The Hexameric Helicase DnaB Adopts a Nonplanar Conformation during Translocation (original) (raw)
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Unraveling DNA helicases. Motif, structure, mechanism and function (vol 271, pg 1849, 2004)
DNA helicases are molecular ÔmotorÕ enzymes that use the energy of NTP hydrolysis to separate transiently energetically stable duplex DNA into single strands. They are therefore essential in nearly all DNA metabolic transactions. They act as essential molecular tools for the cellular machinery. Since the discovery of the first DNA helicase in Escherichia coli in 1976, several have been isolated from both prokaryotic and eukaryotic systems. DNA helicases generally bind to ssDNA or ssDNA/dsDNA junctions and translocate mainly unidirectionally along the bound strand and disrupt the hydrogen bonds between the duplexes. Most helicases contain conserved motifs which act as an engine to drive DNA unwinding. Crystal structures have revealed an underlying common structural fold for their function. These structures suggest the role of the helicase motifs in catalytic function and offer clues as to how these proteins can translocate and unwind DNA. The genes containing helicase motifs may have evolved from a common ancestor. In this review we cover the conserved motifs, structural information, mechanism of DNA unwinding and translocation, and functional aspects of DNA helicases.
Unraveling DNA helicases. Motif, structure, mechanism and function
European Journal of Biochemistry, 2004
DNA helicases are molecular ÔmotorÕ enzymes that use the energy of NTP hydrolysis to separate transiently energetically stable duplex DNA into single strands. They are therefore essential in nearly all DNA metabolic transactions. They act as essential molecular tools for the cellular machinery. Since the discovery of the first DNA helicase in Escherichia coli in 1976, several have been isolated from both prokaryotic and eukaryotic systems. DNA helicases generally bind to ssDNA or ssDNA/dsDNA junctions and translocate mainly unidirectionally along the bound strand and disrupt the hydrogen bonds between the duplexes. Most helicases contain conserved motifs which act as an engine to drive DNA unwinding. Crystal structures have revealed an underlying common structural fold for their function. These structures suggest the role of the helicase motifs in catalytic function and offer clues as to how these proteins can translocate and unwind DNA. The genes containing helicase motifs may have evolved from a common ancestor. In this review we cover the conserved motifs, structural information, mechanism of DNA unwinding and translocation, and functional aspects of DNA helicases.
Nucleic Acids Research, 2008
DNA helicases are motor proteins that play essential roles in DNA replication, repair and recombination. In the replicative hexameric helicase, the fundamental reaction is the unwinding of duplex DNA; however, our understanding of this function remains vague due to insufficient structural information. Here, we report two crystal structures of the DnaB-family replicative helicase from Geobacillus kaustophilus HTA426 (GkDnaC) in the apo-form and bound to single-stranded DNA (ssDNA). The GkDnaC-ssDNA complex structure reveals that three symmetrical basic grooves on the interior surface of the hexamer individually encircle ssDNA. The ssDNA-binding pockets in this structure are directed toward the N-terminal domain collar of the hexameric ring, thus orienting the ssDNA toward the DnaG primase to facilitate the synthesis of short RNA primers. These findings provide insight into the mechanism of ssDNA binding and provide a working model to establish a novel mechanism for DNA translocation at the replication fork.
