Dda helicase tightly couples translocation on single-stranded DNA to unwinding of duplex DNA: Dda is an optimally active helicase

Helicases utilize the energy of ATP hydrolysis to unwind double-stranded DNA while translocating on the DNA. Mechanisms for melting the duplex have been characterized as active or passive, depending on whether the enzyme actively separates the base pairs or simply sequesters single-stranded DNA (ssDNA) that forms due to thermal fraying. Here, we show that Dda translocates unidirectionally on ssDNA at the same rate at which it unwinds double-stranded DNA in both ensemble and single-molecule experiments. Further, the unwinding rate is largely insensitive to the duplex stability and to the applied force. Thus, Dda transduces all of its translocase activity into DNA unwinding activity so that the rate of unwinding is limited by the rate of translocation and that the enzyme actively separates the duplex. Active and passive helicases have been characterized by dividing the velocity of DNA unwinding in base pairs per second (V_un) by the velocity of translocation on ssDNA in nucleotides per second (V_trans). If the resulting fraction is 0.25, then a helicase is considered to be at the lower end of the “active” range. In the case of Dda, the average DNA unwinding velocity was 257 ± 42 bp/s, and the average translocation velocity was 267 ± 15 nt/s. The V_un/V_trans value of 0.96 places Dda in a unique category of being an essentially “perfectly” active helicase.     

Measurement of steady-state kinetic parameters for DNA unwinding by the bacteriophage T4 Dda helicase: use of peptide nucleic acids to trap single-stranded DNA products of helicase reactions

Measurement of steady-state rates of unwinding of double-stranded oligonucleotides by helicases is hampered due to rapid reannealing of the single-stranded DNA products. Including an oligonucleotide in the reaction mixture which can hybridize with one of the single strands can prevent reannealing. However, helicases bind to single-stranded DNA, therefore the additional oligonucleotide can sequester the enzyme, leading to slower observed rates for unwinding. To circumvent this problem, the oligonucleotide that serves as a trap was replaced with a strand of peptide nucleic acid (PNA). Fluorescence polarization was used to determine that a 15mer PNA strand does not bind to the bacteriophage T4 Dda helicase. Steady-state kinetic parameters of unwinding catalyzed by Dda were determined by using PNA as a trapping strand. The substrate consisted of a partial duplex with 15 nt of single-stranded DNA and 15 bp. In the presence of 250 nM substrate and 1 nM Dda, the rate of unwinding in the presence of the DNA trapping strand was 0.30 nM s^–1 whereas the rate was 1.34 nM s^–1 in the presence of the PNA trapping strand. PNA prevents reannealing of single-stranded DNA products, but does not sequester the helicase. This assay will prove useful in defining the complete kinetic mechanism for unwinding of oligonucleotide substrates by this helicase.     

Simultaneous binding to the tracking strand,displaced strand and the duplex of a DNA fork enhances unwinding by Dda helicase

Interactions between helicases and the tracking strand of a DNA substrate are well-characterized; however, the role of the displaced strand is a less understood characteristic of DNA unwinding. Dda helicase exhibited greater processivity when unwinding a DNA fork compared to a ss/ds DNA junction substrate. The lag phase in the unwinding progress curve was reduced for the forked DNA compared to the ss/ds junction. Fewer kinetic steps were required to unwind the fork compared to the ss/ds junction, suggesting that binding to the fork leads to disruption of the duplex. DNA footprinting confirmed that interaction of Dda with a fork leads to two base pairs being disrupted whereas no disruption of base pairing was observed with the ss/ds junction. Neutralization of the phosphodiester backbone resulted in a DNA-footprinting pattern similar to that observed with the ss/ds junction, consistent with disruption of the interaction between Dda and the displaced strand. Several basic residues in the 1A domain which were previously proposed to bind to the incoming duplex DNA were replaced with alanines, resulting in apparent loss of interaction with the duplex. Taken together, these results suggest that Dda interaction with the tracking strand, displaced strand and duplex coordinates DNA unwinding.     

UvsX recombinase and Dda helicase rescue stalled bacteriophage T4 DNA replication forks in vitro

The rescue of stalled replication forks via a series of steps that include fork regression, template switching, and fork restoration often has been proposed as a major mechanism for accurately bypassing non-coding DNA lesions. Bacteriophage T4 encodes almost all of the proteins required for its own DNA replication, recombination, and repair. Both recombination and recombination repair in T4 rely on UvsX, a RecA-like recombinase. We show here that UvsX plus the T4-encoded helicase Dda suffice to rescue stalled T4 replication forks in vitro. This rescue is based on two sequential template-switching reactions that allow DNA replication to bypass a non-coding DNA lesion in a non-mutagenic manner.     

