Helicase Reaction Research Articles

A novel high throughput screening assay for HCV NS3 helicase activity

A novel assay for measurement of Hepatitis C virus (HCV) NS3 helicase activity was developed using Flashplate™ technology. This assay involves the use of a DNA duplex substrate and recombinant HCV NS3 produced in Escherichia coli. The DNA duplex consisted of a pair of oligonucleotides, one biotinylated, the other radiolabeled at their respective 5′ termini. This DNA duplex was immobilized, via the biotin molecule, on the surface of a neutravidin-coated SMP103 Flashplate™ (NEN Life Science Products). Helicase activity results in the release of the radiolabeled oligonucleotide, which translates in signal reduction with respect to control wells. Biochemical characterization of the HCV NS3 helicase activity was performed using this assay. We demonstrated that the NS3-mediated unwinding is proportional to both the amount of DNA substrate in the well, and to the NS3 concentration in the reaction. Most of the NS3-mediated unwinding was achieved in the initial 60 min of incubation. As expected the reactions were ATP-dependent and found to be affected by the concentration of MgCl 2, MnCl 2, KCl, EDTA, and by pH. We found this assay to be highly reproducible since only slight variation was observed when a total of 68 helicase reactions were performed on one plate. Therefore, this Flashplate™ helicase assay is fast, convenient and reproducible. These criteria make it suitable for high throughput screening of potential NS3 helicase inhibitors.

Strand-separating activity of hepatitis C virus helicase in the absence of ATP.

HCV helicase [E(wt)] catalyzed strand separation of a short DNA duplex (F21:HF31) formed from a 5'-hexachlorofluorescein-tagged 31-mer (HF31) and a 3'-fluorescein-tagged 21-mer (F21) complementary to the 5'-end of HF31. Strand separation was monitored by the fluorescence increase associated with the formation of F21 from F21:HF31. In the presence of ATP, the strand-separating activity was catalytic. In the absence of ATP and with E(wt) concentrations greater than that of F21:HF31, a biphasic fluorescence increase was observed at 25 degrees C. The late phase of this reaction was assigned to the separation of F21 from F21:HF31. The ATP-independent strand-separating reaction occurred more rapidly in the absence of Mg(2+) than in its presence. This result correlated with a lower T(m) value of F21:HF31 in the absence of 3.5 mM Mg(2+) than in its presence (45 vs 63 degrees C). The stoichiometry for the strand-separating reaction in the absence of ATP was 8 mol of E(wt) per mole of F21:HF31 separated into single-stranded F21 and HF31. The dissociation constants of HCV helicase for F21, HF31, and F21:HF31 in the absence of Mg(2+) were 0.6 +/- 0.4, 6 +/- 1, and 7.3 +/- 0.9 nM, respectively. Histidinyl-tagged E(wt) [hE(wt)] and a mutant enzyme [hE(V432A)] were prepared. hE(wt) and E(wt) bound F21 and HF31 with similar affinities and had similar ATP-dependent helicase activities, whereas hE(V432A) bound F21 and HF31 with affinities similar to that of E(wt) but had greatly reduced ATP-dependent helicase activities. In contrast to E(wt) and hE(wt), hE(V432A) did not support the ATP-independent strand-separating reaction. Consequently, the ATP-independent strand-separating reaction was not only the result of the high affinity of the enzyme for single-stranded DNA. The enzyme preferentially used duplex DNA with a 3'-tail for the ATP-dependent helicase reaction. In contrast, the enzyme strand-separated blunt-ended, 5'-tailed, and 3'-tailed duplex DNA equally effectively in the ATP-independent strand-separating reaction.

Biochemical characterization of adeno-associated virus rep68 DNA helicase and ATPase activities.

