The QRxGRxGRxxxG motif of the vaccinia virus DExH box RNA helicase NPH-II is required for ATP hydrolysis and RNA unwinding but not for RNA binding.

Vaccinia virus NPH-II is an essential nucleic acid-dependent nucleoside triphosphate that catalyzes unidirectional unwinding of duplex RNA containing a 3' tail. NPH-II is the prototypal RNA helicase of the DExH box protein family, which is defined by several shared sequence motifs. The contribution of the conserved QRKGRVGRVNPG region to enzyme activity was assessed by alanine-scanning mutagenesis. Ten mutated versions of NPH-II were expressed in vaccinia virus-infected BSC-40 cells and purified by nickel affinity chromatography and glycerol gradient sedimentation. The mutated proteins were characterized with respect to RNA helicase, nucleic acid-dependent ATPase, and RNA binding functions. Individual alanine substitutions at invariant residues Q-491, G-494, R-495, G-497, R-498, and G-502 caused severe defects in RNA unwinding that correlated with reduced rates of ATP hydrolysis. None of these mutations affected the binding of NPH-II to single-strand RNA or to the tailed duplex RNA used as a helicase substrate. Mutation of the strictly conserved position R-492 inhibited ATPase and helicase activities and also caused a modest decrement in RNA binding. Alanine mutations at the nonconserved position N-500 and the weakly conserved residue P-501 had no apparent effect on any activity associated with NPH-II, whereas a mutation at the weakly conserved position K-493 reduced helicase to one-third and ATPase to two-thirds of the activity of wild-type required for ATP hydrolysis and RNA unwinding but not for RNA binding. Because mutations in the HRxGRxxR motif of the prototypal DEAD box RNA helicase eIF-4A abolish or severely inhibit RNA binding, we surmise that the contribution of conserved helicase motifs to overall protein function is context dependent.     

Helix-destabilizing properties of the adenovirus DNA-binding protein.

The adenovirus DNA-binding protein (DBP) is a multifunctional protein that is essential for viral DNA replication. DBP binds both single-stranded and double-stranded DNA as well as RNA in a sequence-independent manner. Previous studies showed that DBP does not promote melting of duplex poly(dA-dT) in contrast to prokaryotic single-strand-binding proteins. However, here we show that DBP can displace oligonucleotides annealed to single-stranded M13 DNA. Depending upon the DBP concentration, strands of at least 200 nucleotides can be unwound. Although unwinding of short (17-bp), fully duplex DNA is facilitated by DBP, unwinding of larger (28-bp) duplexes is only possible if single-stranded protruding ends are present. These protruding ends must be at least 4 nucleotides long for optimal unwinding, and both 5' and 3' single-stranded overhangs suffice. DBP-promoted strand displacement is sensitive to MgCl2 and NaCl and not dependent upon ATP. Our results suggest that DBP, through formation of a protein chain on the displaced strand, may destabilize duplex DNA ahead of the replication fork, thereby assisting in strand displacement during replication.     

Escherichia coli Rep protein and helicase IV. Distributive single-stranded DNA-dependent ATPases that catalyze a limited unwinding reaction in vitro.

Rep protein and helicase IV, two DNA-dependent adenosine 5'-triphosphatases with helicase activity, have been purified from Escherichia coli and characterized. Both enzymes exhibit a distributive interaction with single-stranded DNA as DNA-dependent ATPases in a reaction that is relatively resistant to increasing NaCl concentration and sensitive to the addition of E. coli single-stranded DNA binding protein (SSB). The helicase reaction catalyzed by each protein has been characterized using a direct unwinding assay and partial duplex DNA substrates. Both Rep protein and helicase IV catalyzed the unwinding of a duplex region 71 bp in length. However, unwinding of a 119-bp or 343-bp duplex region was substantially reduced compared to unwinding of the 71-bp substrate. At each concentration of protein examined, the number of base pairs unwound was greatest using the 71-bp substrate, intermediate with the 119-bp substrate and lowest using the 343-bp substrate. The addition of E. coli SSB did not increase the fraction of the 343-nucleotide fragment unwound by Rep protein. However, the addition of SSB did stimulate the unwinding reaction catalyzed by helicase IV approximately twofold. In addition, ionic strength conditions which stabilize duplex DNA (i.e. addition of MgCl2 or NaCl), markedly inhibited the helicase reaction catalyzed by either Rep protein or helicase IV while having little effect on the ATPase reaction. Thus, these two enzymes appear to share a common biochemical mechanism for unwinding duplex DNA which can be described as limited unwinding of duplex DNA. Taken together these data suggest that, in vitro, and in the absence of additional proteins, neither Rep protein nor helicase IV catalyzes a processive unwinding reaction.     

