DnaB helicase activity is modulated by DNA geometry and force

The replicative helicase for Escherichia coli is DnaB, a hexameric, ring-shaped motor protein that encircles and translocates along ssDNA, unwinding dsDNA in advance of its motion. The microscopic mechanisms of DnaB are unknown; further, prior work has found that DnaB's activity is modified by other replication proteins, indicating some mechanistic flexibility. To investigate these issues, we quantified translocation and unwinding by single DnaB molecules in three tethered DNA geometries held under tension. Our data support the following conclusions: 1), Unwinding by DnaB is enhanced by force-induced destabilization of dsDNA. 2), The magnitude of this stimulation varies with the geometry of the tension applied to the DNA substrate, possibly due to interactions between the helicase and the occluded ssDNA strand. 3), DnaB unwinding and (to a lesser extent) translocation are interrupted by pauses, which are also dependent on force and DNA geometry. 4), DnaB moves slower when a large tension is applied to the helicase-bound strand, indicating that it must perform mechanical work to compact the strand against the applied force. Our results have implications for the molecular mechanisms of translocation and unwinding by DnaB and for the means of modulating DnaB activity.     

Structural basis for DNA strand separation by a hexameric replicative helicase

Hexameric helicases are processive DNA unwinding machines but how they engage with a replication fork during unwinding is unknown. Using electron microscopy and single particle analysis we determined structures of the intact hexameric helicase E1 from papillomavirus and two complexes of E1 bound to a DNA replication fork end-labelled with protein tags. By labelling a DNA replication fork with streptavidin (dsDNA end) and Fab (5′ ssDNA) we located the positions of these labels on the helicase surface, showing that at least 10 bp of dsDNA enter the E1 helicase via a side tunnel. In the currently accepted ‘steric exclusion’ model for dsDNA unwinding, the active 3′ ssDNA strand is pulled through a central tunnel of the helicase motor domain as the dsDNA strands are wedged apart outside the protein assembly. Our structural observations together with nuclease footprinting assays indicate otherwise: strand separation is taking place inside E1 in a chamber above the helicase domain and the 5′ passive ssDNA strands exits the assembly through a separate tunnel opposite to the dsDNA entry point. Our data therefore suggest an alternative to the current general model for DNA unwinding by hexameric helicases.     

DnaB drives DNA branch migration and dislodges proteins while encircling two DNA strands

DnaB is a ring-shaped, hexameric helicase that unwinds the E. coli DNA replication fork while encircling one DNA strand. This report demonstrates that DnaB can also encircle both DNA strands and then actively translocate along the duplex. With two strands positioned inside its central channel, DnaB translocates with sufficient force to displace proteins tightly bound to DNA with no resultant DNA unwinding. Thus, DnaB may clear proteins from chromosomal DNA. Furthermore, while encircling two DNA strands, DnaB can drive branch migration of a synthetic Holliday junction with heterologous duplex arms, suggesting that DnaB may be directly involved in DNA recombination in vivo. DnaB binds to just one DNA strand during branch migration. T7 phage gp4 protein also drives DNA branch migration, suggesting this activity generalizes to other ring-shaped helicases.     

Mcm4,6,7 uses a "pump in ring" mechanism to unwind DNA by steric exclusion and actively translocate along a duplex

Mcm4,6,7 is a ring-shaped heterohexamer and the putative eukaryotic replication fork helicase. In this study, we examine the mechanism of Mcm4,6,7. Mcm4,6,7 binds to only one strand of a duplex during unwinding, corresponding to the leading strand of a replication fork. Mcm4,6,7 unwinding stops at a nick in either strand. The Mcm4,6,7 ring also actively translocates along duplex DNA, enabling the protein to drive branch migration of Holliday junctions. The Mcm4,6,7 mechanism is very similar to DnaB, except the proteins translocate with opposite polarity along DNA. Mcm4,6,7 and DnaB have different structural folds and evolved independently; thus, the similarity in mechanism is surprising. We propose a "pump in ring" mechanism for both Mcm4,6,7 and DnaB, wherein a single-stranded DNA pump is situated within the central channel of the ring-shaped helicase, and unwinding is the result of steric exclusion. In this example of convergent evolution, the "pump in ring" mechanism was probably selected by eukaryotic and bacterial replication fork helicases in order to restrict unwinding to replication fork structures, stop unwinding when the replication fork encounters a nick, and actively translocate along duplex DNA to accomplish additional activities such as DNA branch migration.     