The adeno-associated virus (AAV) nonstructural proteins Rep68 and Rep78 are site-specific DNA binding proteins, ATP-dependent site-specific endonucleases, helicases, and ATPases. These biochemical activities are required for viral DNA replication and control of viral gene expression. In this study, we characterized the biochemical properties of the helicase and ATPase activities of homogeneously pure Rep68. The enzyme exists as a monomer in solution at the concentrations used in this study (<380 nM), as judged by its mobility in sucrose density gradients. Using a primed single-stranded (ss) circular M13 substrate, the helicase activity had an optimum pH of 7 to 7.5, an optimum temperature of 45 degreesC, and an optimal divalent-cation concentration of 5 mM MgCl2. Several nucleoside triphosphates could serve as cofactors for Rep68 helicase activity, and the order of preference was ATP = GTP > CTP = dATP > UTP > dGTP. The Km values for ATP in both the DNA helicase reaction and the site-specific trs endonuclease reaction were essentially the same, approximately 180 microM. Both reactions were sigmoidal with respect to ATP concentration, suggesting that a dimer or higher-order multimer of Rep68 is necessary for both DNA helicase activity and terminal resolution site (trs) nicking activity. Furthermore, when the enzyme itself was titrated in the trs endonuclease and ATPase reactions, both activities were second order with respect to enzyme concentration. This suggests that a dimer of Rep68 is the active form for both the ATPase and nicking activities. In contrast, DNA helicase activity was linear with respect to enzyme concentration. When bound to ssDNA, the enzyme unwound the DNA in the 3'-to-5' direction. DNA unwinding occurred at a rate of approximately 345 bp per min per monomeric enzyme molecule. The ATP turnover rate was approximately 30 to 50 ATP molecules per min per enzyme molecule. Surprisingly, the presence of DNA was not required for ATPase activity. We estimated that Rep translocates processively for more than 1,300 bases before dissociating from its substrate in the absence of any accessory proteins. DNA helicase activity was not significantly stimulated by substrates that have the structure of a replication fork and contain either a 5' or 3' tail. Rep68 binds only to ssDNA, as judged by inhibition of the DNA helicase reaction with ss or double-stranded (ds) DNA. Consistent with this observation, no helicase activity was detected on blunt-ended ds oligonucleotide substrates unless they also contained an ss 3' tail. However, if a blunt-ended ds oligonucleotide contained the 22-bp Rep binding element sequence, Rep68 was capable of unwinding the substrate. This means that Rep68 can function both as a conventional helicase for strand displacement synthesis and as a terminal-repeat-unwinding protein which catalyzes the conversion of a duplex end to a hairpin primer. Thus, the properties of the Rep DNA helicase activity suggest that Rep is involved in all three of the key steps in AAV DNA replication: terminal resolution, reinitiation, and strand displacement.

The Herpes Simplex Virus Type 1 Helicase-primase: ANALYSIS OF HELICASE ACTIVITY

The rate of unwinding of duplex DNA by the herpes simplex virus type 1 (HSV-1)-encoded helicase-primase (primosome) was determined by measuring the rate of appearance of single strands from a circular duplex DNA containing a 40-nucleotide 5' single-stranded tail, i.e. a preformed replication fork, in the presence of the HSV-1 single strand DNA-binding protein, infected cell protein 8 (ICP8). With this substrate, the rate at low ionic strength was highly sensitive to Mg2+ concentration. The Mg2+ dependence was a reflection of both the requirement for ICP8 for helicase activity and the ability of ICP8 to reverse the helicase reaction as a consequence of its capacity to anneal homologous single strands at Mg2+ concentrations in excess of 3 mM. The rate of unwinding of duplex DNA by the HSV-1 primosome was also determined indirectly by measuring the rate of leading strand synthesis with a preformed replication fork as template in the presence of the T7 DNA polymerase. The value of 60-65 base pairs unwound/s by both methods is consistent with the rate of 50 base pairs/s estimated for the rate of fork movement in vivo during replication of pseudorabies virus, another herpesvirus. Interaction with the helicase-primase did not increase its helicase activity.

Isolation and characterization of a processive DNA helicase from the fission yeast Schizosaccharomyces pombe that translocates in a 5'-to-3' direction.

We report here the isolation and characterization of a novel DNA helicase from extracts of the fission yeast Schizosaccharomyces pombe. The enzyme, called DNA helicase II, also contains an intrinsic DNA-dependent ATPase activity. Both the helicase and ATPase activities co-purified with a 63 kDa polypeptide on an SDS/polyacrylamide gel. The protein has a sedimentation coefficient of 4.8 S and a Stokes radius of 36 A (3.6 nm); from these data the native molecular mass was calculated to be 65 kDa. The enzyme translocates in a 5'-to-3' direction with respect to the substrate strand to which it is bound. Unwinding reactions carried out in the presence of increasing enzyme showed a sigmoidal curve, suggesting either co-operative interactions between monomers or multimerization of DNA helicase II in the presence of single-stranded DNA and/or ATP. This enzyme favoured adenosine nucleotides (ATP and dATP) as its energy source, but utilized to limited extents GTP, CTP, dGTP and dCTP. Non-hydrolysable ATP analogues did not support helicase activity. Kinetic analyses showed that the unwinding reaction was rapid, being complete after 50-100 s of incubation. Addition of unlabelled substrates to the helicase reaction after preincubation of the enzyme with substrate did not significantly diminish unwinding. The ATPase activity of DNA helicase II increased proportionally with increasing lengths of single-stranded DNA cofactor. In the presence of circular DNA, ATP hydrolysis continued to increase up to the longest time tested (3 h), whereas it ceased to increase after 5-10 min in the presence of shorter oligonucleotides. The initial rate of ATP hydrolysis during the first 5 min of incubation time was not affected by DNA species used. These data indicate that the enzyme does not dissociate from the single-stranded DNA once it is bound and is therefore highly processive.