Multiple full-length NS3 molecules are required for optimal unwinding of oligonucleotide DNA in vitro

NS3 (nonstructural protein 3) from the hepatitis C virus is a 3' --> 5' helicase classified in helicase superfamily 2. The optimally active form of this helicase remains uncertain. We have used unwinding assays in the presence of a protein trap to investigate the first cycle of unwinding by full-length NS3. When the enzyme was in excess of the substrate, NS3 (500 nM) unwound >80% of a DNA substrate containing a 15-nucleotide overhang and a 30-bp duplex (45:30-mer; 1 nM). This result indicated that the active form of NS3 that was bound to the DNA prior to initiation of the reaction was capable of processive DNA unwinding. Unwinding with varying ratios of NS3 to 45:30-mer allowed us to investigate the active form of NS3 during the first unwinding cycle. When the substrate concentration slightly exceeded that of the enzyme, little or no unwinding was observed, indicating that if a monomeric form of the protein is active, then it exhibits very low processivity. Binding of NS3 to the 45:30-mer was measured by electrophoretic mobility shift assays, resulting in K(D) = 2.7 +/- 0.4 nM. Binding to individual regions of the substrate was investigated by measuring the K(D) for a 15-mer oligonucleotide as well as a 30-mer duplex. NS3 bound tightly to the 15-mer (K(D) = 1.3 +/- 0.2 nM) and, surprisingly, fairly tightly to the double-stranded 30-mer (K(D) = 11.3 +/- 1.3 nM). However, NS3 was not able to rapidly unwind a blunt-end duplex. Thus, under conditions of optimal unwinding, the 45:30-mer is initially saturated with the enzyme, including the duplex region. The unwinding data are discussed in terms of a model whereby multiple molecules of NS3 bound to the single-stranded DNA portion of the substrate are required for optimal unwinding.     

Binding specificity determines polarity of DNA unwinding by the Sgs1 protein of S. cerevisiae.

Saccharomyces cerevisiae Sgs1 protein is a member of the RecQ DNA helicase family which also includes the products of the human Bloom's syndrome and Werner's syndrome genes. We have studied the substrate specificity of a recombinant Sgs1 helicase (amino acid residues 400-1268 of the Sgs1 protein). Sgs1 shows a strong preference for binding branched DNA substrates, including duplex structures with a 3' single-stranded overhang and DNA junctions with multiple branches. Duplex DNA with a 5' rather than a 3' single-stranded tail is not recognized or unwound by Sgs1. DNase I and hydroxyl radical footprinting of the Sgs1-DNA complex shows that the protein binds specifically to the junction of a double-stranded DNA and its 3' overhang. Binding and unwinding of duplex DNA with a 3' overhang are much reduced if the backbone polarity of the 3' overhang is reversed in the junction region, but are unaffected if polarity reversal occurs four nucleotides away from the junction. These results indicate that the 3' to 5' polarity of unwinding by the recombinant Sgs1 protein is a direct consequence of the binding of the helicase to the single-stranded/double-stranded DNA junction and its recognition of the polarity of the single-stranded DNA at the junction. The recombinant Sgs1 also unwinds four-way junctions (synthetic Holliday junctions), a result that may be significant in terms of its role in suppressing DNA recombination in vivo.     

An oligomeric form of E. coli UvrD is required for optimal helicase activity.