Coupling dTTP hydrolysis with DNA unwinding by the DNA helicase of bacteriophage T7

The DNA helicase encoded by gene 4 of bacteriophage T7 assembles on single-stranded DNA as a hexamer of six identical subunits with the DNA passing through the center of the toroid. The helicase couples the hydrolysis of dTTP to unidirectional translocation on single-stranded DNA and the unwinding of duplex DNA. Phe(523), positioned in a β-hairpin loop at the subunit interface, plays a key role in coupling the hydrolysis of dTTP to DNA unwinding. Replacement of Phe(523) with alanine or valine abolishes the ability of the helicase to unwind DNA or allow T7 polymerase to mediate strand-displacement synthesis on duplex DNA. In vivo complementation studies reveal a requirement for a hydrophobic residue with long side chains at this position. In a crystal structure of T7 helicase, when a nucleotide is bound at a subunit interface, Phe(523) is buried within the interface. However, in the unbound state, it is more exposed on the outer surface of the helicase. This structural difference suggests that the β-hairpin bearing the Phe(523) may undergo a conformational change during nucleotide hydrolysis. We postulate that upon hydrolysis of dTTP, Phe(523) moves from within the subunit interface to a more exposed position where it contacts the displaced complementary strand and facilitates unwinding.     

Non-Watson-Crick interactions between PNA and DNA inhibit the ATPase activity of bacteriophage T4 Dda helicase

Peptide nucleic acid (PNA) is a DNA mimic in which the nucleobases are linked by an N-(2-aminoethyl) glycine backbone. Here we report that PNA can interact with single-stranded DNA (ssDNA) in a non-sequence-specific fashion. We observed that a 15mer PNA inhibited the ssDNA-stimulated ATPase activity of a bacteriophage T4 helicase, Dda. Surprisingly, when a fluorescein-labeled 15mer PNA was used in binding studies no interaction was observed between PNA and Dda. However, fluorescence polarization did reveal non-sequence-specific interactions between PNA and ssDNA. Thus, the inhibition of ATPase activity of Dda appears to result from depletion of the available ssDNA due to non-Watson–Crick binding of PNA to ssDNA. Inhibition of the ssDNA-stimulated ATPase activity was observed for several PNAs of varying length and sequence. To study the basis for this phenomenon, we examined self-aggregation by PNAs. The 15mer PNA readily self-aggregates to the point of precipitation. Since PNAs are hydrophobic, they aggregate more than DNA or RNA, making the study of this phenomenon essential for understanding the properties of PNA. Non-sequence-specific interactions between PNA and ssDNA were observed at moderate concentrations of PNA, suggesting that such interactions should be considered for antisense and antigene applications.     

Chemically modified DNA substrates implicate the importance of electrostatic interactions for DNA unwinding by Dda helicase

Helicase-catalyzed disruption of double-stranded nucleic acid is vital to DNA replication, recombination, and repair in all forms of life. The relative influence of specific chemical interactions between helicase and the substrate over a series of multistep catalytic events is still being defined. To this end, three modified DNA oligonucleotides were designed to serve as substrates for the bacteriophage T4 helicase, Dda. A 5'-DNA-PNA-DNA-3' chimera was synthesized, thereby, conferring both a loss of charge and altering the conformational flexibility of the oligonucleotide. The second modified oligonucleotide possessed a single methylphosphonate replacement on the phosphate backbone, creating a gap in the charge distribution of the substrate. The third modification introduced an abasic site into the oligonucleotide sequence. This abasic site retains the charge distribution of the normal DNA substrate yet alters the conformational flexibility of the oligonucleotide. The loss of a base also serves to disrupt the hydrogen-bonding lattice, the intramolecular base-stacking interactions, as well as the intermolecular base-stacking interactions between aromatic amino acid side chains and the substrate. Our results indicate that a gap in the charge distribution along the backbone of the substrate has a more pronounced effect upon helicase-catalyzed unwinding than does the loss of a single base. While all three substrates exhibited some degree of inhibition, analysis of both pre-steady-state and excess enzyme experiments places a greater value upon the electrostatic interactions between helicase and the substrate.     