Herpes Simplex Virus Type-1 Single-strand DNA-binding Protein (ICP8) Enhances the Ability of the Viral DNA Helicase-primase to Unwind Cisplatin-modified DNA

The herpes simplex virus type-1 UL5, UL8, and UL52 genes encode an essential heterotrimeric DNA helicase-primase that is responsible for concomitant DNA unwinding and primer synthesis at the viral DNA replication fork. The viral single-strand DNA-binding protein (ICP8) can stimulate DNA unwinding by the helicase-primase as a result of a physical interaction that is mediated by the UL8 subunit. In this study, we investigated the ability of the helicase-primase to unwind a fork-like substrate that contains an intrastrand d(GpG) DNA cross-link produced by the antitumor drug cisplatin. We also examined the ability of ICP8 to modulate the effect of the cisplatin lesion. The data show that the lesion inhibited the helicase-primase when located on the DNA strand along which it translocates. However, the lesion did not represent a permanent obstacle to its progression. In contrast, the adduct did not affect the helicase-primase when located on the opposite DNA strand. ICP8 specifically stimulated DNA unwinding by the helicase-primase. Coating concentrations of ICP8 were necessary for optimal unwinding of damaged DNA. Addition of competitor DNA to helicase reactions led to substantial reduction of DNA unwinding by the helicase-primase, suggesting that the enzyme is distributive. ICP8 did not abolish the competition, indicating that it did not stimulate the helicase by increasing its processivity. Rather, ICP8 may stimulate DNA unwinding and enable bypass of cisplatin damaged DNA by recruiting the helicase-primase to the DNA.

Characterization of a helicase, DHEL III, from Drosophila melanogaster embryonic nuclei.

Nuclear extracts prepared from Drosophila preblastoderm embryos contain a nucleoside 5'-triphosphate-dependent helicase that unwinds circular or linear partial duplex DNA substrates and moves along the DNA in 3' to 5' direction. The helicase reaction is supported with both nucleoside and deoxynucleoside 5'-triphosphates and requires divalent cations. Optimum activity in vitro is achieved with dATP or ATP and with MgCl2. In glycerol density gradients, embryonic enzyme migrates as a single peak of around 90kDa and it is the most prominent DNA-unwinding activity detectable in nuclear extracts prepared from embryos 0-2 hours after egg laying. In embryos collected 2-4 hours after egg laying this DNA unwinding activity increases and then gradually decreases in embryos collected 4-8 and 8-16 hours after egg laying.

DNA helicase activity of the hepatitis C virus nonstructural protein 3.

Hepatitis C virus (HCV) nonstructural protein 3 (NS3) is a known RNA helicase, an enzyme that unwinds RNA x DNA and RNA x RNA duplexes. We have now deciphered the biochemical characteristics of the HCV NS3 DNA helicase activity. Recombinant NS3 was expressed in Escherichia coli, purified to near homogeneity, and tested for DNA helicase activity. The optimal conditions for DNA unwinding (for example, the preferred pH and magnesium ion concentration) were similar to those for RNA unwinding. The DNA helicase activity was very sensitive to potassium ion concentration, while DNA binding and DNA-stimulated ATPase activities were not. The direction of DNA unwinding was determined to be 3' to 5'. All four ribonucleoside triphosphates (ATP, GTP, CTP, UTP) and deoxynucleoside triphosphates (dATP, dGTP, dCTP, dTTP) could serve as energy sources, but GTP and dGTP were less efficient than the others. When nucleotide analog inhibitors were added to the DNA helicase reaction, the overall order of inhibitory capacity was: adenosine 5'-O-(3-thiotriphosphate) > adenylyl-imidodiphosphate and adenylyl-(beta,gamma-methylene)-diphosphate > AMP. DNA helicase activity was inhibited strongly by ssDNA and ssRNA, but was little affected by dsDNA. The ATPase activity was stimulated greatly by ssDNA and ssRNA, but not by dsDNA. The NS3 protein could unwind up to 500 base pairs of duplex DNA. The possible multifunctional nature of the NS3 protein is discussed and compared with that of Simian virus 40 large T antigen.