On translocation mechanism of ring-shaped helicase along single-stranded DNA

The ring-shaped helicases represent one important group of helicases that can translocate along single-stranded (ss) DNA and unwinding double-stranded (ds) DNA by using the energy derived from NTP binding and hydrolysis. Despite intensive studies, the mechanism by which the ring-shaped helicase translocates along ssDNA and unwinds dsDNA remains undetermined. In order to understand their chemomechanical-coupling mechanism, two models on NTPase activities of the hexamers in the presence of DNA have been studied here. One model is assumed that, of the six nucleotide-binding sites, three are noncatalytic and three are catalytic. The other model is assumed that all the six nucleotide-binding sites are catalytic. In terms of the sequential NTPase activity around the ring and the previous determined crystal structure of bacteriophage T7 helicase it is shown that the obtained mechanical behaviors such as the ssDNA-translocation size and DNA-unwinding size per dTTPase cycle using the former model are in good quantitative agreement with the previous experimental results for T7 helicase. Moreover, the acceleration of DNA unwinding rate with the stimulation of DNA synthesis by DNA polymerase can also be well explained by using the former model. In contrast, the ssDNA-translocation size and DNA-unwinding size per dTTPase cycle obtained by using the latter model are not consistent with the experimental results for T7 helicase. Thus it is preferred that the former model is the appropriate one for the T7 helicase. Furthermore, using the former model some dynamic behaviors such as the rotational speeds of DNA relative to the T7 helicase when translocation along ssDNA and when unwinding dsDNA have been predicted, which are expected to test in order to further verify the model.     

Single-molecule studies reveal dynamics of DNA unwinding by the ring-shaped T7 helicase

Helicases are molecular motors that separate DNA strands for efficient replication of genomes. We probed the kinetics of individual ring-shaped T7 helicase molecules as they unwound double-stranded DNA (dsDNA) or translocated on single-stranded DNA (ssDNA). A distinctive DNA sequence dependence was observed in the unwinding rate that correlated with the local DNA unzipping energy landscape. The unwinding rate increased approximately 10-fold (approaching the ssDNA translocation rate) when a destabilizing force on the DNA fork junction was increased from 5 to 11 pN. These observations reveal a fundamental difference between the mechanisms of ring-shaped and nonring-shaped helicases. The observed force-velocity and sequence dependence are not consistent with a simple passive unwinding model. However, an active unwinding model fully supports the data even though the helicase on its own does not unwind at its optimal rate. This work offers insights into possible ways helicase activity is enhanced by associated proteins.     

Mechanistic studies of the T4 DNA (gp41) replication helicase: functional interactions of the C-terminal Tails of the helicase subunits with the T4 (gp59) helicase loader protein

We compare the activities of the wild-type (gp41WT) and mutant (gp41delta C20) forms of the bacteriophage T4 replication helicase. In the gp41delta C20 mutant the helicase subunits have been genetically truncated to remove the 20 residue C-terminal tail peptide domains present in the wild-type enzyme. Here, we examine the interactions of these helicase forms with the T4 gp59 helicase loader and the gp32 single-stranded DNA binding proteins, both of which are physically and functionally coupled with the helicase in the T4 DNA replication complex. We show that the wild-type and mutant forms of the helicase do not differ in their ability to assemble into dimers and hexamers, nor in their interactions with gp61 (the T4 primase). However they do differ in their gp59-stimulated unwinding activities and in their abilities to translocate along a ssDNA strand that has been coated with gp32. We demonstrate that functional coupling between gp59 and gp41 involves direct interactions between the C-terminal tail peptides of the helicase subunits and the loading protein, and measure the energetics and kinetics of these interactions. This work helps to define a gp41-gp59 assembly pathway that involves an initial interaction between the C-terminal tails of the helicases and the gp59 loader proteins, followed by a conformational change of the helicase subunits that exposes new interaction surfaces, which can then be trapped by the gp59 protein. Our results suggest that the gp41-gp59 complex is then poised to bind ssDNA portions of the replication fork. We suggest that one of the important functions of gp59 may be to aid in the exposure of the ssDNA binding sites of the helicase subunits, which are otherwise masked and regulated by interactions with the helicase carboxy-terminal tail peptides.     

