Selective bypass of a lagging strand roadblock by the eukaryotic replicative DNA helicase

The eukaryotic replicative DNA helicase, CMG, unwinds DNA by an unknown mechanism. In some models, CMG encircles and translocates along one strand of DNA while excluding the other strand. In others, CMG encircles and translocates along duplex DNA. To distinguish between these models, replisomes were confronted with strand-specific DNA roadblocks in Xenopus egg extracts. An ssDNA translocase should stall at an obstruction on the translocation strand but not the excluded strand, whereas a dsDNA translocase should stall at obstructions on either strand. We found that replisomes bypass large roadblocks on the lagging strand template much more readily than on the leading strand template. Our results indicate that CMG is a 3' to 5' ssDNA translocase, consistent with unwinding via "steric exclusion." Given that MCM2-7 encircles dsDNA in G1, the data imply that formation of CMG in S phase involves remodeling of MCM2-7 from a dsDNA to a ssDNA binding mode.     

The type I restriction endonuclease EcoR124I, couples ATP hydrolysis to bidirectional DNA translocation

Type I restriction endonuclease holoenzymes contain methylase (M), restriction (R) and specificity (S) subunits, present in an M2:R2:S1 stoichiometry. These enzymes bind to specific DNA sequences and translocate dsDNA in an ATP-dependent manner toward the holoenzyme anchored at the recognition sequence. Once translocation is impeded, DNA restriction, which functions to protect the host cell from invading DNA, takes place. Translocation and DNA cleavage are afforded by the two diametrically opposed R-subunits. To gain insight into the mechanism of translocation, a detailed characterization of the ATPase activity of EcoR124I was done. Results show that following recognition sequence binding, ATP hydrolysis-coupled, bidirectional DNA translocation by EcoR124I ensues, with the R-subunits transiently disengaging, on average, every 515 bp. Macroscopic processivity of 2031(+/-184)bp is maintained, as the R-subunits remain in close proximity to the DNA through association with the methyltransferase. Transient uncoupling of ATP hydrolysis from translocation results in 3.1(+/-0.4) ATP molecules being hydrolyzed per base-pair translocated per R-subunit. This is the first clear demonstration of the coupling of ATP hydrolysis to dsDNA translocation, albeit inefficient. Once translocation is impeded on supercoiled DNA, the DNA is cleaved. DNA cleavage inactivates the EcoR124I holoenzyme partially and reversibly, which explains the stoichiometric behaviour of type I restriction enzymes. Inactivated holoenzyme remains bound to the DNA at the recognition sequence and immediately releases the nascent ends. The release of nascent ends was demonstrated using a novel, fluorescence-based, real-time assay that takes advantage of the ability of the Escherichia coli RecBCD enzyme to unwind restricted dsDNA. The resulting unwinding of EcoR124I-restricted DNA by RecBCD reveals coordination between the restriction-modification and recombination systems that functions to destroy invading DNA efficiently. In addition, we demonstrate the displacement of EcoR124I following DNA cleavage by the translocating RecBCD enzyme, resulting in the restoration of catalytic function to EcoR124I.     

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.     

A DNA helicase from human cells.

We have initiated the characterization of the DNA helicases from HeLa cells, and we have observed at least 4 molecular species as judged by their different fractionation properties. One of these only, DNA helicase I, has been purified to homogeneity and characterized. Helicase activity was measured by assaying the unwinding of a radioactively labelled oligodeoxynucleotide (17 mer) annealed to M13 DNA. The apparent molecular weight of helicase I on SDS polyacrylamide gel electrophoresis is 65 kDa. Helicase I reaction requires a divalent cation for activity (Mg2+ greater than Mn2+ greater than Ca2+) and is dependent on hydrolysis of ATP or dATP. CTP, GTP, UTP, dCTP, dGTP, dTTP, ADP, AMP and non-hydrolyzable ATP analogues such as ATP gamma S are unable to sustain helicase activity. The helicase activity has an optimal pH range between pH8.0 to pH9.0, is stimulated by KCl or NaCl up to 200mM, is inhibited by potassium phosphate (100mM) and by EDTA (5mM), and is abolished by trypsin. The unwinding is also inhibited competitively by the coaddition of single stranded DNA. The purified fraction was free of DNA topoisomerase, DNA ligase and nuclease activities. The direction of unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound. The enzyme also catalyses the ATP-dependent unwinding of a DNA:RNA hybrid consisting of a radioactively labelled single stranded oligodeoxynucleotide (18 mer) annealed on a longer RNA strand. The enzyme does not require a single stranded DNA tail on the displaced strand at the border of duplex regions; i.e. a replication fork-like structure is not required to perform DNA unwinding. The purification of the other helicases is in progress.     