Kinetics of the RNA-DNA helicase activity of Escherichia coli transcription termination factor rho. 2. Processivity, ATP consumption, and RNA binding.

The RNA-binding and RNA-DNA helicase activities of the Escherichia coli transcription termination factor rho have been investigated using natural RNA molecules that are 255 and 391 nucleotide residues in length and that contain the trp t' rho-dependent termination sequence of E. coli. Helicase substrates were prepared from these RNA molecules by annealing one or more DNA oligomers to complementary sequences located at or near the 3'-ends of the RNA molecules to form defined RNA-DNA hybrid sequences ranging in length from 20 to 100 bp. By comparing the fraction of the RNA molecules bound to rho with the fraction of bound DNA oligomers removed from the RNA during one round of the helicase reaction, we have shown that rho translocates processively at 37 degrees C in buffer containing 50 mM KCl. Helicase reactions and ATPase measurements were performed in parallel in the presence of RNA molecules containing RNA-DNA hybrids of various lengths, and we show that both the rate of translocation of the rho hexamer along the RNA chain and the rate of ATP consumption are similar, whether or not DNA is hybridized to the RNA transcript. By combining measurements of translocation and ATPase rates, we estimate that rho consumes approximately 1-2 ATP molecules in translocating over 1 nucleotide residue of the RNA chain at 37 degrees C in 50 mM KCl. The ATPase activity of rho remains the same after one round of the helicase reaction, indicating that rho appears to hydrolyze ATP at the same rate, whether it is translocating along the RNA, separating RNA-DNA hybrids, or bound at the 3'-end of the RNA substrate. We also show that rho binds cooperatively ( approximately 2-4 rho hexamers per RNA chain) to the RNA substrates under our standard helicase reaction conditions. However, cooperative binding is not essential for helicase activity, since this binding stoichiometry can be reduced to approximately 1.5 rho hexamers per 255-nucleotide residue RNA chain by blocking approximately 100 nt of either end of the rho binding site of the helicase substrate with complementary DNA oligonucleotides, with no change in helicase properties. The implications of these results for models of rho helicase function and for the role of rho in termination are discussed.

Targeted versus non-targeted DNA helicase activity of the RuvA and RuvB proteins of Escherichia coli

The RuvA and RuvB proteins of Escherichia coli promote the branch migration of Holliday junctions in vitro. To understand the relationship between branch migration and the intrinsic 5'-->3' DNA helicase activity of RuvAB, the requirements and substrate specificity of the helicase reaction have been studied in more detail. We find that RuvAB-mediated DNA unwinding and branch migration reactions show similar requirements for Mg2+ and ATP and are inhibited to a similar extent by ADP and ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)). The helicase activity, measured by the dissociation of a short fragment from circular single-stranded DNA, requires both RuvA and RuvB and is stimulated by subsaturating concentrations of single-strand binding protein (SSB). In contrast, saturating concentrations of SSB are inhibitory. Using substrates that contain a DNA junction, which permits the specific binding of RuvA, we find that the RuvA and RuvB proteins promote two types of helicase reactions: nonspecific reactions, which are sensitive to inhibition by stoichiometric amounts of SSB, and junction-targeted reactions, which are not inhibited by SSB. Using three-armed structures, we observe that junction-targeted reactions display a polarity and result in asymmetric product formation. Junction-specific binding and the subsequent initiation of DNA unwinding are likely to represent early steps in the process of branch migration.

