase.

FIG 1.

Milestones of archaeal MCM helicase research. Blue, genetic studies; black, bioinformatics analysis; red, biochemical studies; green, structural studies.

Many of these studies focused on the archaeal DNA replication machinery both as a source for biotechnology reagents (e.g., thermostable DNA polymerases for PCR) and as a group of microorganisms with a unique replication process. When the complete genomes of several archaeal species were determined, bioinformatics studies suggested that although archaea are prokaryotes with a circular chromosome, like bacteria, their replication machinery is more similar to that of eukarya (Table 1) (for reviews on the archaeal replication machinery, see references 2 to 4). In the following years, biochemical, structural, and genetic studies demonstrated the relationship between the archaeal and eukaryal DNA replication machineries. These studies also revealed that although, in general, the archaeal replication process is more similar to that of eukarya, some aspects are more like those in bacteria, and others are specific for archaea (Table 1). For example, the replicative helicase in archaea, the MCM protein, is a homolog of the eukaryotic MCM but not the bacterial DnaB protein, and it translocates on DNA in the 3′ to 5′ direction as does the eukaryotic helicase. The bacterial DnaB translocates in the 5′ to 3′ direction (Table 2). Another example is the DNA sliding clamp. While the bacterial protein, the β-subunit of DNA polymerase III, forms homodimers (5), the eukaryotic and archaeal protein, proliferating cell nuclear antigen (PCNA), forms homotrimers (5, 6). It is worth noting, however, that all three clamps have similar three-dimensional structures and all have a pseudo 6-fold symmetry (7). However, some features of the replication machinery are specific for archaea, such as the archaeon-specific DNA polymerase D, found in some species as the only essential DNA polymerase (8) (Table 1).

TABLE 1.

A comparison of the common features of chromosomal DNA replication in Escherichia coli, Saccharomyces cerevisiae/Homo sapiens, and Euryarchaeota

Parameter	Value(s) fora:
	E. coli	S. cerevisiae/H. sapiens	Euryarchaea
Chromosome type	Circular	Linear	Circular
Replication origin	Single	Multiple	Single or multiple
Prereplication complex
Origin recognition	DnaA (1)	ORC (6)	Cdc6 (Orc1) (≥1)b
Helicase	DnaB (1)c	MCM (6)	MCM (1)
Helicase loader	DnaC (1)c	ORC (6), Cdc6 (1)	Cdc6 (Orc1) (≥1)b
Preinitiation complex
Cdc45		Cdc45 (1)	GAN (Cdc45, RecJ) (1)
GINS		GINS (4)	GINS (1–2)
CMG/GMG complexd		+	+
Single-stranded DNA binding protein	SSB (1)	RPA (3)	RPA (1–3)
Replisome assembly
Primase	DnaG (1)	Polα/primase (4)e,f	Primase (2)
Sliding clamp	β-Clamp (1)	PCNA (1)	PCNA (1)
Clamp loader	τ-Complex (5)	RFC (5)	RFC (2)
DNA polymerase
Leading strand	PolC (3)	Polε (4)f	PolB (1),g PolD (2)
Lagging strand	PolC (3)	Polδ (4)f	PolB (1),g PolD (2)
Okazaki fragment maturation
Primer removal	PolI (1)	Fen1 (1), Dna2 (1)	Fen1 (1)
Gap filling	PolI (1)	Polδ (4)	PolB (1),g PolD (2)
Ligation	NAD+ dependent (1)	ATP dependent (1)	ATP dependent (1)h

^aThe number of different proteins forming the active unit is shown in parentheses. The comparison includes the Euryarchaea as representative archaea. There are many lineages and kingdoms, each with a slightly different set of replication proteins. Bacterial or bacterium-like features are underlined; factors specific for archaea are shown in boldface; other data represent eukaryotic or eukaryote-like features. RPA, replication protein A; RFC, replication factor C.

^bThe genomes of species belonging to Methanococcales and Methanopyrales do not contain genes encoding Cdc6 (Orc1) homologs.

^cIn bacteria the helicase and helicase loader are considered to be part of not the prereplication complex but, rather, the preinitiation complex. As this paper is about archaea, these proteins were included under prereplication complex.