Direct and random routing of a molecular motor protein at a DNA junction

With the aim of investigating how motor proteins negotiate DNA nanostructures, we produced test circuits based on recombination intermediates in which 1D translocation across a Holliday junction (HJ) could be assessed by subsequent triplex displacement signals on each DNA arm. Using the EcoR124I restriction-modification enzyme, a 3′–5′ double-strand DNA (dsDNA) translocase, we could show that the motor will tend to follow its translocated strand across a junction. Nonetheless, as the frequency of junction bypass events increases, the motor will occasionally jump tracks.     

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.     

Characterization of the helicase activity of the Escherichia coli RecBCD enzyme using a novel helicase assay

We describe an assay to measure the extent of enzymatic unwinding of DNA by a DNA helicase. This assay takes advantage of the quenching of the intrinsic protein fluorescence of Escherichia coli SSB protein upon binding to ssDNA and is used to characterize the DNA unwinding activity of recBCD enzyme. Unwinding in this assay is dependent on the presence of recBCD enzyme and linear dsDNA, is consistent with the known properties of recBCD enzyme, and closely parallels other methods for measuring recBCD enzyme helicase activity. The effects of varying temperature, substrate concentrations, enzyme concentration, and mono- and divalent salt concentrations on the helicase activity of recBCD enzyme were characterized. The apparent Km values for recBCD enzyme helicase activity on linear M13 dsDNA molecules at 25 degrees C are 0.6 nM dsDNA molecules and 130 microM ATP, respectively. The apparent turnover number for unwinding is approximately 15 microM base pairs s-1 (microM recBCD enzyme)-1. When this rate is corrected for the observed stoichiometry of recBCD enzyme binding to dsDNA, kcat for helicase activity corresponds to an unwinding rate of approximately 250 base pairs of DNA s-1 (functional recBCD complex)-1 at 25 degrees C. At 37 degrees C, the apparent Km value for dsDNA molecules was the same as that at 25 degrees C, but the apparent turnover number became 56 microM base pairs s-1 (microM recBCD enzyme)-1 [or 930 base pairs s-1 (functional recBCD complex)-1 when corrected for observed stoichiometry]. With increasing NaCl concentration, kcat peaks at 100 mM, and the apparent Km value for dsDNA increases by 3-fold at 200 mM NaCl. In the presence of 5 mM calcium acetate, the apparent Km value is increased by 3-fold, and kcat decreased by 20-30%. We have also shown that recBCD enzyme molecules are able to catalytically unwind additional dsDNA substrates subsequent to initiation, unwinding, and dissociation from a previous dsDNA molecule.     

Regulation of translocation polarity by helicase domain 1 in SF2B helicases

Structurally similar superfamily I (SF1) and II (SF2) helicases translocate on single-stranded DNA (ssDNA) with defined polarity either in the 5'-3' or in the 3'-5' direction. Both 5'-3' and 3'-5' translocating helicases contain the same motor core comprising two RecA-like folds. SF1 helicases of opposite polarity bind ssDNA with the same orientation, and translocate in opposite directions by employing a reverse sequence of the conformational changes within the motor domains. Here, using proteolytic DNA and mutational analysis, we have determined that SF2B helicases bind ssDNA with the same orientation as their 3'-5' counterparts. Further, 5'-3' translocation polarity requires conserved residues in HD1 and the FeS cluster containing domain. Finally, we propose the FeS cluster-containing domain also provides a wedge-like feature that is the point of duplex separation during unwinding.     

