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At least 19 recordsLinked to original sources

PubMed1987

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.

Adenosine Triphosphatases↗

PubMed1990

Mapping of helicase and helicase substrate-binding domains on simian virus 40 large T antigen.

We generated fragments of simian virus 40 large tumor antigen (T antigen) by tryptic digestion and assayed them for helicase activity and helicase substrate (mostly single-stranded DNA)-binding activity in order to map the domain sites on the protein. The N-terminal 130 amino acids were not required for either activity, since a 76-kilodalton (kDa) fragment (amino acids 131 to 708) was just as active as intact T antigen. To map the helicase domain further, smaller tryptic fragments were generated. A 66-kDa fragment (131 to about 616) retained some activity, whereas a slightly smaller 62-kDa fragment (137 or 155 to 616) had none. This suggests that the minimal helicase domain maps from residue 131 to approximately residue 616. To map the helicase substrate-binding domain, we tested various fragments in a substrate-binding assay. The smallest fragment for which we could clearly demonstrate activity was a 46-kDa fragment (131 to 517). To determine the relationship between the helicase substrate domain and the origin-binding domain (131 to 257, minimal core region; 131 to 371, optimal region), we performed binding experiments with competitor DNAs present. We found that origin-containing double-stranded DNA was an excellent competitor of the binding of the helicase substrate to T antigen, suggesting that the two domains overlap. Therefore, full helicase activity requires at least a partial origin-binding domain as well as an active ATPase domain. Additionally, we found that the helicase substrate was a poor competitor of origin-binding activity, indicating that T antigen has a much higher affinity to origin sequences than to the helicase substrate.

Animals↗

PubMed1989

Identification of Escherichia coli DNA helicase IV with the use of a DNA helicase activity gel.

A DNA helicase activity gel was developed based on the assumption that DNA helicases could unwind double-stranded DNA in a polyacrylamide matrix. The production of single-stranded DNA was detected by staining the activity gel with acridine orange and visualizing the gel under long-wave UV light. The products of DNA helicase activities appeared as red bands within a green fluorescent background. A novel DNA helicase, called helicase IV, was detected in crude extracts of Escherichia coli with the use of the helicases activity gel assay. The new DNA helicase was purified to near homogeneity. The chromatographic properties and the sequence of its 11 amino-terminal residues proved that helicase IV was distinct from all of the previously described DNA helicases from E. coli.

Amino Acid Sequence↗

PubMed1985

Production of antibodies directed against Escherichia coli helicase III and the molecular cloning of the helicase III gene.

Helicase III from Escherichia coli has been purified to near homogeneity using single-stranded DNA-dependent adenosine nucleoside 5'-triphosphatase activity as an assay to monitor the purification. The denatured form of this 18.5-kilodalton polypeptide, isolated on a preparative polyacrylamide gel run in the presence of sodium dodecyl sulfate, has been used as an antigen to direct the production of rabbit anti-helicase III antibodies. The antibodies obtained fail to inhibit directly either the helicase activity or the DNA-dependent adenosine nucleoside 5'-triphosphatase activity of helicase III. However, when the antigen-antibody complex is removed from solution by binding to Staphylococcus aureus cells with subsequent sedimentation, there is excellent correlation between the loss of both enzymatic activities and the loss of the helicase III polypeptide. The anti-helicase III antibodies have been used as a reagent to probe immunologically a library of E. coli DNA fragments inserted into the plasmid pBR322 for expression of the helicase III antigen. The gene encoding helicase III has been localized on a 2.0-kilobase pair PvuII-EcoRI fragment. Bacterial cells harboring a multicopy plasmid containing this fragment overproduce the helicase III antigen approximately 100-fold.

Adenosine Triphosphatases↗

PubMed1989

Trypsin cleavage in the COOH terminus of the bacteriophage T4 gene 41 DNA helicase alters the primase-helicase activities of the T4 replication complex in vitro.