^dThe archaeal CMG complex is also called GMG. The plus sign indicates the presence of the complex.

^ePolα/primase is a complex of four subunits that include polymerase and primase activity.

^fAll three replicative polymerases in eukarya (Polα, Polε, and Polδ) belong to family B.

^gIn some archaeal species, PolB is not essential for cell viability.

^hMost archaeal ligases use ATP, but some use NAD⁺ as a cofactor.

TABLE 2.

Comparison of the replicative helicases from the three domains of life

Characteristic	Bacteria	Eukarya	Archaea
Protein(s)	DnaB	MCM2 to MCM7	MCM
Essential for viability	Yes	Yes	Yes
Oligomeric structure	Homohexamer	Heterohexamer	Homododecamer
Direction of translocation on ssDNA	5′ to 3′	3′ to 5′	3′ to 5′
Additional factors required for activity in vitro	None	Cdc45 and GINSa	Noneb
In vitro processivity (bp)
Alone	400	0c	4,500
Replication complex	86,000	500	NDd
Binds to ssDNA and dsDNA	Yes	Yes	Yes
Translocates on ssDNA and dsDNA	Yes	Yes	Yes
Unwinds DNA-RNA hybrid	Yes	Yes	Yes

^aUnder some conditions, the eukaryotic MCM possesses in vitro activity on its own.

^bIn most species.

^cFor the MCM2-MCM7 complex.

^dND, not determined.

THE REPLICATIVE HELICASE OF BACTERIA AND EUKARYA

In bacteria, the replicative helicase is the DnaB protein, which forms a homohexameric ring with helicase activity and is essential for DNA replication and cell viability (reference 9 and references therein). In eukarya, the MCM protein is a family of six related proteins, MCM2 to MCM7, that are essential for chromosomal DNA replication (10–12). All six proteins belong to the AAA+ family of ATPases (ATPases associated with diverse cellular activities) and contain all the hallmarks of other members of the family (13, 14). Based on amino acid sequence analysis, the largest conserved portion of the six proteins is a region of about 300 amino acids that contains the domains involved in ATPase activity. A region of about 250 residues, N terminal to the catalytic part, is also conserved among the six eukaryotic MCM proteins. Outside these regions, the eukaryotic MCM proteins show no similarity with each other, and each contains long, diverse N- and C-terminal regions (15).

Although the eukaryotic proteins MCM2 to MCM7 contain all the elements of a DNA helicase, in vivo, the MCM2-MCM7 complex is tightly associated with two additional factors, the Cdc45 protein and the heterotetrameric GINS complex. Together, these form the CMG (Cdc45, MCM, and GINS) complex that functions as the replicative helicase in eukarya (10, 11). Each of the components of the CMG complex are essential for cell viability (Table 1).

ALL ARCHAEAL GENOMES ENCODE MCM HOMOLOGS

When the genome sequences of several archaeal species were analyzed, some proteins were annotated as putative helicases. Edgell and Doolittle were the first to recognize the presence of MCM homologs in the archaeal genomes (Fig. 1) (16). Subsequent studies showed that all archaeal species contain at least one homolog of an MCM protein (17), and this was suggested to function as the replicative helicase. The archaeal MCM proteins, however, are shorter than the eukaryotic enzymes. Most are about 650 amino acids in length and include a 250-residue N-terminal portion and an approximately 300-amino-acid catalytic region (Fig. 2). Both of these regions are similar to the eukaryotic proteins MCM2 to MCM7. The enzymes also contain ∼100-amino-acid C-terminal regions suggested to fold into a helix-turn-helix (HTH) motif (17, 18) (Fig. 2). The C-terminal region is thought to play a regulatory function (19, 20). In several archaeal species with multiple MCM homologs, some are longer than 650 amino acids. However, in the few cases in which the enzymes were studied, it was found that only the MCM proteins that are similar to all other archaeal MCMs are essential for cell viability (21, 22).

FIG 2.

Schematic representation of the archaeal MCM protein. The N-terminal region is responsible for DNA binding and protein multimerization, the AAA+ region is the catalytic portion, and the C-terminal region is unique to the archaeal MCM and is a predicted helix-turn-helix motif. The three major regions of the protein are shown at the top, and some of the structural motifs are shown at the bottom. aa, amino acid.