Characterisation of Bacillus stearothermophilus PcrA helicase: evidence against an active rolling mechanism.

PcrA from Bacillus stearothermophilus is a DNA helicase for which, despite the availability of a crystal structure, there is very little biochemical information. We show that the enzyme has a broad nucleotide specificity, even being able to hydrolyse ethenonucleotides, and is able to couple the hydrolysis to unwinding of DNA substrates. In common with the Escherichia coli helicases Rep and UvrD, PcrA is a 3'-5' helicase but at high protein concentrations it can also displace a substrate with a 5' tail. However, in contrast to Rep and UvrD, we do not see any evidence for dimerisation of the protein even in the presence of DNA. The enzyme shows a specificity for the DNA substrate in gel mobility assays, with the preferred substrate being one with both single and double stranded regions of DNA. We propose that these data, together with existing structural evidence, support an inchworm rather than a rolling model for 3'-5' helicase activity.     

The AddAB helicase/nuclease forms a stable complex with its cognate chi sequence during translocation

The Bacillus subtilis AddAB enzyme possesses ATP-dependent helicase and nuclease activities, which result in the unwinding and degradation of double-stranded DNA (dsDNA) upon translocation. Similar to its functional counterpart, the Escherichia coli RecBCD enzyme, it also recognizes and responds to a specific DNA sequence, referred to as Chi (chi). Recognition of chi triggers attenuation of the 3'- to 5'-nuclease, which permits the generation of recombinogenic 3'-overhanging, single-stranded DNA (ssDNA), terminating at chi. Although the RecBCD enzyme briefly pauses at chi, no specific binding of RecBCD to chi during translocation has been documented. Here, we show that the AddAB enzyme transiently binds to its cognate chi sequence (chi(Bs): 5'-AGCGG-3') during translocation. The binding of AddAB enzyme to the 3'-end of the chi(Bs)-specific ssDNA results in protection from degradation by exonuclease I. This protection is gradually reduced with time and lost upon phenol extraction, showing that the binding is non-covalent. Addition of AddAB enzyme to processed, chi(Bs)-specific ssDNA that had been stripped of all protein does not restore nuclease protection, indicating that AddAB enzyme binds to chi(Bs) with high affinity only during translocation. Finally, protection of chi(Bs)-specific ssDNA is still observed when translocation occurs in the presence of competitor chi(Bs)-carrying ssDNA, showing that binding occurs in cis. We suggest that this transient binding of AddAB to chi(Bs) is an integral part of the AddAB-chi(Bs) interaction and propose that this molecular event underlies a general mechanism for regulating the biochemical activities and biological functions of RecBCD-like enzymes.     

DNA binding by the substrate specificity (wedge) domain of RecG helicase suggests a role in processivity

RecG differs from most helicases acting on branched DNA in that it is thought to catalyze unwinding via translocation of a monomer on dsDNA, with a wedge domain facilitating strand separation. Conserved phenylalanines in the wedge are shown to be critical for DNA binding. When detached from the helicase domains, the wedge bound a Holliday junction with high affinity but failed to bind a replication fork structure. Further stabilizing contacts are identified in full-length RecG, which may explain fork binding. Detached from the wedge, the helicase region unwound junctions but had extremely low substrate affinity, arguing against the "classical inchworm" mode of translocation. We propose that the processivity of RecG on branched DNA substrates is dependent on the ability of the wedge to establish strong binding at the branch point. This keeps the helicase motor in contact with the substrate, enabling it to drive dsDNA translocation with high efficiency.     

Vinylphosphonate internucleotide linkages inhibit the activity of PcrA DNA helicase

During the past 5 years a great deal of structural and biochemical information has given us a detailed insight into the molecular mechanism of action of the PcrA DNA helicase and challenged previous notions about the molecular mechanism of action of helicases in general. Despite this wealth of information the mechanisms of the interaction of helicases with their DNA substrates and their unidirectional translocation along ssDNA are poorly understood. In this study, we synthesized a chemically modified DNA substrate with reduced backbone rotational flexibility and minimal steric hindrance and studied its effect on the activity of the monomeric 3'-5' DNA helicase, PcrA. Our results show that a single modification on the backbone of the translocating strand is sufficient to inhibit the activity of PcrA helicase, suggesting that rotational flexibility of the backbone is important for efficient unwinding.     