Model for helicase translocating along single-stranded DNA and unwinding double-stranded DNA

A model is proposed for non-hexameric helicases translocating along single-stranded (ss) DNA and unwinding double-stranded (ds) DNA. The translocation of a monomeric helicase along ssDNA in weakly-ssDNA-bound state is driven by the Stokes force that is resulted from the conformational change following the transition of the nucleotide state. The unwinding of dsDNA is resulted mainly from the bending of ssDNA induced by the strong binding force of helicase with dsDNA. The interaction force between ssDNA and helicases in weakly-ssDNA-bound state determines whether monomeric helicases such as PcrA can unwind dsDNA or dimeric helicases such as Rep are required to unwind dsDNA.     

T7 DNA helicase: a molecular motor that processively and unidirectionally translocates along single-stranded DNA

DNA helicases are molecular motors that use the energy from NTP hydrolysis to drive the process of duplex DNA strand separation. Here, we measure the translocation and energy coupling efficiency of a replicative DNA helicase from bacteriophage T7 that is a member of a class of helicases that assembles into ring-shaped hexamers. Presteady state kinetics of DNA-stimulated dTTP hydrolysis activity of T7 helicase were measured using a real time assay as a function of ssDNA length, which provided evidence for unidirectional translocation of T7 helicase along ssDNA. Global fitting of the kinetic data provided an average translocation rate of 132 bases per second per hexamer at 18 degrees C. While translocating along ssDNA, T7 helicase hydrolyzes dTTP at a rate of 49 dTTP per second per hexamer, which indicates that the energy from hydrolysis of one dTTP drives unidirectional movement of T7 helicase along two to three bases of ssDNA. One of the features that distinguishes this ring helicase is its processivity, which was determined to be 0.99996, which indicated that T7 helicase travels on an average about 75kb of ssDNA before dissociating. We propose that the ability of T7 helicase to translocate unidirectionally along ssDNA in an efficient manner plays a crucial role in DNA unwinding.     

Interaction of Rep and DnaB on DNA

Genome duplication requires not only unwinding of the template but also the displacement of proteins bound to the template, a function performed by replicative helicases located at the fork. However, accessory helicases are also needed since the replicative helicase stalls occasionally at nucleoprotein complexes. In Escherichia coli, the primary and accessory helicases DnaB and Rep translocate along the lagging and leading strand templates, respectively, interact physically and also display cooperativity in the unwinding of model forked DNA substrates. We demonstrate here that this cooperativity is displayed only by Rep and not by other tested helicases. ssDNA must be exposed on the leading strand template to elicit this cooperativity, indicating that forks blocked at protein-DNA complexes contain ssDNA ahead of the leading strand polymerase. However, stable Rep-DnaB complexes can form on linear as well as branched DNA, indicating that Rep has the capacity to interact with ssDNA on either the leading or the lagging strand template at forks. Inhibition of Rep binding to the lagging strand template by competition with SSB might therefore be critical in targeting accessory helicases to the leading strand template, indicating an important role for replisome architecture in promoting accessory helicase function at blocked replisomes.     