Pre-steady-state chemical quenched-flow techniques were used to study DNA unwinding catalyzed by Escherichia coli UvrD helicase (helicase II), a member of the SF1 helicase superfamily. Single turnover experiments, with respect to unwinding of a DNA oligonucleotide, were used to examine the DNA substrate and UvrD concentration requirements for rapid DNA unwinding by pre-bound UvrD helicase. In excess UvrD at low DNA concentrations (1 nM), the bulk of the DNA is unwound rapidly by pre-bound UvrD complexes upon addition of ATP, but with time-courses that display a distinct lag phase for formation of fully unwound DNA, indicating that unwinding occurs in discrete steps, with a "step size" of four to five base-pairs as previously reported. Optimum unwinding by pre-bound UvrD-DNA complexes requires a 3' single-stranded (ss) DNA tail of 36-40 nt, whereas productive complexes do not form readily on DNA with 3'-tail lengths 相似文献   

Mechanism and substrate specificity of telomeric protein POT1 stimulation of the Werner syndrome helicase

Loss of the RecQ helicase WRN protein causes the cancer-prone progeroid disorder Werner syndrome (WS). WS cells exhibit defects in DNA replication and telomere preservation. The telomeric single-stranded binding protein POT1 stimulates WRN helicase to unwind longer telomeric duplexes that are otherwise poorly unwound. We reasoned that stimulation might occur by POT1 recruiting and retaining WRN on telomeric substrates during unwinding and/or by POT1 loading on partially unwound ssDNA strands to prevent strand re-annealing. To test these possibilities, we used substrates with POT1-binding sequences in the single-stranded tail, duplex or both. POT1 binding to ssDNA tails did not alter WRN activity on nontelomeric duplexes or recruit WRN to telomeric ssDNA. However, POT1 bound tails inhibited WRN activity on telomeric duplexes with a single 3'-ssDNA tail, which mimic telomeric ends in the open conformation. In contrast, POT1 bound tails stimulated WRN unwinding of forked telomeric duplexes. This indicates that POT1 interaction with the ssDNA/dsDNA junction regulates WRN activity. Furthermore, POT1 did not enhance retention of WRN on telomeric forks during unwinding. Collectively, these data suggest POT1 promotes the apparent processivity of WRN helicase by maintaining partially unwound strands in a melted state, rather than preventing WRN dissociation from the substrate.     

Extent of single-stranded DNA required for efficient TraI helicase activity in vitro

The IncF plasmid protein TraI functions during bacterial conjugation as a site- and strand-specific DNA transesterase and a highly processive 5' to 3' DNA helicase. The N-terminal DNA transesterase domain of TraI localizes the protein to nic and cleaves this site within the plasmid transfer origin. In the cell the C-terminal DNA helicase domain of TraI is essential for driving the 5' to 3' unwinding of plasmid DNA from nic to provide the strand destined for transfer. In vitro, however, purified TraI protein cannot enter and unwind nicked plasmid DNA and instead requires a 5' tail of single-stranded DNA at the duplex junction. In this study we evaluate the extent of single-stranded DNA adjacent to the duplex that is required for efficient TraI-catalyzed DNA unwinding in vitro. A series of linear partial duplex DNA substrates containing a central stretch of single-stranded DNA of defined length was created and its structure verified. We found that substrates containing >or=27 nucleotides of single-stranded DNA 5' to the duplex were unwound efficiently by TraI, whereas substrates containing 20 or fewer nucleotides were not. These results imply that during conjugation localized unwinding of >20 nucleotides at nic is necessary to initiate unwinding of plasmid DNA strands.     

The MER3 DNA helicase catalyzes the unwinding of holliday junctions

The MER3 protein of Saccharomyces cerevisiae is required for crossover in meiosis and has been suggested to act at the initiation of homologous pairing and the resolution of Holliday junctions. The purified MER3 protein is a DNA helicase that translocates along single-stranded DNA in the 3' to 5' direction displacing annealed DNA fragments. Here, MER3 was found to be able to unwind various double-stranded DNA (dsDNA) substrates, including a 30-bp dsDNA with a 20-nucleotide 3'-overhang, a 30-bp dsDNA with a 20-nucleotide 5'-overhang, a 50-bp dsDNA with blunt ends, and a Holliday junction with 25-bp arms, each of which had a blunt end. Efficient unwinding of the 3'-overhang substrate appeared to initiate by the binding of MER3 to the 3' single-stranded tail in a reaction that required six or more unpaired bases. Unwinding of the blunt end and 5'-overhang substrates appeared to initiate at the blunt ends of these substrates. Unwinding of the Holliday junction was more efficient than the unwinding of the blunt and 5'-overhang substrates and was influenced by Mg(2+) concentrations that cause changes in the structure of the junction. Possible roles for Holliday junction unwinding in meiotic crossover are discussed.     