Biochemical analysis of mutants with changes in the origin-binding domain of simian virus 40 tumor antigen

The role of the origin-binding domain of simian virus 40 large tumor antigen (T antigen) in the initiation of virus DNA replication was investigated by analyzing the biochemical activities of a series of mutants with single-site substitutions in this region. These activities include origin-specific and nonspecific DNA binding, melting of the imperfect palindromic sequence, untwisting of the AT-rich region, unwinding of origin-containing DNA, helicase activity, and the ability to oligomerize normally in response to ATP. Three classes of T-antigen mutants that are unable to support virus replication in monkey cells are described. Class 1 mutants are unable to bind to the origin of DNA replication but are able to bind to DNA nonspecifically. Class 2 mutants exhibit defective binding to both types of DNA. As expected, mutants in these first two classes are unable to unwind origin DNA. Surprisingly, however, these mutants possess significant levels of melting and untwisting activities, suggesting that these reactions may not be solely dependent on the ability of the protein to recognize origin sequences. Most class 1 mutants oligomerize normally in response to ATP, indicating that their DNA-binding defects are not due to structural alterations but probably to a failure to directly recognize origin sequences. In contrast, class 2 mutants exhibit defective oligomerization. Class 3 mutants bind to origin and nonorigin DNA at near wild-type levels and melt and untwist origin DNA normally but exhibit defective oligomerization and unwinding. These mutants are, however, perfectly able to carry out the helicase reaction, indicating that their unwinding defect is at some step after melting but before a nonspecific helicase is used to separate parental strands during replication. These results therefore suggest that proper oligomerization to correctly position the molecules on the DNA may be more important in initiating unwinding than in bringing about efficient DNA binding, inducing structural changes in the DNA, or carrying out the helicase reaction.

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.

Bound Lac repressor protein differentially inhibits the unwinding reactions catalyzed by DNA helicases.

A partial duplex DNA substrate containing the Lac repressor binding site, within the duplex region, was constructed to examine the effect of bound Lac repressor on the unwinding reaction catalyzed by several DNA helicases. The substrate contained 90 base pairs of double-stranded DNA and, in the absence of Lac repressor, was effectively unwound by each of the seven helicases tested. The unwinding reactions catalyzed by Escherichia coli Rep protein, bacteriophage T4 Dda protein and E. coli DNA helicase I were not inhibited by the presence of bound Lac repressor. Both SV40 T antigen and E. coli helicase II were partially inhibited by bound repressor at the highest repressor concentrations tested. The helicase reactions catalyzed by E. coli DnaB protein and helicase IV were substantially inhibited by the presence of bound protein. When the length of the duplex region was increased to 323 base pairs the inhibition spectrum caused by bound Lac repressor on the unwinding reactions catalyzed by DnaB protein, helicase I and helicase II was essentially the same as that observed using the shorter partial duplex molecule. Inhibition of the unwinding reaction was due to the presence of bound Lac repressor as evidenced by the substantially weaker inhibition of helicase IV by Lac repressor in the presence of IPTG. In addition, we have shown that Rep protein displaces the bound repressor protein during the course of an unwinding reaction.

Escherichia coli DNA helicase I catalyzes a site- and strand-specific nicking reaction at the F plasmid oriT

A site- and strand-specific nick, introduced in the F plasmid origin of transfer, initiates conjugal DNA transfer during bacterial conjugation. Recently, molecular genetic studies have suggested that DNA helicase I, which is known to be encoded on the F plasmid, may be involved in this nicking reaction (Traxler, B. A., and Minkley, E. G., Jr. (1988) J. Mol. Biol. 204, 205-209). We have demonstrated this site- and strand-specific nicking event using purified helicase I in an in vitro reaction. The nicking reaction requires a superhelical DNA substrate containing the F plasmid origin of transfer, Mg2+ and helicase I. The reaction is protein concentration-dependent but, under the conditions used, only 50-70% of the input DNA substrate is converted to the nicked species. Genetic data (Everett, R., and Willetts, N. (1980) J. Mol. Biol. 136, 129-150) have also suggested the involvement of a second F-encoded protein, the TraY protein, in the oriT nicking reaction. Unexpectedly, the in vitro nicking reaction does not require the product of the F plasmid traY gene. The implications of this result are discussed. The phosphodiester bond interrupted by helicase I has been shown to correspond exactly to the site nicked in vivo suggesting that helicase I is the site- and strand-specific nicking enzyme that initiates conjugal DNA transfer. Thus, helicase I is a bifunctional protein which catalyzes site- and strand-strand specific nicking of the F plasmid in addition to the previously characterized duplex DNA unwinding (helicase) reaction.