Mechanism of DNA translocation in a replicative hexameric helicase
Nature, 2006
Information Methods Complex preparation A fragment encoding BPV1 E1 306-577 was generated by PCR amplification from an expression plasmid template kindly provided by Dr. Arne Stenlund (CSHL), and cloned into the BamHI and XHOI sites of pGEX-4T-1 (Amersham). The GST-fusion protein was expressed in E. coli and purified in a manner analogous to methods described previously for other E1 GSTfusion proteins 1. Following elution of E1 306-577 as a monomer from a Superdex 200 gel-filtration column, the protein was concentrated to 10 mg/mL in the presence of 5 mM ADP and 200 mM MgCl 2. Crystallization and data collection The sample was mixed with excess oligo-dT oligonucleotide (13-mer), and immediately used for crystallization trials. Crystals grew at 17º C by the hanging drop method with a well solution consisting of 100 mM KCl. Crystals grew as clusters, and were separated by cleavage with the tip of a needle. Single crystals were quickly swished through a solution consisting of 1:2 ethylene glycol:well solution and flash frozen in a stream of cold nitrogen gas (100 K) at beamline X26C of the NSLS for evaluation of diffraction. The most favorable sample was transferred to liquid nitrogen and moved to beamline X29 for data collection. For this triclinic crystal, data were collected in a 360º sweep of 0.5º oscillations. Data collection statistics are shown in Supplementary Table S2. Structure determination and refinement The structure was solved by molecular replacement with the program PHASER 2 by placing 12 copies of a hybrid search model consisting of the previously reported helicase domain of HPV18 E1 3 (pdb code: 1TUE) and the helicase domain of BPV E1 (Enemark, Takara & Joshua-Tor, unpublished structure of E1 368-577 refined to 1.65 Å). Density-modification of the molecular replacement phases by RESOLVE 4 including 12-fold NCS-averaging yielded an electrondensity map with obvious α-helices for the oligomerization domain (E1 310-377), and allowed the construction of a poly-alanine model for this region with the program O 5. The calculation of NCS operators for the two domains (oligomerization and helicase domains) from the preliminary model revealed significant differences. Multi-domain NCS averaging (2 sets of 12-fold averaging) with solvent flattening was carried out with the program DMMULTI to generate a fully traceable electron density map with side-chains included. The structure was refined with NCS restraints with the program CNS 6 with rebuilding of the structure within the program O. The structure had very strong difference density at each of the P-loop regions (Supplementary Fig. S6) and at the center of the channels (Supplementary Fig. S7). These differences were not modeled until all obvious model errors had been removed. ADP molecules were readily apparent in some of the subunits and were added to the model first. ADP parameters were obtained from the Hic-Up server 7. After further refinement, additional ADP molecules were added to the model. Oligonucleotides consisting of 6 dTs were apparent in the center of each hexamer with obvious phosphate groups. Oligo-dT molecules with both directions of polarity were constructed and refined. The polarity that yielded a lower Rfree value and fit the electron
Journal of Molecular Biology, 2002
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 8C. 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 75 kb 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.
Processive and Unidirectional Translocation of Monomeric UvsW Helicase on Single-Stranded DNA †
Biochemistry, 2009
UvsW protein from bacteriophage controls the transition from origin-dependent to originindependent initiation of replication through the unwinding of R-loops bound to the T4 origins of replication. UvsW has also been implicated through genetic and biochemical experiments to play a role in DNA repair processes such as replication fork regression and Holliday junction branch migration. UvsW is capable of unwinding a wide variety of substrates, many of which contain only duplex DNA without single-stranded regions. Based on this observation, it has been suggested that UvsW is a dsDNA translocase. In this work we examine the ability of UvsW to translocate on ssDNA. Kinetic analysis indicates that the rate of ATP hydrolysis is strongly dependent on the length of the ssDNA lattice, whereas the K M -DNA remains relatively constant, demonstrating that UvsW translocates on ssDNA in an ATP-dependent fashion. Experiments using streptavidin blocks or poly-dT sequences located at either end of the ssDNA substrate indicate that UvsW translocates in a 3' to 5' direction. Mutant competition and heparin trapping experiments reveal that UvsW is extremely processive during ATP-driven translocation with a half-life on the order of several minutes. Finally, functional assays provide evidence that UvsW is monomeric while translocating on ssDNA. The ability of UvsW to unwind DNA duplexes is likely to be mechanistically linked to its ability to processively translocate on ssDNA in a 3' to 5' unidirectional fashion.
PLOS One, 2009
Replication initiation is a crucial step in genome duplication and homohexameric DnaB helicase plays a central role in the replication initiation process by unwinding the duplex DNA and interacting with several other proteins during the process of replication. N-terminal domain of DnaB is critical for helicase activity and for DnaG primase interactions. We present here the crystal structure of the N-terminal domain (NTD) of H. pylori DnaB (HpDnaB) helicase at 2.2 Å resolution and compare the structural differences among helicases and correlate with the functional differences. The structural details of NTD suggest that the linker region between NTD and C-terminal helicase domain plays a vital role in accurate assembly of NTD dimers. The sequence analysis of the linker regions from several helicases reveals that they should form four helix bundles. We also report the characterization of H. pylori DnaG primase and study the helicase-primase interactions, where HpDnaG primase stimulates DNA unwinding activity of HpDnaB suggesting presence of helicase-primase cohort at the replication fork. The protein-protein interaction study of C-terminal domain of primase and different deletion constructs of helicase suggests that linker is essential for proper conformation of NTD to interact strongly with HpDnaG. The surface charge distribution on the primase binding surface of NTDs of various helicases suggests that DnaB-DnaG interaction and stability of the complex is most probably charge dependent. Structure of the linker and helicase-primase interactions indicate that HpDnaB differs greatly from E.coli DnaB despite both belong to gram negative bacteria.