The 3'-tail of a forked-duplex sterically determines whether one or two DNA strands pass through the central channel of a replication-fork helicase

DnaB helicase is a ring-shaped hexamer that unwinds DNA at a replication fork. To understand how this protein interacts with DNA during unwinding, DnaB from Thermus aquaticus was incubated with chemically modified forked-duplex DNA substrates and the unwinding rates were measured. Unwinding was inhibited by modifications made to the 5'-tail, but not the 3'-tail, suggesting that the helicase interacts with the 5'-tail but not the 3'-tail during unwinding. Using oligonucleotides of mixed polarity, it was confirmed that DnaB translocates in the 5' to 3' direction as it unwinds DNA. A substrate was synthesized that contained two duplexes in tandem. Experiments involving various modifications of this tandem duplex demonstrated that when the 3'-tail is short, two stands of DNA pass through the central channel of DnaB with no resultant unwinding. Thus, the role of the 3'-tail in stimulating unwinding has been elucidated. The 3'-tail does not bind to DnaB during unwinding, but sterically determines whether one or two DNA strands pass through the central channel of DnaB. Furthermore, a new substrate for DnaB locomotion has been discovered. DnaB may actively translocate in the 5' to 3' direction along single-stranded DNA, even when a complementary strand is also present within the protein's central channel. This new mode of action may regulate DnaB activity by inhibiting unwinding at regions of DNA that are not forked. Furthermore, this new function for DnaB may coordinate abortion of leading and lagging strand replication if a nick is encountered on the leading strand.     

Electron microscopic analysis of DNA forks generated by Escherichia coli DNA helicase II

T7 phage DNA eroded with lambda exonuclease (to create 3'-protruding strands) or exonuclease III (to create 5'-protruding strands) was treated under unwinding assay conditions with DNA helicase II. Single-stranded DNA-binding protein (of Escherichia coli or phage T4) was added to disentangle the denatured DNA and the complexes were examined in the electron microscope. DNA helicase II complexes filtered through a gel column before assay retain the ability to generate forks suggesting that DNA helicase II unwinds in a preformed complex by translocating along the bound DNA strand. The enzyme initiates preferentially at the ends of the lambda-exonuclease-treated duplexes and is found at a fork on the initially protruding strand. It also initiates at the ends of the exonuclease-III-treated duplexes where, as with approximately 5% of the forks traceable back to a single-stranded gap, it is found on the initially recessed strand. The results are consistent with the view that DNA helicase II unwinds in the 3'-5' direction relative to the bound strand. They also confirm that the enzyme can initiate at the end of a fully base-paired strand. At a fork, DNA helicase II is bound as a tract of molecules of approximately 110 nm in length. Tracts of enzyme assemble from non-cooperatively bound molecules in the presence of ATP. During unwinding, DNA helicase II apparently can translocate to the displaced strand which conceivably can deplete the leading strand of the enzyme. Continued adsorption of enzyme to DNA might replenish forks arrested by strand switch of the unwinding enzyme.     

DNA helicase IV from HeLa cells.

Human DNA helicase IV, a novel enzyme, was purified to homogeneity from HeLa cells and characterized. The activity was measured by assaying the unwinding of 32P labeled 17-mer annealed to M13 ss DNA. From 440g of HeLa cells we obtained 0.31 mg of pure protein. Helicase IV was free of DNA topoisomerases, DNA ligase and nuclease activities. The apparent molecular weight is 100 kDa. It requires a divalent cation for activity (Mg2+ = Mn2+ = Zn2+) and the hydrolysis of only ATP or dATP. The activity is destroyed by trypsin and is inhibited by 200 mM KCl or NaCl, 100 mM potassium phosphate, 45 mM ammonium sulfate, 5 mM EDTA, 20 microM ss M13 DNA or 20 microM poly [G] (as phosphate). The enzyme unwinds DNA by moving in the 5' to 3' direction along the bound strand, a polarity opposite to that of the previously described human DNA helicase I (Tuteja et al Nucleic Acids Res. 18, 6785-6792, 1990). It requires more than 84 bases of single-stranded DNA in order to exert its unwinding activity and does not require a replication fork-like structure. Like human DNA helicase I the enzyme can also unwind RNA-DNA hybrid.     