The Escherichia coli dnaB replication protein is a DNA helicase

Genetic and biochemical analyses indicate that the Escherichia coli dnaB replication protein functions in the propagation of replication forks in the bacterial chromosome. We have found that the dnaB protein is a DNA helicase that is capable of unwinding extensive stretches of double-stranded DNA. We constructed a partially duplex DNA substrate, containing two preformed forks of single-stranded DNA, which was used to characterize this helicase activity. The dnaB helicase depends on the presence of a hydrolyzable ribonucleoside triphosphate, is maximally stimulated by a combination of E. coli single-stranded DNA-binding protein and E. coli primase, is inhibited by antibody directed against dnaB protein, and is inhibited by prior coating of the single-stranded regions of the helicase substrate with the E. coli single-stranded DNA-binding protein. It was determined that the dnaB protein moves 5' to 3' along single-stranded DNA, apparently in a processive fashion. To invade the duplex portion of the helicase substrate, the dnaB protein requires a 3'-terminal extension of single-stranded DNA in the strand to which it is not bound. Under optimal conditions at 30 degrees C, greater than 1 kilobase pair of duplex DNA can be unwound within 30 s. Based on these findings and other available data, we propose that the dnaB protein is the primary replicative helicase of E. coli and that it actively and processively migrates along the lagging strand template, serving both to unwind the DNA duplex in advance of the leading strand and to potentiate synthesis by the bacterial primase of RNA primers for the nascent (Okazaki) fragments of the lagging strand.     

Probing the relationship between RNA-stimulated ATPase and helicase activities of HCV NS3 using 2'-O-methyl RNA substrates

The hepatitis C virus (HCV) NS3 protein contains an amino terminal protease (NS3 aa. 1-180) and a carboxyl terminal RNA helicase (NS3 aa. 181-631). NS3 functions as a heterodimer of NS3 and NS4A (NS3/4A). NS3 helicase, a nucleic acid stimulated ATPase, can unwind RNA, DNA, and RNA:DNA duplexes, provided that at least one strand of the duplex contains a single-stranded 3' overhang (this strand of the duplex is referred to as the 3' strand). We have used 2'-O-methyl RNA (MeRNA) substrates to study the mechanism of NS3 helicase activity and to probe the relationship between its helicase and RNA-stimulated ATPase activities. NS3/4A did not unwind double-stranded (ds) MeRNA. NS3/4A unwinds hybrid RNA:MeRNA duplex containing MeRNA as the 5' strand but not hybrid duplex containing MeRNA as the 3' strand. The helicase activity of NS3/4A was 50% inhibited by 40 nM single-stranded (ss) RNA but only 35% inhibited by 320 nM ss MeRNA. Double-stranded RNA was 17 times as effective as double-stranded MeRNA in inhibiting NS3/4A helicase activity, while the apparent affinity of NS3/4A for ds MeRNA differed from ds RNA by only 2.4-fold. However ss MeRNA stimulated NS3/4A ATPase activity similar to ss RNA. These results indicate that the helicase mechanism involves 3' to 5' procession of the NS3 helicase along the 3' strand and only weak association of the enzyme with the displaced 5' strand. Further, our findings show that maximum stimulation of NS3 ATPase activity by ss nucleic acid is not directly related to procession of the helicase along the 3' strand.     