DNA helicase and duplex DNA fragment unwinding activities of polyoma and simian virus 40 large T antigen display similarities and differences

We have characterized the biochemical activities of purified polyoma (Py) large T antigen (T Ag) that was capable of mediating the replication of a plasmid containing the Py origin (ori(+) DNA) in mouse cell extracts. We report here that like the T Ag encoded by simian virus 40 (SV40), Py T Ag has DNA helicase and double-stranded DNA fragment unwinding activities. Py T Ag displaced DNA fragments greater than 1,600 nucleotides which were annealed to complementary sequences in single-stranded M13 by translocating in the 3' to 5' direction. Both helicase and double-stranded DNA fragment unwinding reactions were completely dependent upon NTP hydrolysis, displaying a strong preference for ATP and dATP. At low T Ag concentrations, significantly more Py ori(+) DNA fragment was unwound compared with a fragment lacking the replication origin. However, at higher ratios of Py T Ag to DNA, equivalent to those used in replication reactions, unwinding of both ori-containing and -lacking fragments was equally efficient. This is in contrast to SV40 T Ag which exhibited a more stringent requirement for SV40 origin sequences under similar conditions. Furthermore, some of the nucleotides that supported the helicase and unwinding activities of Py T Ag were different from those for the same SV40 T Ag reactions. We have also observed that in contrast to the very poor replication of linear SV40 ori(+) DNA by SV40 T Ag in human cell extracts, linear Py ori(+) DNA was replicated efficiently in mouse cell extracts by Py T Ag. However, despite the fact that linear Py ori(+), SV40 ori(+), and ori(-) DNA fragments could be unwound with comparable efficiency by Py T Ag, only fragments containing the Py replication origin were replicated in vitro. These results suggest that the initiation of DNA synthesis at the Py origin of replication requires features in addition to unwinding of the template.

Differential ATP requirements distinguish the DNA translocation and DNA unwinding activities of the Escherichia coli PRI A protein.

The Escherichia coli primosome is a mobile multiprotein DNA replication-priming apparatus that assembles at a specific site (termed a primosome assembly site (PAS] on single-stranded DNA-binding protein-coated single-stranded DNA. The PRI A protein (factor Y, protein n') is a PAS sequence-specific (d)ATPase as well as a DNA helicase and is believed to direct the assembly of the primosome at a PAS. In this report, the PRI A DNA helicase reaction is dissected in vitro, by use of a strand displacement assay, into three steps with distinct ATP requirements. First, the PRI A protein gains entry to the DNA via an ATP-independent, PAS sequence-specific binding event. Second, the PRI A protein translocates along the single-stranded DNA in the 3'----5' direction at a maximal rate of 90 nucleotides/s. DNA translocation requires ATP hydrolysis. The ATP concentration required to support half of the maximal translocation rate is 100 microM, which is identical to the Km for ATP of the PRI A protein DNA-dependent ATPase activity. Finally, the PRI A protein unwinds duplex DNA. The ATP concentration required for duplex DNA unwinding depends upon the length of the duplex region to be unwound. Displacement of a 24-nucleotide long oligomer required no more ATP than that required for the translocation of PRI A protein along single-stranded DNA, whereas displacement of a 390-nucleotide long DNA fragment required a 10-fold higher concentration of ATP than that required for oligomer displacement.

Specificity and efficiency of rho-factor helicase activity depends on magnesium concentration and energy coupling to NTP hydrolysis.

The RNA-DNA helicase activity of Escherichia coli transcription termination factor rho can be significantly enhanced at lower potassium chloride and magnesium acetate concentrations than previously used. Decreasing the potassium chloride concentration from 150 to 50 mM increases the rate of release at least 4-fold, while at lower magnesium concentrations less ATP is required for maximal duplex disruption. For all concentrations tested (between 0.1 and 5 mM), the optimal magnesium and ATP concentrations are interdependent; a roughly equimolar ratio gives the maximal rate of RNA release, although peak height and breadth vary. Surprisingly, rho behaves differently with an RNA-RNA duplex, which cannot be efficiently disrupted at magnesium concentrations below 1 mM. Above 2.0 mM, release does occur efficiently suggesting that Mg2+ promotes some structural transition in the RNA-RNA helix to a rho-susceptible conformation. In addition to Mg2+, helicase activity requires hydrolysis of nucleoside triphosphates, but for all four standard NTPs the rates of NTP hydrolysis do not correlate uniformly with the rates of RNA release. Based on the ratio of the rate of RNA release to the rate of NTP hydrolysis, rho utilizes ATP most efficiently. The 2-4-fold weaker coupling of hydrolysis to duplex disruption for the other three NTPs demonstrates that NTP utilization is not, on its own, sufficient for efficient helicase activity. The less efficient coupling with GTP, CTP, and UTP correlates with conformational differences in the protein complex as probed by mild trypsin digestion. The implications of our findings for substrate specificity and energy coupling in the helicase reaction are discussed.