The bacteriophage T4 gene 41 protein is a 5' to 3' DNA helicase which unwinds DNA ahead of the growing replication fork and, together with the T4 gene 61 protein, also functions as a primase to initiate DNA synthesis on the lagging strand. Proteolytic cleavage by trypsin approximately 20 amino acids from the COOH terminus of the 41 protein produces 41T, a 51,500-dalton fragment (possibly still associated with small COOH-terminal fragments) which still retains the ssDNA-stimulated GTPase (ATPase) activity, the 61 protein-stimulated DNA helicase activity, and the ability to act with 61 protein to synthesize pentaribonucleotide primers. In the absence of the T4 gene 32 ssDNA binding protein, the primase-helicase composed of the tryptic fragment (41T) and 61 proteins efficiently primes DNA synthesis on circular ssDNA templates by the T4 DNA polymerase and the three T4 polymerase accessory proteins. In contrast, the 41T protein is defective as a helicase or a primase component on 32 protein-covered DNA. Thus, unlike the intact protein, 41T does not support RNA-dependent DNA synthesis on 32 protein-covered ssDNA and does not stimulate strand displacement DNA synthesis on a nicked duplex DNA template. High concentrations of 32 protein strongly inhibit RNA primer synthesis with either 41 T or intact 41 protein. The 44/62 and 45 polymerase accessory proteins (and even the 44/62 proteins to some extent) substantially reverse the 32 protein inhibition of RNA primer synthesis with intact 41 protein but not with 41T protein. We propose that the COOH-terminal region of the 41 protein is required for its interaction with the T4 polymerase accessory proteins, permitting the synthesis and utilization of RNA primers and helicase function within the T4 replication complex. When this region is altered, as in 41T protein, the protein is unable to assemble a functional primase-helicase in the replication complex. An easy and rapid purification of T4 41 protein produced by a plasmid encoding this gene (Hinton, D. M., Silver, L. L., and Nossal, N. G. (1985) J. Biol. Chem. 260, 12851-12857) is also described.

Bacteriophage phi X 174↗

PubMed1992

The six conserved helicase motifs of the UL5 gene product, a component of the herpes simplex virus type 1 helicase-primase, are essential for its function.

The UL5 protein of herpes simplex virus type 1, one component of the viral helicase-primase complex, contains six sequence motifs found in all members of a superfamily of DNA and RNA helicases. Although this superfamily contains more than 20 members ranging from bacteria to mammalian cells and their viruses, the importance of these motifs has not been addressed experimentally for any one of them. In this study, we have examined the functional significance of these six motifs for the UL5 protein through the introduction of site-specific mutations resulting in single amino acid substitutions of the most highly conserved residues within each motif. A transient replication complementation assay was used to test the effect of each mutation on the function of the UL5 protein in viral DNA replication. In this assay, a mutant UL5 protein expressed from an expression clone is used to complement a replication-deficient null mutant with a mutation in the UL5 gene for the amplification of herpes simplex virus origin-containing plasmids. Eight mutations in conserved regions and three similar mutations in nonconserved regions of the UL5 gene were analyzed, and the results indicate that all six conserved motifs are essential to the function of UL5 protein in viral DNA replication; on the other hand, mutations in nonconserved regions are tolerated. These data provide the first direct evidence for the importance of these conserved regions in any member of the superfamily of DNA and RNA helicases. In addition, three motif mutations were introduced into the viral genome, and the phenotypic analyses of these mutants are consistent with results from the transient replication complementation assay. The ability of these three mutant UL5 proteins to form specific interactions with other members of the helicase-primase complex, UL8 and UL52, indicates that the functional domains required for replication activity of UL5 are separable from domains responsible for protein-protein interactions. It is anticipated that this type of structure-function analysis will lead to the identification of protein domains that contribute not only to the enzymatic activities of helicase or primase but also to protein-protein interactions within members of the complex.

Amino Acid Sequence↗

PubMed1991

Herpes simplex-1 helicase-primase. Identification of two nucleoside triphosphatase sites that promote DNA helicase action.

Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase comprised of the products of the UL5, UL8, and UL52 genes (Crute, J. J., and Lehman, I. R. (1991) J. Biol. Chem. 266, 4484-4488). A steady state kinetic analysis of the enzyme isolated from HSV-1-infected CV-1 cells or insect cells expressing the enzyme after infection with recombinant baculoviruses has shown it to possess two sites capable of hydrolyzing nucleoside triphosphates in a DNA-dependent manner. One site (Site I) hydrolyzes both ATP and GTP; the second (Site II) hydrolyzes only ATP. These two sites are contained within a subassembly of the helicase-primase formed by coexpression of the UL5 and UL52 genes in insect cells. Sites I and II are activated by separate DNA effector sites, both of which support DNA helicase action. These findings are likely to be of importance in understanding how helicases in general catalyze the unwinding of duplex DNA and, in particular, how the helicase-primase functions at the HSV-1 replication fork.

Adenosine Triphosphatases↗

PubMed1989

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.

Acetates↗

PubMed1988

Further characterization of DNA helicase activity of mouse DNA-dependent adenosinetriphosphatase B (DNA helicase B).