THE BIOCHEMICAL PROPERTIES OF THE ARCHAEAL MCM PROTEINS

The first report on the biochemical properties of the archaeal MCM was a talk given by James Chong, then a postdoc in Bruce Stillman’s laboratory, at the 1999 Cold Spring Harbor Laboratory Conference on Eukaryotic DNA Replication (15 to 19 September 1999, Cold Spring Harbor, NY). This presentation and subsequent publications from three groups focused on the initial characterization of the MCM protein from Methanothermobacter thermautotrophicus (then called Methanobacterium thermoautotrophicum ΔH) (23–25). These early studies showed that the protein is a 3′-to-5′ ATP-dependent DNA helicase, binds to single-stranded and double-stranded DNA (ssDNA and dsDNA, respectively), has a processivity of several hundred bases, and forms a homododecameric structure in solution (Table 2).

Research on the biochemical properties of the archaeal MCM proteins was expanded to enzymes from other species and kingdoms. These studies illuminated the diverse activities of the helicase, the role of specific residues and domains in MCM function, and factors involved in the regulation of helicase activity. The similarities and differences between MCM homologs from different species were also examined. These studies explored the processivity of the enzymes (26) and regions involved in DNA binding including the Zn finger motif (27) and the N-terminal portion (28). The studies also demonstrated the ability of the helicase to translocate along ssDNA and dsDNA (29), the ability to displace proteins from DNA during translocation (30), and the ability to displace RNA from DNA-RNA hybrid duplexes while translocating on the DNA strand (31) (Table 2). Many of these activities are consistent with MCM serving as the archaeal replicative helicase as they are shared by the eukaryotic MCM and the bacterial replicative helicase DnaB (32).

In eukarya, under most experimental conditions, the MCM helicase is not active on its own. Only the CMG complex possesses helicase activity, and the CMG complex is the active helicase in vivo (33, 34). The situation in archaea, however, is more complex. While most of the archaeal MCM proteins studied are active on their own (23), some require additional factors for appreciable helicase activity (35). And in some cases, opposite effects can be observed with the proteins from different species. For example, while the initiator protein Cdc6 (also referred to as Orc1) stimulates the in vitro helicase activity of MCM from some species (for example Thermoplasma acidophilum [35]), it inhibits the activity of others (for example, M. thermautotrophicus [36]). Another example of MCM-interacting enzymes that affect helicase activity is the MCM association with the archaeal GINS and GAN proteins (also referred to as the archaeal Cdc45 protein or RecJ). In some species the GMG (GAN, MCM, and GINS) complex (also referred to as the archaeal CMG) has no effect on helicase activity in vitro although all three components are present in all archaeal species (37). In other species, however, the complex stimulates helicase activity (38, 39).

Single-molecule analysis studies were also employed to determine the properties of the helicase. Single-molecule fluorescence resonance energy transfer (smFRET) studies identified the interactions between the MCM protein and the DNA substrate and showed that the helicase interacts better with a fork substrate than with a substrate with only a 3′ overhanging ssDNA region (40). The processivity of the helicase was also determined using a high-temperature single-molecule bead tether assay to study the speed and processivity of several archaeal enzymes. These studies revealed that, in vitro, archaeal MCMs from some species possess a processivity of several thousand bases without the need for accessory factors (Table 2) (26).

MCM STRUCTURE

The three-dimensional structures of the MCM proteins were determined using different techniques. The first observation on the structure of the MCM complex came from low-resolution size exclusion chromatography studies reported in the first few publications on the M. thermautotrophicus protein (23, 24). These studies suggested that, in solution, the helicase forms a double hexameric ring structure. This was exciting as it strongly suggested that the MCM protein is the replicative helicase. This stemmed from knowledge that the bacterial replicative helicase, DnaB, and the large tumor antigen (T-Ag) of simian virus 40 (SV40) are single polypeptides that form dodecameric rings that encircle DNA (reference 9 and references therein).

