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.     

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.     

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.     

Archaeal Hel308 domain V couples DNA binding to ATP hydrolysis and positions DNA for unwinding over the helicase ratchet

Hel308 and PolQ are paralogues with roles promoting genome stability in archaea and higher eukaryotes. The context in which they act is not clear, although Hel308 helicase from archaea may interact with abnormal replication forks. The atomic structure of archaeal Hel308 from Archaeoglobus fulgidus in complex with DNA was recently reported and has given insights into the mechanisms of superfamily-2 helicases generally. An intriguing aspect of the structure was the positioning of a C-terminal domain V relative to single-stranded DNA and to the helicase ratchet domain IV. We have mutagenised a triplet of arginine residues in domain V of archaeal Hel308 to assess the effects on DNA binding, unwinding, and ATPase activities. Our observations can now be interpreted in light of the atomic structure. We describe crucial roles for domain V as a brake on ATP hydrolysis by coupling it to binding single-stranded DNA and in positioning DNA relative to the helicase ratchet domain IV for efficient unwinding of forked DNA.     

Mechanism of DnaB helicase of Escherichia coli: structural domains involved in ATP hydrolysis, DNA binding, and oligomerization.

We describe the delineation of three distinct structural domains of the DnaB helicase of Escherichia coli: domain alpha, amino acid residues (aa) 1-156; domain beta, aa 157-302; and domain gamma, aa 303-471. Using mutants with deletion in these domains, we have examined their role(s) in hexamer formation, DNA-dependent ATPase, and DNA helicase activities. The mutant DnaBbetagamma protein, in which domain alpha was deleted, formed a hexamer; whereas the mutant DnaBalphabeta, in which domain gamma was deleted, could form only dimers. The dimerization of DnaBalphabeta was Mg(2+) dependent. These data suggest that the oligomerization of DnaB helicase involves at least two distinct protein-protein interaction sites; one of these sites is located primarily within domain beta (site 1), while the other interaction site is located within domain gamma (site 2). The mutant DnaBbeta, a polypeptide of 147 aa, where both domains alpha and gamma were deleted, displayed a completely functional ATPase activity. This domain, thus, constitutes the "central catalytic domain" for ATPase activity. The ATPase activity of DnaBalphabeta was kinetically comparable to that of DnaBbeta, indicating that domain alpha had little or no influence on the ATPase activity. In both cases, the ATPase activities were DNA independent. DnaBbetagamma had a DNA-dependent ATPase activity that was kinetically comparable to the ATPase activity of wild-type DnaB protein (wtDnaB), indicating a specific role for C-terminal domain gamma in enhancement of the ATPase activity of domain beta as well as in DNA binding. Mutant DnaBbetagamma, which lacked domain alpha, was devoid of any helicase activity pointing to a significant role for domain alpha. The major findings of this study are (i) domain beta contained a functional ATPase active site; (ii) domain gamma appeared to be the DNA binding domain and a positive regulator of the ATPase activity of domain beta; (iii) although domain alpha did not have any significant effect on the ATPase, DNA binding activities, or hexamer formation, it definitely plays a pivotal role in transducing the energy of ATP hydrolysis to DNA unwinding by the hexamer; and (iv) all three domains are required for helicase activity.     

The N-terminal domain of TWINKLE contributes to single-stranded DNA binding and DNA helicase activities

The TWINKLE protein is a hexameric DNA helicase required for replication of mitochondrial DNA. TWINKLE displays striking sequence similarity to the bacteriophage T7 gene 4 protein (gp4), which is a bi-functional primase-helicase required at the phage DNA replication fork. The N-terminal domain of human TWINKLE contains some of the characteristic sequence motifs found in the N-terminal primase domain of the T7 gp4, but other important motifs are missing. TWINKLE is not an active primase in vitro and the functional role of the N-terminal region has remained elusive. In this report, we demonstrate that the N-terminal part of TWINKLE is required for efficient binding to single-stranded DNA. Truncations of this region reduce DNA helicase activity and mitochondrial DNA replisome processivity. We also find that the gp4 and TWINKLE are functionally distinct. In contrast to the phage protein, TWINKLE binds to double-stranded DNA. Moreover, TWINKLE forms stable hexamers even in the absence of Mg²⁺ or NTPs, which suggests that an accessory protein, a helicase loader, is needed for loading of TWINKLE onto the circular mtDNA genome.     

Coupled ATP and DNA binding of adeno-associated virus Rep40 helicase

Adeno-associated virus 2 Rep40 helicase is involved in packaging single-stranded genomic DNA into virions. ATPase activity was stimulated 5-10-fold by DNA, depending upon assay conditions. The concentration dependence of Rep40 ATPase activity in the absence and presence of DNA indicates that the monomer is inactive and that the active enzyme is at least a dimer. Binding to oligonucleotides, examined by fluorescence anisotropy, was positively cooperative and required ATP or ATPgammaS; ADP and AMPPCP did not promote binding. The cooperativity and the nucleotide requirement were also demonstrated by surface plasmon resonance. Although the Rep40 behaves as a monomer in solution, it binds to DNA as an oligomer. The requirement of a nucleotide for DNA binding and the stimulation of ATPase activity by DNA indicate that the two processes are linked. Glutaraldehyde cross-linking generated a species that migrates as a trimer on sodium dodecyl sulfate (SDS) gel electrophoresis; ATPS promoted the formation of this species and higher order oligomers. The predominant cross-linked species was a trimer in the absence of ATPgammaS, regardless of whether duplex or single-stranded DNA was present. In the presence of duplex or single-stranded DNA and ATPgammaS, glutaraldehyde cross-linking generated a species that behaved as a dimer on SDS gel elctrophoresis. Sucrose-gradient velocity sedimentation of Rep40 gave an S20,w of 3 in the absence of ligands or in the presence of a 26 bp duplex DNA. The S20,w was 3.5 in the presence of ATPgammaS and 7 and 7.6 in the presence of DNA and ATPgammaS.     