Purification and characterization of a new DNA-dependent ATPase with helicase activity from Escherichia coli

A previously unreported single-stranded DNA-dependent nucleoside 5'-triphosphatase with DNA unwinding activity has been purified from extracts of Escherichia coli lacking the F factor. Fractions of the purified enzyme contain a major polypeptide of Mr = 75,000 which contains the active site(s) for both ATP hydrolysis and helicase activity. This is consistent with the results of gel filtration chromatography which indicate a native molecular mass of 75 kDa. The 75-kDa helicase has a preference for ATP (dATP) as a substrate in the hydrolysis reaction and requires the presence of a single-stranded DNA cofactor. The helicase reaction catalyzed by the enzyme has been characterized using an in vitro strand displacement assay. The 75-kDa helicase displaces a 71-nucleotide DNA fragment in an enzyme concentration-dependent and time-dependent reaction. The helicase reaction depends on the presence of a hydrolyzable nucleoside 5'-triphosphate (NTP) suggesting that NTP hydrolysis is required for the unwinding activity. In addition, the enzyme can displace a 343-nucleotide DNA fragment albeit less efficiently. The direction of the unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound. The molecular size of this helicase and the direction of the unwinding reaction are similar to both helicase II and Rep protein. However, the 75-kDa helicase has been shown to be distinct from both helicase II and Rep protein using immunological, physical, and genetic criteria. The discovery of a new helicase brings the total number of helicases found in E. coli cell extracts (lacking F factor) to five.     

A residue of motif III positions the helicase domains of motor subunit HsdR in restriction-modification enzyme EcoR124I

Type I restriction-modification enzymes differ significantly from the type II enzymes commonly used as molecular biology reagents. On hemi-methylated DNAs type I enzymes like the EcoR124I restriction-modification complex act as conventional adenine methylases at their specific target sequences, but unmethylated targets induce them to translocate thousands of base pairs through the stationary enzyme before cleaving distant sites nonspecifically. EcoR124I is a superfamily 2 DEAD-box helicase like eukaryotic double-strand DNA translocase Rad54, with two RecA-like helicase domains and seven characteristic sequence motifs that are implicated in translocation. In Rad54 a so-called extended region adjacent to motif III is involved in ATPase activity. Although the EcoR124I extended region bears sequence and structural similarities with Rad54, it does not influence ATPase or restriction activity as shown in this work, but mutagenesis of the conserved glycine residue of its motif III does alter ATPase and DNA cleavage activity. Through the lens of molecular dynamics, a full model of HsdR of EcoR124I based on available crystal structures allowed interpretation of functional effects of mutants in motif III and its extended region. The results indicate that the conserved glycine residue of motif III has a role in positioning the two helicase domains.     

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.     

The interrelationship of helicase and nuclease domains during DNA translocation by the molecular motor EcoR124I

The type I restriction-modification enzyme EcoR124I comprises three subunits with the stoichiometry HsdR₂/HsdM₂/HsdS₁. The HsdR subunits are archetypical examples of the fusion between nuclease and helicase domains into a single polypeptide, a linkage that is found in a great many other DNA processing enzymes. To explore the interrelationship between these physically linked domains, we examined the DNA translocation properties of EcoR124I complexes in which the HsdR subunits had been mutated in the RecB-like nuclease motif II or III. We found that nuclease mutations can have multiple effects on DNA translocation despite being distinct from the helicase domain. In addition to reductions in DNA cleavage activity, we also observed decreased translocation and ATPase rates, different enzyme populations with different characteristic translocation rates, a tendency to stall during initiation and altered HsdR turnover dynamics. The significance of these observations to our understanding of domain interactions in molecular machines is discussed.