Dual functions of single-stranded DNA-binding protein in helicase loading at the bacteriophage T4 DNA replication fork

Semi-conservative DNA synthesis reactions catalyzed by the bacteriophage T4 DNA polymerase holoenzyme are initiated by a strand displacement mechanism requiring gp32, the T4 single-stranded DNA (ssDNA)-binding protein, to sequester the displaced strand. After initiation, DNA helicase acquisition by the nascent replication fork leads to a dramatic increase in the rate and processivity of leading strand DNA synthesis. In vitro studies have established that either of two T4-encoded DNA helicases, gp41 or dda, is capable of stimulating strand displacement synthesis. The acquisition of either helicase by the nascent replication fork is modulated by other protein components of the fork including gp32 and, in the case of the gp41 helicase, its mediator/loading protein gp59. Here, we examine the relationships between gp32 and the gp41/gp59 and dda helicase systems, respectively, during T4 replication using altered forms of gp32 defective in either protein-protein or protein-ssDNA interactions. We show that optimal stimulation of DNA synthesis by gp41/gp59 helicase requires gp32-gp59 interactions and is strongly dependent on the stability of ssDNA binding by gp32. Fluorescence assays demonstrate that gp59 binds stoichiometrically to forked DNA molecules; however, gp59-forked DNA complexes are destabilized via protein-protein interactions with the C-terminal "A-domain" fragment of gp32. These and previously published results suggest a model in which a mobile gp59-gp32 cluster bound to lagging strand ssDNA is the target for gp41 helicase assembly. In contrast, stimulation of DNA synthesis by dda helicase requires direct gp32-dda protein-protein interactions and is relatively unaffected by mutations in gp32 that destabilize its ssDNA binding activity. The latter data support a model in which protein-protein interactions with gp32 maintain dda in a proper active state for translocation at the replication fork. The relationship between dda and gp32 proteins in T4 replication appears similar to the relationship observed between the UL9 helicase and ICP8 ssDNA-binding protein in herpesvirus replication.     

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.     

Unzipping mechanism of the double-stranded DNA unwinding by a hexameric helicase: the effect of the 3' arm and the stability of the dsDNA on the unwinding activity of the Escherichia coli DnaB helicase

The effect of two structural elements of a replication DNA fork substrate, the length of the 3' arm of the fork and the stability of the double-stranded DNA (dsDNA) part, on the kinetics of the dsDNA unwinding by the Escherichia coli hexameric helicase DnaB protein has been examined under single turnover conditions using the rapid quench-flow technique. The length of the 3' arm of the replication fork, i.e. the number of nucleotides in the arm, is a major structural factor that controls the unwinding rate and processivity of the helicase. The data show the existence of an optimal length of the 3' arm where there is the highest unwinding rate and processivity, indicating that during the unwinding process, the helicase transiently interacts with the 3' arm at a specific distance on the arm with respect to the duplex part of the DNA. Moreover, the area on the enzyme that engages in interactions has also a discrete size. For DNA substrates with the 3' arm containing 14, or less, nucleotide residues, the DnaB helicase becomes a completely distributive enzyme. However, the 3' arm is not a "specific activating cofactor" in the unwinding reaction. Rather, the 3' arm plays a role as a mechanical fulcrum for the enzyme, necessary to provide support for the advancing large helicase molecule on the opposite strand of the DNA. Binding of ATP is necessary to engage the 3' arm with the DnaB helicase, but it does not change the initial distribution of complexes of the enzyme with the DNA fork substrate. Stability of the dsDNA has a significant effect on the unwinding rate and processivity. The unwinding rate constant is a decreasing linear function of the fractional content of GC base-pairs in the dsDNA, indicating that the activation of the unwinding step is proportional to the stability of the nucleic acid.     