Escherichia coli helicase II (uvrD) protein can completely unwind fully duplex linear and nicked circular DNA

We have examined the duplex DNA unwinding (helicase) properties of the Escherichia coli helicase II protein (uvrD gene product) over a wide range of protein concentrations and solution conditions using a variety of duplex DNA substrates including fully duplex blunt ended and nicked circular molecules. We find that helicase II protein is able to initiate on and completely unwind fully duplex DNA molecules without the requirement for a covalently attached 3' single-stranded DNA tail. This DNA unwinding activity is dependent upon Mg2+ and ATP and requires that the amount of protein be in excess of that needed to saturate the resulting single-stranded DNA. Unwinding experiments on fully duplex blunt ended DNA with lengths of 341, 849, 1625, and 2671 base pairs indicate that unwinding occurs at the same high ratios of helicase II protein/nucleotide, independent of DNA length (50% unwinding requires approximately 0.6 helicase II monomers/nucleotide in 2.5 mM MgCl2, 10% glycerol, pH 7.5, 37 degrees C). Helicase II protein is also able to unwind completely a nicked circular DNA molecule containing 2671 base pairs. At lower but still high molar ratios of helicase II protein to DNA, duplex DNA molecules containing a single-stranded (ss) region attached to a 3' end of the duplex are preferentially unwound in agreement with the results obtained by S. W. Matson [1986) J. Biol. Chem. 261, 10169-10175). This preferential unwinding of duplex DNA with an attached 3' ssDNA most likely reflects the availability of a high affinity site (ssDNA) with the proper orientation for initiation; however, this may not reflect the type of DNA molecule upon which helicase II protein initiates DNA unwinding in vivo. The effects of changes in NaCl, NaCH3COO, and MgCl2 concentration on the ability of helicase II protein to unwind fully duplex DNA and duplex DNA with a 3' ssDNA tail have also been examined. Although the unwinding of fully duplex and nicked circular DNA molecules reported here occurs at higher helicase II protein to DNA ratios than have been previously used in most studies of this protein in vitro, this activity is likely to be relevant to the function of this protein in vivo since very high levels of helicase II protein accumulate in E. coli during the SOS response to DNA damage (approximately 2-5 x 10(4) copies/cell).(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for a functional dimeric form of the PcrA helicase in DNA unwinding

PcrA helicase, a member of the superfamily 1, is an essential enzyme in many bacteria. The first crystal structures of helicases were obtained with PcrA. Based on structural and biochemical studies, it was proposed and then generally believed that PcrA is a monomeric helicase that unwinds DNA by an inchworm mechanism. But a functional state of PcrA from unwinding kinetics studies has been lacking. In this work, we studied the kinetic mechanism of PcrA-catalysed DNA unwinding with fluorometric stopped-flow method under both single- and multiple-turnover conditions. It was found that the PcrA-catalysed DNA unwinding depended strongly on the PcrA concentration as well as on the 3′-ssDNA tail length of the substrate, indicating that an oligomerization was indispensable for efficient unwinding. Study of the effect of ATP concentration on the unwinding rate gave a Hill coefficient of ~2, suggesting strongly that PcrA functions as a dimer. It was further determined that PcrA unwound DNA with a step size of 4 bp and a rate of ~9 steps per second. Surprisingly, it was observed that PcrA unwound 12-bp duplex substrates much less efficiently than 16-bp ones, highlighting the importance of protein-DNA duplex interaction in the helicase activity. From the present studies, it is concluded that PcrA is a dimeric helicase with a low processivity in vitro. Implications of the experimental results for the DNA unwinding mechanism of PcrA are discussed.     

Biochemical characterization of the DNA substrate specificity of Werner syndrome helicase

Werner syndrome is a hereditary premature aging disorder characterized by genome instability. The product of the gene defective in WS, WRN, is a helicase/exonuclease that presumably functions in DNA metabolism. To understand the DNA structures WRN acts upon in vivo, we examined its substrate preferences for unwinding. WRN unwound a 3'-single-stranded (ss)DNA-tailed duplex substrate with streptavidin bound to the end of the 3'-ssDNA tail, suggesting that WRN does not require a free DNA end to unwind the duplex; however, WRN was completely blocked by streptavidin bound to the 3'-ssDNA tail 6 nucleotides upstream of the single-stranded/double-stranded DNA junction. WRN efficiently unwound the forked duplex with streptavidin bound just upstream of the junction, suggesting that WRN recognizes elements of the fork structure to initiate unwinding. WRN unwound two important intermediates of replication/repair, a 5'-ssDNA flap substrate and a synthetic replication fork. WRN was able to translocate on the lagging strand of the synthetic replication fork to unwind duplex ahead of the fork. For the 5'-flap structure, WRN specifically displaced the 5'-flap oligonucleotide, suggesting a role of WRN in Okazaki fragment processing. The ability of WRN to target DNA replication/repair intermediates may be relevant to its role in genome stability maintenance.     