Distinct roles for ATP binding and hydrolysis at individual subunits of an archaeal clamp loader

Circular clamps are utilised by replicative polymerases to enhance processivity. The topological problem of loading a toroidal clamp onto DNA is overcome by ATP-dependent clamp loader complexes. Different organisms use related protein machines to load clamps, but the mechanisms by which they utilise ATP are surprisingly different. Using mutant clamp loaders that are deficient in either ATP binding or hydrolysis in different subunits, we show how the different subunits of an archaeal clamp loader use ATP binding and hydrolysis in distinct ways at different steps in the loading process. Binding of nucleotide by the large subunit and three of the four small subunits is sufficient for clamp loading. However, ATP hydrolysis by the small subunits is required for release of PCNA to allow formation of the complex between PCNA and the polymerase, while hydrolysis by the large subunit is required for catalytic clamp loading.     

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     

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.     

ATP hydrolysis and DNA binding by the Escherichia coli RecF protein.

The Escherichia coli RecF protein possesses a weak ATP hydrolytic activity. ATP hydrolysis leads to RecF dissociation from double-stranded (ds)DNA. The RecF protein is subject to precipitation and an accompanying inactivation in vitro when not bound to DNA. A mutant RecF protein that can bind but cannot hydrolyze ATP (RecF K36R) does not readily dissociate from dsDNA in the presence of ATP. This is in contrast to the limited dsDNA binding observed for wild-type RecF protein in the presence of ATP but is similar to dsDNA binding by wild-type RecF binding in the presence of the nonhydrolyzable ATP analog, adenosine 5'-O-(3-thio)triphosphate (ATPgammaS). In addition, wild-type RecF protein binds tightly to dsDNA in the presence of ATP at low pH where its ATPase activity is blocked. A transfer of RecF protein from labeled to unlabeled dsDNA is observed in the presence of ATP but not ATPgammaS. The transfer is slowed considerably when the RecR protein is also present. In competition experiments, RecF protein appears to bind at random locations on dsDNA and exhibits no special affinity for single strand/double strand junctions when bound to gapped DNA. Possible roles for the ATPase activity of RecF in the regulation of recombinational DNA repair are discussed.     

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.     

Escherichia coli PriA protein, two modes of DNA binding and activation of ATP hydrolysis

Escherichia coli PriA protein plays crucial roles in processing of arrested replication forks. PriA serves as a sensor/stabilizer for an arrested replication fork and eventually promotes restart of DNA replication through assembly of a primosome. PriA carries a 3' terminus binding pocket required for its high affinity binding to a specific arrested fork as well as for its biological functions. We show here that PriA binds to DNA in a manner either dependent on or independent of 3' terminus recognition. The former mode of binding requires the 3' terminus binding pocket present at the N-terminal half of the 181-residue DNA binding domain and exhibits specific bipartite interaction on the template DNA. The latter mode is independent of the pocket function, but requires the C-terminal half of the same domain. ATP hydrolysis activity of PriA can be stimulated in vitro by either of the two binding modes. We propose architecture of PriA bound to various arrested replication fork structures and discuss its implication in helicase activation and ATP hydrolysis.     

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.     

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.     

Steric exclusion and wrapping of the excluded DNA strand occurs along discrete external binding paths during MCM helicase unwinding

The minichromosome maintenance (MCM) helicase complex is essential for the initiation and elongation of DNA replication in both the eukaryotic and archaeal domains. The archaeal homohexameric MCM helicase from Sulfolobus solfataricus serves as a model for understanding mechanisms of DNA unwinding. In this report, the displaced 5'-tail is shown to provide stability to the MCM complex on DNA and contribute to unwinding. Mutations in a positively charged patch on the exterior surface of the MCM hexamer destabilize this interaction, alter the path of the displaced 5'-tail DNA and reduce unwinding. DNA footprinting and single-molecule fluorescence experiments support a previously unrecognized wrapping of the 5'-tail. This mode of hexameric helicase DNA unwinding is termed the steric exclusion and wrapping (SEW) model, where the 3'-tail is encircled by the helicase while the displaced 5'-tail wraps around defined paths on the exterior of the helicase. The novel wrapping mechanism stabilizes the MCM complex in a positive unwinding mode, protects the displaced single-stranded DNA tail and prevents reannealing.     

Mutations altering the interplay between GkDnaC helicase and DNA reveal an insight into helicase unwinding

Replicative helicases are essential molecular machines that utilize energy derived from NTP hydrolysis to move along nucleic acids and to unwind double-stranded DNA (dsDNA). Our earlier crystal structure of the hexameric helicase from Geobacillus kaustophilus HTA426 (GkDnaC) in complex with single-stranded DNA (ssDNA) suggested several key residues responsible for DNA binding that likely play a role in DNA translocation during the unwinding process. Here, we demonstrated that the unwinding activities of mutants with substitutions at these key residues in GkDnaC are 2-4-fold higher than that of wild-type protein. We also observed the faster unwinding velocities in these mutants using single-molecule experiments. A partial loss in the interaction of helicase with ssDNA leads to an enhancement in helicase efficiency, while their ATPase activities remain unchanged. In strong contrast, adding accessory proteins (DnaG or DnaI) to GkDnaC helicase alters the ATPase, unwinding efficiency and the unwinding velocity of the helicase. It suggests that the unwinding velocity of helicase could be modulated by two different pathways, the efficiency of ATP hydrolysis or protein-DNA interaction.