The DNA helicase activity of DNA-dependent ATPase B purified from mouse FM3A cells [Seki, M., Enomoto, T., Hanaoka, F., & Yamada, M. (1987) Biochemistry 26, 2924-2928] has been further characterized. The helicase activity was assayed with partially duplex DNA substrates in which oligonucleotides to be released by the enzyme were radiolabeled. Oligonucleotides with or without phosphate at the 5' termini or with a deoxy- or dideoxyribose at the 3'-terminal nucleotides were displaced by this enzyme with essentially the same efficiency and with the same ATP (and dATP) and Mg2+ requirements. Thus, there was no strict structure requirement for both ends of duplex regions of substrates to be unwound by the enzyme. Shorter strands were released more readily than longer strands up to the length of 140 bases. The attachment of the enzyme to a single-stranded DNA region was a prerequisite for the neighboring duplex to be unwound; the enzyme-catalyzed unwinding was inhibited competitively by the coaddition of single-stranded DNAs which act as cofactors of the ATPase activity. Their activities as the inhibitor of helicase were well correlated with those as the cofactor of ATPase. The helicase B was found to migrate along single-stranded DNA in the 5' to 3' direction by the use of single strands with short duplex regions at both 3' and 5' ends as substrate. A possible role of this enzyme in DNA replication in mammalian cells is discussed.

Adenosine Triphosphatases↗

PubMed1990

Herpes simplex virus helicase-primase: the UL8 protein is not required for DNA-dependent ATPase and DNA helicase activities.

The herpes simplex virus type 1 helicase-primase complex consists of the products of the UL5, UL8 and UL52 genes. We have expressed these proteins in insect cells using baculovirus vectors and studied the requirements for enzymatic activities associated with the DNA unwinding function of the complex. In agreement with a recent report (Dodson, M.S., Crute, J.J., Bruckner, R.C. and Lehman, I.R. 1989, J. Biol. Chem. 264, 20835-20838) we find that DNA-dependent ATPase and DNA helicase activities are assembled in vivo in insect cells triply infected with viruses expressing the UL5, UL8 and UL52 proteins. Moreover, these activities were also detected in cells in which only the UL5 and UL52 products were expressed indicating that the presence of the UL8 protein is essential for neither the ATPase nor helicase activity of the complex.

Adenosine Triphosphatases↗

PubMed1991

Association of DNA helicase and primase activities with a subassembly of the herpes simplex virus 1 helicase-primase composed of the UL5 and UL52 gene products.

Herpes simplex virus 1 encodes a helicase-primase that is composed of the products of the UL5, UL8, and UL52 genes. A stable subassembly consisting of only the UL5 and UL52 gene products has been purified to near homogeneity from insect cells doubly infected with baculovirus recombinant for these two genes. The purified subassembly has the DNA-dependent ATPase, DNA-dependent GTPase, DNA helicase, and DNA primase activities that are characteristic of the three-subunit holoenzyme. The purified UL8 gene product, although required for viral DNA replication, neither exhibits these enzymatic activities nor stably associates with either the UL5 or the UL52 gene product.

Adenosine Triphosphatases↗

PubMed1991

Purification and characterization of two DNA helicases from calf thymus nuclei.

Two DNA helicases from calf thymus nuclei have been purified and characterized. The two proteins, designated as nuclear DNA helicase I and II, were copurified on Bio-Rex 70, DEAE-Sepharose, phosphocellulose and subsequently separated from each other on a heparin-Sepharose column. Final purification of DNA helicase I was achieved on single-stranded DNA-cellulose and that of DNA helicase II on ATP-agarose. On denaturing polyacrylamide gels, nuclear DNA helicase I displayed two polypeptide bands of 170 and 200 kDa; nuclear DNA helicase II also consisted of two bands, in this case of 100 and 130 kDa. Both enzymes catalyzed the unwinding of a DNA primer from a single-stranded DNA template but had different nucleotide requirements for their energy supply. While nuclear DNA helicase I preferred to hydrolyze ATP or dATP to support its unwinding activity, nuclear DNA helicase II could utilize all four rNTPs or dNTPs, though ATP or dATP were still preferred to other nucleotides. ADP, AMP, or adenosine 5'-O-(thiotriphosphate) could not be used by either enzyme in the unwinding reactions. A divalent cation was essential for activity of both enzymes. Nuclear DNA helicase I required 3-5 mM Mg2+ or 1 mM Mn2+ for optimal unwinding. In contrast, nuclear DNA helicase II displayed high activity even at very low concentrations of Mg2+. Nuclear DNA helicase I was stimulated by NaCl, KCl, or potassium acetate up to concentrations of 150 mM; in contrast, nuclear DNA helicase II was strongly inhibited at salt concentrations over 75 mM. Both DNA helicases had an associated ATPase activity. However, while the ATPase activity of nuclear DNA helicase I was stimulated only in presence of single-stranded DNA, the ATPase activity of the nuclear DNA helicase II was stimulated by single-stranded DNA and, even more efficiently, by RNA. Finally, the translocation of both DNA helicases had a polarity from 3' to 5' with respect to the single-stranded DNA template to which the enzymes were bound. A comparison of these DNA helicases with the other reported mammalian DNA helicases will be given. The significance of the association of the two DNA helicases during the process of the purification will be discussed.