These observations were followed by electron microscopy (EM) studies of the full-length protein from M. thermautotrophicus. These studies showed that the protein can adopt different oligomeric structures depending on protein concentration and buffer conditions. These structures include hexamers, heptamers, octamers, dodecamers, open rings, and filaments (41, 42). Although the enzyme can form multiple structures, it was suggested that, at least in vitro, only the hexamers possess helicase activity (43). EM studies also showed that, when provided with long dsDNA, the DNA wraps around the hexameric ring (44). This wrapping was suggested to play a role during the initiation of replication.

The first high-resolution structures of the MCM were X-ray structures of the N-terminal portion of the M. thermautotrophicus protein (45, 46), followed by the N-terminal part of the protein from other species (47, 48) (Fig. 3). The structures revealed a hexameric arrangement, with each monomer folded into two distinct domains: domain A and domain B/C. The structures opened the door for detailed biochemical, functional, and structure-function studies of the different domains, regions, and residues of the N-terminal region. These studies elucidated the role of the N-terminal portion in MCM multimerization, ssDNA and dsDNA binding, and ATPase activity (28). The structures also revealed a loop, not identified by sequence analysis, that is highly conserved among archaeal and eukaryal MCM proteins. This loop was shown to play an important role in communication between the N-terminal DNA binding region and the ATPase activity of the catalytic portion (49).

FIG 3.

Structures of the archaeal MCM protein N-terminal regions. (A) Ribbon diagrams of Methanothermobacter thermautotrophicus (PDB code 1LTL), Sulfolobus solfataricus (PDB code 2VL6), Thermoplasma acidophilum (PDB code 4ME3), and Pyrococcus furiosus (PDB code 4POF) viewed from the N-terminal face. For M. thermautotrophicus and S. solfataricus, crystallographic symmetry was applied to reconstruct the hexamer, while for T. acidophilum the hexamer was constructed by superposition with the crystallized P. furiosus hexamer. (B) The same structures viewed from right of the N-terminal face. (C) Calculated solvent-accessible surfaces colored by electrostatic potential: blue, positively charged; red, negatively charged; white, neutral.

In addition, the solution structure of the N-terminal part of the protein was also determined using small-angle neutron scattering (SANS) and revealed a large movement of domain A with respect to the other domain (50).

The structures of the N-terminal portion were followed by an X-ray structure of the nearly full-length MCM protein from Sulfolobus solfataricus (51). This structure, although it did not include the entire protein and was of low resolution, was instrumental in advancing the research on the MCM proteins (52). As had been predicted by amino acid sequence analysis, the structure confirmed the presence of all conserved motifs found in other AAA+ proteins. However, several motifs not identified by sequence analysis were also observed. The structure revealed four β-hairpins per monomer, three located within the main channel and one on the exterior of the hexamer. Mutational analysis of the latter elucidated its role in DNA binding and helicase activity (52, 53). The structure of the full-length protein in the presence of ssDNA was also determined (54) (Fig. 4). The structure suggested that, like DnaB, the helicase moves with a step of two nucleotides per MCM subunit. A structure of a chimeric MCM protein that included the N-terminal portion of the S. solfataricus protein and the catalytic domains of Pyrococcus furiosus was also determined using X-ray crystallography (55).

FIG 4.

The structure of the full-length Sulfolobus solfataricus MCM protein in the presence of ssDNA. (A) Ribbon diagram (PDB code 6MII) viewed from the N-terminal face. The ssDNA molecule is shown in gray. (B) Calculated protein solvent-accessible surface colored by electrostatic potential viewed from the right of the N-terminal face. Two monomers are omitted to show the internal surface of the helicase channel. (C) Enlargement of the ssDNA (gray) within the helicase channel.

The solution structure of the full-length protein from M. thermautotrophicus was also determined using SANS (56) and suggested that all 12 AAA+ domains lie at approximately the same distance from the axis. The results also indicated that domain A of the N-terminal portion of each monomer is next to the AAA+ region for all 12 monomers.

GENETIC STUDIES

Two decades ago, the ability to study archaeal proteins in vivo was very limited due to the lack of robust genetic tools. This changed, however, when in the past decade genetic methods were developed for several archaeal species (57–60). Genetic studies show that all archaeal species depend on a single MCM protein for chromosomal replication. Here, archaea are similar to bacteria, where a single protein, DnaB, is multimerized to assemble the active helicase (Table 2). However, the archaeal helicase is biochemically and structurally similar to that of eukarya (Table 2).