Flexibility of the rings: structural asymmetry in the DnaB hexameric helicase

DnaB is the primary replicative helicase in Escherichia coli and the hexameric DnaB ring has previously been shown to exist in two states in the presence of nucleotides. In one, all subunits are equivalent, while in the other, there are two different subunit conformations resulting in a trimer of dimers. Under all conditions that we have used for electron microscopy, including the absence of nucleotide, some rings exist as trimers of dimers, showing that the symmetry of the DnaB hexamer can be broken prior to nucleotide binding. Three-dimensional reconstructions reveal that the N-terminal domain of DnaB makes two very different contacts with neighboring subunits in the trimer of dimers, but does not form a predicted dimer with a neighboring N-terminal domain. Within the trimer of dimers, the helicase domain exists in two alternate conformations, each of which can form symmetrical hexamers depending upon the nucleotide cofactor used. These results provide new information about the modular architecture and domain dynamics of helicases, and suggest, by comparison with the hexameric bacteriophage T7 gp4 and SV40 large T-antigen helicases, that a great structural and mechanistic diversity may exist among the hexameric helicases.     

大肠杆菌RecQ解旋酶的表达纯化和生物学活性研究

RecQ家族解旋酶是DNA解旋酶中高度保守的一个重要家族,在维持染色体的稳定性中起着重要的作用.人类RecQ家族解旋酶突变会导致几种与癌症有关的疾病.本研究旨在诱导大肠杆菌RecQ解旋酶体外表达,并应用生物化学和生物物理学技术研究大肠杆菌RecQ解旋酶的生物学活性. 体外诱导表达获得纯度达90% 以上并具有高活性的大肠杆菌重组RecQ解旋酶,其可溶性好;经生物学活性分析显示具有DNA结合活性、ATP依赖的DNA解链活性、DNA依赖的ATP酶活性. 较之双链DNA（dsDNA）,大肠杆菌RecQ解旋酶更容易与单链DNA(ssDNA)结合( P<0.01 ),但与长度不同的dsDNA的结合特性有差异(P<0.01)而与ssDNA没有差异(P>0.05);大肠杆菌RecQ解旋酶对3种dsDNA的解链速率不同(P<0.05);大肠杆菌RecQ解旋酶的ATP酶活性与辅助因子ssDNA长度也呈正相关(P<0.01). 这些研究结果将有助于阐明大肠杆菌RecQ解旋酶的分子作用机制,并为研究RecQ解旋酶家族其它成员的结构与功能提供帮助.     

Structure and primase-mediated activation of a bacterial dodecameric replicative helicase

Replicative helicases are essential ATPases that unwind DNA to initiate chromosomal replication. While bacterial replicative DnaB helicases are hexameric, Helicobacter pylori DnaB (HpDnaB) was found to form double hexamers, similar to some archaeal and eukaryotic replicative helicases. Here we present a structural and functional analysis of HpDnaB protein during primosome formation. The crystal structure of the HpDnaB at 6.7 Å resolution reveals a dodecameric organization consisting of two hexamers assembled via their N-terminal rings in a stack-twisted mode. Using fluorescence anisotropy we show that HpDnaB dodecamer interacts with single-stranded DNA in the presence of ATP but has a low DNA unwinding activity. Multi-angle light scattering and small angle X-ray scattering demonstrate that interaction with the DnaG primase helicase-binding domain dissociates the helicase dodecamer into single ringed primosomes. Functional assays on the proteins and associated complexes indicate that these single ringed primosomes are the most active form of the helicase for ATP hydrolysis, DNA binding and unwinding. These findings shed light onto an activation mechanism of HpDnaB by the primase that might be relevant in other bacteria and possibly other organisms exploiting dodecameric helicases for DNA replication.     