Adenosine Triphosphatases↗

PubMed1992

Three new DNA helicases from Saccharomyces cerevisiae.

At least six DNA helicases have been identified during fractionation of extracts from the yeast Saccharomyces cerevisiae. Three of those, DNA helicases B, C, and D, have been further purified and characterized. DNA helicases B and C co-purified with DNA polymerase delta through several chromatographic steps, but were separated from the polymerase by hydrophobic chromatography. DNA helicase D co-purified with Replication Factor C over seven chromatographic steps, and was only separated from it by glycerol gradient centrifugation in the presence of 0.2 M NaCl. All three helicases are DNA dependent ATPases with Km values for ATP of 190 microM, 325 microM, and 60 microM for DNA helicases B, C, and D, respectively. Their DNA helicase activities are comparable. They are 5'-3' helicases and have pH optima of 6.5-7 and Mg2+ optima of 1-2 mM. However, they differ in the nucleotide requirement for helicase action. Whereas all three helicases preferred ATP, dATP, UTP, CTP, and dCTP as cofactors, DNA helicase C also used GTP, but not dTTP. On the other hand, DNA helicase D used dTTP, but not GTP, and DNA helicase B used neither nucleotide as cofactor. These studies allowed us to conclude that DNA helicases B, C, and D are not only distinct enzymes, but also different from two previously identified yeast DNA helicases, the RAD3 protein and ATPase III.

Adenosine Triphosphatases↗

PubMed1992

Four different DNA helicases from calf thymus.

Using a strand displacement assay we have followed DNA helicase activities during the simultaneous isolation of several enzymes from calf thymus such as DNA polymerases alpha, delta, and epsilon, proliferating cell nuclear antigen, and replication factor A. Thus we were able to discriminate and isolate four different DNA helicases called A, B, C, and D. DNA helicase A is identical with the enzyme described earlier (Thömmes, P., and Hübscher, U. (1990) J. Biol. Chem. 265, 14347-14354). The four enzymes can be distinguished by (i) their putative molecular weights after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (ii) glycerol gradient sedimentation under low and high salt conditions, (iii) sensitivity to salt, (iv) binding to DNA, (v) nucleoside- and deoxynucleoside 5'-triphosphate requirements, and (vi) by their direction of movement. DNA helicase A unwinds in the 3'----5' direction on the DNA it was bound to, while DNA helicases B, C, and D do so in the 5'----3' direction. DNA helicase D, and to some extent DNA helicases B and C, are able to unwind long substrates of more than 400 nucleotides. Replication factor A, a single-stranded heterotrimeric DNA binding protein involved in cellular DNA replication and DNA repair stimulates the DNA helicases. The stimulatory effect is most pronounced on DNA helicase A, where replication factor A enables this helicase to unwind longer substrates. DNA helicases B, C, and D are also stimulated by replication factor A. The effect of replication factor A appears to be specific since corresponding single-stranded DNA binding proteins from Escherichia coli and bacteriophage T4 have no or even a negative effect on the four DNA helicases. Heterologous human replication factor A has no stimulatory effect on any of the four DNA helicases suggesting a species specificity of these interactions. Thus it appears that mammalian cells possess, as does E. coli, a variety of different enzymes that can transiently abolish the double helical DNA structure in the cell.

Adenosine Triphosphatases↗

PubMed1992

Inhibition of DNA helicase II unwinding and ATPase activities by DNA-interacting ligands. Kinetics and specificity.