DNA Structure Specificity Conferred on a Replicative Helicase by Its Loader
Journal of Biological Chemistry, 2009
Prokaryotic and eukaryotic replicative helicases can translocate along single-stranded and double-stranded DNA, with the central cavity of these multimeric ring helicases being able to accommodate both forms of DNA. Translocation by such helicases along single-stranded DNA results in the unwinding of forked DNA by steric exclusion and appears critical in unwinding of parental strands at the replication fork, whereas translocation over double-stranded DNA has no well-defined role. We have found that the accessory factor, DnaC, that promotes loading of the Escherichia coli replicative helicase DnaB onto singlestranded DNA may also act to confer DNA structure specificity on DnaB helicase. When present in excess, DnaC inhibits DnaB translocation over double-stranded DNA but not over singlestranded DNA. Inhibition of DnaB translocation over doublestranded DNA requires the ATP-bound form of DnaC, and this inhibition is relieved during translocation over single-stranded DNA indicating that stimulation of DnaC ATPase is responsible for this DNA structure specificity. These findings demonstrate that DnaC may provide the DNA structure specificity lacking in DnaB, limiting DnaB translocation to bona fide replication forks. The ability of other replicative helicases to translocate along single-stranded and double-stranded DNA raises the possibility that analogous regulatory mechanisms exist in other organisms.
Helicase-catalyzed DNA unwinding
The Journal of biological chemistry, 1993
DNA helicases are ubiquitous and multiple helicases have been identified in a number of prokaryotes and eukaryotes. Although it is clear that not all helicases function identically, many of these enzymes possess similar properties that appear to be of general importance for their mechanism of action. For example, the assembly states of most (possibly all) helicases are oligomeric. The prime consequence of an oligomeric helicase is that it possesses multiple DNA binding sites, a feature that is required for any "active" mechanism of DNA unwinding, since it enables a helicase to bind both ss- and duplex DNA or two strands of ss-DNA simultaneously at an unwinding fork. Modulation of the relative affinities of ss- versus duplex DNA for these multiple binding sites through ATP binding and hydrolysis, as has been observed for the E. coli Rep dimer, can provide a mechanism for translocation and processive unwinding of DNA. Along with studies of DNA unwinding, further understandin...
DNA synthesis provides the driving force to accelerate DNA unwinding by a helicase
Nature, 2005
Helicases are molecular motors that use the energy of nucleoside 5 0 -triphosphate (NTP) hydrolysis to translocate along a nucleic acid strand and catalyse reactions such as DNA unwinding. The ring-shaped helicase 1 of bacteriophage T7 translocates along single-stranded (ss)DNA at a speed of 130 bases per second 2 ; however, T7 helicase slows down nearly tenfold when unwinding the strands of duplex DNA 3 . Here, we report that T7 DNA polymerase, which is unable to catalyse strand displacement DNA synthesis by itself, can increase the unwinding rate to 114 base pairs per second, bringing the helicase up to similar speeds compared to its translocation along ssDNA. The helicase rate of stimulation depends upon the DNA synthesis rate and does not rely on specific interactions between T7 DNA polymerase and the carboxy-terminal residues of T7 helicase. Efficient duplex DNA synthesis is achieved only by the combined action of the helicase and polymerase. The strand displacement DNA synthesis by the DNA polymerase depends on the unwinding activity of the helicase, which provides ssDNA template. The rapid trapping of the ssDNA bases by the DNA synthesis activity of the polymerase in turn drives the helicase to move forward through duplex DNA at speeds similar to those observed along ssDNA.