Robust translocation along a molecular monorail: the NS3 helicase from hepatitis C virus traverses unusually large disruptions in its track

The NS3 helicase is essential for replication of the hepatitis C virus. This multifunctional Superfamily 2 helicase protein unwinds nucleic acid duplexes in a stepwise, ATP-dependent manner. Although kinetic features of its mechanism are beginning to emerge, little is known about the physical determinants for NS3 translocation along a strand of nucleic acid. For example, it is not known whether NS3 can traverse covalent or physical discontinuities on the tracking strand. Here we provide evidence that NS3 translocates with a mechanism that is different from its well-studied relative, the Vaccinia helicase NPH-II. Like NPH-II, NS3 translocates along the loading strand (the strand bearing the 3'-overhang) and it fails to unwind substrates that contain nicks, or covalent discontinuities in the loading strand. However, unlike NPH-II, NS3 readily unwinds RNA duplexes that contain long stretches of polyglycol, which are moieties that bear no resemblance to nucleic acid. Whether located on the tracking strand, the top strand, or both, long polyglycol regions fail to disrupt the function of NS3. This suggests that NS3 does not require the continuous formation of specific contacts with the ribose-phosphate backbone as it translocates along an RNA duplex, which is an observation consistent with the large NS3 kinetic step size (18 base-pairs). Rather, once NS3 loads onto a substrate, the helicase can translocate along the loading strand of an RNA duplex like a monorail train following a track. Bumps in the track do not significantly disturb NS3 unwinding, but a break in the track de-rails the helicase.     

Choosing between DNA and RNA: the polymer specificity of RNA helicase NPH-II

NPH-II is a prototypical member of the DExH/D subgroup of superfamily II helicases. It exhibits robust RNA helicase activity, and a detailed kinetic framework for unwinding has been established. However, like most SF2 helicases, there is little known about its mode of substrate recognition and its ability to differentiate between RNA and DNA substrates. Here, we employ a series of chimeric RNA–DNA substrates to explore the molecular determinants for NPH-II specificity on RNA and to determine if there are conditions under which DNA is a substrate. We show that efficient RNA helicase activity depends exclusively on ribose moieties in the loading strand and in a specific section of the 3′-overhang. However, we also document the presence of trace activity on DNA polymers, showing that DNA can be unwound under extremely permissive conditions that favor electrostatic binding. Thus, while polymer-specific SF2 helicases control substrate recognition through specific interactions with the loading strand, alternative specificities can arise under appropriate reaction conditions.     

Analysis of the DNA substrate specificity of the human BACH1 helicase associated with breast cancer

We have investigated the DNA substrate specificity of BACH1 (BRCA1-associated C-terminal helicase). The importance of various DNA structural elements for efficient unwinding by purified recombinant BACH1 helicase was examined. The results indicated that BACH1 preferentially binds and unwinds a forked duplex substrate compared with a duplex flanked by only one single-stranded DNA (ssDNA) tail. In support of its DNA substrate preference, helicase sequestration studies revealed that BACH1 can be preferentially trapped by forked duplex molecules. BACH1 helicase requires a minimal 5 ' ssDNA tail of 15 nucleotides for unwinding of conventional duplex DNA substrates; however, the enzyme is able to catalytically release the third strand of the homologous recombination intermediate D-loop structure irrespective of DNA tail status. In contrast, BACH1 completely fails to unwind a synthetic Holliday junction structure. Moreover, BACH1 requires nucleic acid continuity in the 5 ' ssDNA tail of the forked duplex substrate within six nucleotides of the ssDNA-dsDNA junction to initiate efficiently DNA unwinding. These studies provide the first detailed information on the DNA substrate specificity of BACH1 helicase and provide insight to the types of DNA structures the enzyme is likely to act upon to perform its functions in DNA repair or recombination.