Although DNA helicases play important roles in the processing of DNA, little is known about the effects of DNA-interacting ligands on these helicases. Therefore, the effects of a wide variety of DNA-binding ligands on the unwinding and ATPase reactions catalyzed by Escherichia coli DNA helicase II were examined. DNA minor groove binders and simple DNA intercalators did not inhibit helicase II. However, DNA intercalators, such as mitoxantrone and nogalamycin, which position functionalities in the major groove upon binding duplex DNA, were potent inhibitors of helicase II. To determine the mechanism by which mitoxantrone inhibited helicase II, the unwinding and DNA-dependent ATPase activities of helicase II were measured using a spectrum of double- and single-stranded DNA substrates. Using either a 71-base pair (bp) M13mp7 partially duplexed DNA substrate or a 245-bp bluntended, fully duplexed DNA substrate, the apparent Ki value for inhibition by mitoxantrone of both the unwinding and ATPase reactions was approximately 1 microM for both substrates, suggesting that the mechanism of inhibition of helicase II by mitoxantrone is the same for both substrates and requires the presence of double-stranded structure. To strengthen this conclusion, the ability of mitoxantrone to inhibit the DNA-dependent ATPase activity of helicase II was determined using two single-stranded substrates, poly(dT) and the 245-bp substrate after heat denaturation. Using either substrate, mitoxantrone inhibited the ATPase activity of helicase II far less effectively. Thus, these results indicate that the intercalation of mitoxantrone into double-stranded DNA, with accompanying placement of functionalities in the major groove, generates a complex that impedes helicase II, resulting in both inhibition of ATP hydrolysis and unwinding activity. Furthermore, we report here that DNA-binding ligands inhibit the unwinding activity of helicases I and IV and Rep protein from E. coli, demonstrating that the inhibition observed for helicase II is not unique to this enzyme.

Adenosine Triphosphatases↗

PubMed1991

Isolation of a DNA helicase from HeLa cells requiring the multisubunit human single-stranded DNA-binding protein for activity.

A DNA helicase, dependent on the multisubunit human single-stranded DNA binding protein (HSSB), was isolated from HeLa cells. At low levels of helicase, only the multisubunit SSBs, HSSB and yeast SSB, stimulated DNA helicase activity. At high levels of the helicase Escherichia coli SSB partially substituted for HSSB whereas other SSBs such as T4 gene 32 and adenovirus DNA binding protein did not stimulate the enzyme activity. Maximal activation of helicase activity occurred in the presence of one molecule of HSSB for every 20 nucleotides of single-stranded DNA. The addition of E. coli SSB significantly lowered the amount of HSSB required for strand displacement, suggesting that the HSSB plays at least two roles in the activation of the helicase. One is to bind single-stranded DNA, thereby preventing sequestration of the helicase, the other involves the interaction of the HSSB with the helicase. Monoclonal antibodies that interact with the 70- and 34-kDa subunits of HSSB inhibited its stimulation of the helicase activity. The DNA helicase acted catalytically in displacing duplex DNA and translocated in the 3' to 5' direction. The helicase displaced fragments from both ends of a DNA substrate that contained duplex region at both termini, but the 3' to 5' fragment was displaced 20 times faster than the 5' to 3' fragment. Since this helicase also displaced fully duplex DNA, the release of the 5' to 3' fragment may have occurred by entry of the helicase through the duplex end in a 3' to 5' direction.

Adenosine Triphosphatases↗

PubMed1992

Escherichia coli DNA helicases: mechanisms of DNA unwinding.

DNA helicases are ubiquitous enzymes that catalyse the unwinding of duplex DNA during replication, recombination and repair. These enzymes have been studied extensively; however, the specific details of how any helicase unwinds duplex DNA are unknown. Although it is clear that not all helicases unwind duplex DNA in an identical way, many helicases possess similar properties, which are thus likely to be of general importance to their mechanism of action. For example, since helicases appear generally to be oligomeric enzymes, the hypothesis is presented in this review that the functionally active forms of DNA helicases are oligomeric. The oligomeric nature of helicases provides them with multiple DNA-binding sites, allowing the transient formation of ternary structures, such that at an unwinding fork, the helicase can bind either single-stranded and duplex DNA simultaneously or two strands of single-stranded DNA. Modulation of the relative affinities of these binding sites for single-stranded versus duplex DNA through ATP binding and hydrolysis would then provide the basis for a cycling mechanism for processive unwinding of DNA by helicases. The properties of the Escherichia coli DNA helicases are reviewed and possible mechanisms by which helicases might unwind duplex DNA are discussed in view of their oligomeric structures, with emphasis on the E. coli Rep, RecBCD and phage T7 gene 4 helicases.

Binding Sites↗

PubMed1989

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)

Adenosine Triphosphatases↗