Repair of DNA-containing pyrimidine dimers.

Ultraviolet light-induced pyrimidine dimers in DNA are recognized and repaired by a number of unique cellular surveillance systems. The most direct biochemical mechanism responding to this kind of genotoxicity involves direct photoreversal by flavin enzymes that specifically monomerize pyrimidine:pyrimidine dimers monophotonically in the presence of visible light. Incision reactions are catalyzed by a combined pyrimidine dimer DNA-glycosylase:apyrimidinic endonuclease found in some highly UV-resistant organisms. At a higher level of complexity, Escherichia coli has a uvr DNA repair system comprising the UvrA, UvrB, and UvrC proteins responsible for incision. There are several preincision steps governed by this pathway, which includes an ATP-dependent UvrA dimerization reaction required for UvrAB nucleoprotein formation. This complex formation driven by ATP binding is associated with localized topological unwinding of DNA. This same protein complex can catalyze an ATPase-dependent 5'----3'-directed strand displacement of D-loop DNA or short single strands annealed to a single-stranded circular or linear DNA. This putative translocational process is arrested when damaged sites are encountered. The complex is now primed for dual incision catalyzed by UvrC. The remainder of the repair process involves UvrD (helicase II) and DNA polymerase I for a coordinately controlled excision-resynthesis step accompanied by UvrABC turnover. Furthermore, it is proposed that levels of repair proteins can be regulated by proteolysis. UvrB is converted to truncated UvrB* by a stress-induced protease that also acts at similar sites on the E. coli Ada protein. Although UvrB* can bind with UvrA to DNA, it cannot participate in helicase or incision reactions. It is also a DNA-dependent ATPase.

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.

DNA helicase II of Escherichia coli. Characterization of the single-stranded DNA-dependent NTPase and helicase activities.

Escherichia coli helicase II has been purified to near homogeneity from cells harboring a multicopy plasmid containing the structural gene for helicase II, uvrD. In this paper a detailed description of the single-stranded DNA-dependent nucleoside 5'-triphosphatase and helicase reactions catalyzed by helicase II is presented. The results of this study suggest that nucleoside 5'-triphosphate hydrolysis provides the energy required for translocation of the enzyme along single-stranded DNA. Measurements of the rate of ATP hydrolysis using a variety of single-stranded DNAs of known structure and length suggest a processive translocation mechanism for helicase II. Single-stranded DNA coated with either Escherichia coli single-stranded DNA binding protein (SSB) or bacteriophage T4 gene 32 protein fails to support helicase II ATPase activity. Moreover, helicase II is apparently unable to displace a molecule of bound SSB protein from single-stranded DNA when it is encountered in the process of translocation along a single-stranded DNA effector. The helicase reaction has been characterized using an in vitro strand displacement helicase assay. The helicase reaction requires concomitant nucleoside 5'-triphosphatase hydrolysis that is satisfied by the hydrolysis of either rATP or dATP. As the length of duplex DNA present in the partial duplex helicase substrate is increased from 71 base pairs to 343 base pairs, the fraction of duplex DNA molecules that are unwound by helicase II decreases in the absence of any accessory proteins. However, the total number of base pairs of duplex DNA unwound depends primarily on the amount of enzyme added to the helicase reaction and not on the length of the duplex DNA present in the partial duplex DNA substrate. These data suggest the number of base pairs of duplex DNA unwound is directly proportional with the concentration of helicase II in the reaction mixture. In addition, the rate of the unwinding reaction is independent of the length of the duplex DNA available for unwinding. Helicase II has been shown to dissociate from single-stranded DNA molecules infrequently acting as an ATPase. However, the enzyme dissociates from partial duplex helicase substrates more frequently. This suggests a more distributive reaction mechanism on duplex DNA than was observed on single-stranded DNA substrates. The fraction of 343-base pair partial duplex DNA molecules unwound by helicase II can be increased by the addition of appropriate concentrations of E. coli SSB to the reaction. This suggests that helicase II and SSB may act in a concerted reaction to unwind duplex DNA.