DNA helicases displace streptavidin from biotin-labeled oligonucleotides

Helicases are enzymes that use energy derived from nucleoside triphosphate hydrolysis to unwind double-stranded (ds) DNA, a process vital to virtually every phase of DNA metabolism. The helicases used in this study, gp41 and Dda, are from the bacteriophage T4, an excellent system for studying enzymes that process DNA. gp41 is the replicative helicase and has been shown to form a hexamer in the presence of ATP. In this study, protein cross-linking was performed in the presence of either linear or circular single-stranded (ss) DNA substrates to determine the topology of gp41 binding to ssDNA. Results indicate that the hexamer binds ssDNA by encircling it, in a manner similar to that of other hexameric helicases. A new assay was developed for studying enzymatic activity of gp41 and Dda on single-stranded DNA. The rate of dissociation of streptavidin from various biotinylated oligonucleotides was determined in the presence of helicase by an electrophoretic mobility shift assay. gp41 and Dda were found to significantly enhance the dissociation rate of streptavidin from biotin-labeled oligonucleotides in an ATP-dependent reaction. Helicase-catalyzed dissociation of streptavidin from the 3'-end of a biotin-labeled 62-mer oligonucleotide occurred with a first-order rate of 0.17 min-1, which is over 500-fold faster than the spontaneous dissociation rate of biotin from streptavidin. Dda activity leads to even faster displacement of streptavidin from the 3' end of the 62-mer, with a first-order rate of 7.9 s-1. This is more than a million-fold greater than the spontaneous dissociation rate. There was no enhancement of streptavidin dissociation from the 5'-biotin-labeled oligonucleotide by either helicase. The fact that each helicase was capable of dislodging streptavidin from the 3'-biotin label suggests that these enzymes are capable of imparting a force on a molecule blocking their path. The difference in displacement between the 5' and 3' ends of the oligonucleotide is also consistent with the possibility of a 5'-to-3' directional bias in translocation on ssDNA for each helicase.     

Crystal structure of the hexameric replicative helicase RepA of plasmid RSF1010

Unwinding of double-stranded DNA into single-stranded intermediates required for various fundamental life processes is catalyzed by helicases, a family of mono-, di- or hexameric motor proteins fueled by nucleoside triphosphate hydrolysis. The three-dimensional crystal structure of the hexameric helicase RepA encoded by plasmid RSF1010 has been determined by X-ray diffraction at 2.4 A resolution. The hexamer shows an annular structure with 6-fold rotational symmetry and a approximately 17 A wide central hole, suggesting that single-stranded DNA may be threaded during unwinding. Homologs of all five conserved sequence motifs of the DnaB-like helicase family are found in RepA, and the topography of the monomer resembles RecA and the helicase domain of the bacteriophage T7 gp4 protein. In a modeled complex, ATP molecules are located at the subunit interfaces and clearly define adenine-binding and ATPase catalytic sites formed by amino acid residues located on adjacent monomers; most remarkable is the "arginine finger" Arg207 contributing to the active site in the adjacent monomer. This arrangement of active-site residues suggests cooperativity between monomers in ATP hydrolysis and helicase activity of RepA. The mechanism of DNA unwinding remains elusive, as RepA is 6-fold symmetric, contrasting the recently published asymmetric structure of the bacteriophage T7 gp4 helicase domain.     

Substrate requirements for duplex DNA translocation by the eukaryal and archaeal minichromosome maintenance helicases

Replicative DNA helicases are ring-shaped hexamers that play an essential role in DNA synthesis by separating the two strands of chromosomal DNA to provide the single-stranded (ss) substrate for replicative polymerases. Biochemical and structural studies suggest that these helicases translocate along one strand of the duplex, which passes through and interacts with the central channel of these ring-shaped hexamers, and displace the complementary strand. A number of these helicases were shown to also encircle both strands simultaneously and then translocate along double-stranded (ds)DNA. In this report it is shown that the Schizosaccharomyces pombe Mcm4,6,7 complex and archaeal minichromosome maintenance (MCM) helicase from Methanothermobacter thermautotrophicus move along duplex DNA. These two helicases, however, differ in the substrate required to support dsDNA translocation. Although the S. pombe Mcm4,6,7 complex required a 3'-overhang ssDNA region to initiate its association with the duplex, the archaeal protein initiated its transit along dsDNA in the absence of a 3'-overhang region, as well. Furthermore, DNA substrates containing a streptavidin-biotin steric block inhibited the movement of the eukaryotic helicase along ss and dsDNAs but not of the archaeal enzyme. The M. thermautotrophicus MCM helicase, however, was shown to displace a streptavidin-biotin complex from ss, as well as dsDNAs. The possible roles of dsDNA translocation by the MCM proteins during the initiation and elongation phases of chromosomal replication are discussed.