Genetic tools were also used to identify proteins that interact with the MCM complex. For example, the Thermococcus kodakarensis MCM proteins were tagged in vivo, and interacting proteins were identified by protein complex purification followed by mass spectrometry analysis (61). Some of the proteins identified were known to be involved in DNA replication (e.g., DNA polymerase), while others are of unknown function. Only future studies will determine their role, if any, in DNA replication or other cellular processes and the roles of their interactions with MCM.

FUTURE DIRECTIONS

One of the outstanding questions regarding the MCM is how the hexameric ring is loaded onto DNA at the origin of replication. Although the initiator protein, Cdc6, was implicated in the assembly process (62, 63) the mechanism is not known, and several different processes were suggested (64). The newly developed single-molecule approaches may help in addressing this essential question in MCM function.

In the past several years, a large number of new archaeal species, lineages, groups, and supergroups have been identified (for examples, see references 65 and 66). Unfortunately, many of the newly identified organisms cannot be cultured, and the classification is based largely on metagenomics of environmental samples. Therefore, the organisms cannot be studied directly, but their DNA sequences can be used to express recombinant MCM homologs for in vitro analysis. It will be interesting to elucidate the structures and functions of these proteins and to determine their similarities to, and differences from, enzymes of other species.

To date, most of the studies on the archaeal MCM were in vitro or in vivo genetic studies involving tagged proteins and attempts to delete the gene(s) encoding the MCM from the genome. Few other types of in vivo studies have been reported. In the future, in vivo imaging studies of proteins in live cells could determine cellular location and kinetics (for examples, see reference 67). The development of tools for in vivo protein labeling for mesophilic and thermophilic organisms may enable the study of helicase activity and localization within the cell during the different stages of the cell cycle (68). Such tools may also help to determine if the MCM protein is needed only for DNA replication or for other cellular processes.

The studies of the replicative helicases of Archaea, Bacteria, and Eukarya illustrate the similarities and differences between the enzymes in the three domains (Table 2). However, while the DnaB proteins in bacteria and the MCM and CMG complexes in eukarya are quite similar between species, it was shown that archaeal MCM proteins are more diverse. This includes the requirement of additional factors for activity and the mechanisms by which helicase activity is regulated. In addition, to date, most archaeal MCM proteins studied are from thermophilic organisms. It will be of interest to determine if MCM proteins from organisms growing in other extreme environments, such as psychrophiles, are similar to those from thermophiles. Although a great deal has been learned in the last 2 decades, much remains to be discovered about the archaeal replicative helicase.

Biographies

Lori M. Kelman is professor of biotechnology at Montgomery College, Germantown, MD. She received an A.B. in biochemistry from Mount Holyoke College, an M.S. in biology from St. John’s University, an M.B.A. in management from Iona College, and a Ph.D. in molecular biology from Cornell University. Prior to coming to Montgomery College, she was on the faculty of Iona College in New Rochelle, NY. She has performed research at the Rockefeller University, Memorial Sloan-Kettering Cancer Center, the National Institutes of Health, the National Institute of Standards and Technology, and the Institute for Bioscience and Biotechnology Research. She is editor of BIOS, a journal of undergraduate research and the journal of the Beta Beta Beta Biological Society.

William B. O’Dell was born in Newport, TN. He received a B.A. in college scholars honors (concentration, structural chemistry) from the University of Tennessee, Knoxville, in 2009. He completed a Ph.D. in biochemistry (with Flora Meilleur) at North Carolina State University in 2017 while conducting research in neutron protein crystallography in residence at the Neutron Sciences Directorate, Oak Ridge National Laboratory. In 2017, he was awarded a National Research Council Postdoctoral Associateship (with Zvi Kelman) to join the Biomolecular Structure and Function Group, Biomolecular Measurement Division, within the National Institute of Standards and Technology Materials Measurement Laboratory, where he works today as a biologist. He pursues his research interests in protein structure determination using neutron scattering methods and in biological consequences of deuterium isotopic labeling through affiliation with the Biomolecular Labeling Laboratory (BL²) of the Institute for Bioscience and Biotechnology Research.