Model for helicase translocating along single-stranded DNA and unwinding double-stranded DNA

A model is proposed for non-hexameric helicases translocating along single-stranded (ss) DNA and unwinding double-stranded (ds) DNA. The translocation of a monomeric helicase along ssDNA in weakly-ssDNA-bound state is driven by the Stokes force that is resulted from the conformational change following the transition of the nucleotide state. The unwinding of dsDNA is resulted mainly from the bending of ssDNA induced by the strong binding force of helicase with dsDNA. The interaction force between ssDNA and helicases in weakly-ssDNA-bound state determines whether monomeric helicases such as PcrA can unwind dsDNA or dimeric helicases such as Rep are required to unwind dsDNA.     

Dual functions of single-stranded DNA-binding protein in helicase loading at the bacteriophage T4 DNA replication fork

Semi-conservative DNA synthesis reactions catalyzed by the bacteriophage T4 DNA polymerase holoenzyme are initiated by a strand displacement mechanism requiring gp32, the T4 single-stranded DNA (ssDNA)-binding protein, to sequester the displaced strand. After initiation, DNA helicase acquisition by the nascent replication fork leads to a dramatic increase in the rate and processivity of leading strand DNA synthesis. In vitro studies have established that either of two T4-encoded DNA helicases, gp41 or dda, is capable of stimulating strand displacement synthesis. The acquisition of either helicase by the nascent replication fork is modulated by other protein components of the fork including gp32 and, in the case of the gp41 helicase, its mediator/loading protein gp59. Here, we examine the relationships between gp32 and the gp41/gp59 and dda helicase systems, respectively, during T4 replication using altered forms of gp32 defective in either protein-protein or protein-ssDNA interactions. We show that optimal stimulation of DNA synthesis by gp41/gp59 helicase requires gp32-gp59 interactions and is strongly dependent on the stability of ssDNA binding by gp32. Fluorescence assays demonstrate that gp59 binds stoichiometrically to forked DNA molecules; however, gp59-forked DNA complexes are destabilized via protein-protein interactions with the C-terminal "A-domain" fragment of gp32. These and previously published results suggest a model in which a mobile gp59-gp32 cluster bound to lagging strand ssDNA is the target for gp41 helicase assembly. In contrast, stimulation of DNA synthesis by dda helicase requires direct gp32-dda protein-protein interactions and is relatively unaffected by mutations in gp32 that destabilize its ssDNA binding activity. The latter data support a model in which protein-protein interactions with gp32 maintain dda in a proper active state for translocation at the replication fork. The relationship between dda and gp32 proteins in T4 replication appears similar to the relationship observed between the UL9 helicase and ICP8 ssDNA-binding protein in herpesvirus replication.     

Characterization of the bacteriophage T4 gene 41 DNA helicase

The T4 gene 41 protein and the gene 61 protein function together as a primase-helicase within the seven protein bacteriophage T4 multienzyme complex that replicates duplex DNA in vitro. We have previously shown that the 41 protein is a 5' to 3' helicase that requires a single-stranded region on the 5' side of the duplex to be unwound and is stimulated by the 61 protein (Venkatesan, M., Silver L. L., and Nossal, N. G. (1982) J. biol. Chem. 257, 12426-12434). The 41 protein, in turn, is required for pentamer primer synthesis by the 61 protein. We now show that the 41 protein helicase unwinds a partially duplex DNA molecule containing a performed fork more efficiently than a DNA molecule without a fork. Optimal helicase activity requires greater than 29 nucleotides of single-stranded DNA on the 3' side of the duplex (analogous to the leading strand template). This result suggests the 41 protein helicase interacts with the leading strand template as well as the lagging strand template as it unwinds the duplex region at the replication fork. As the single-stranded DNA on the 3' side of a short duplex (51 base pairs) is lengthened, the stimulation of the 41 protein helicase by the 61 protein is diminished. However, both the 61 protein and a preformed fork are essential for efficient unwinding of longer duplex regions (650 base pairs). These findings suggest that the 61 protein promotes both the initial unwinding of the duplex to form a fork and subsequent unwinding of longer duplexes by the 41 protein. A stable protein-DNA complex, detected by a gel mobility shift of phi X174 single-stranded DNA, requires both the 41 and 61 proteins and a rNTP (preferably rATP or rGTP, the nucleotides with the greatest effect on the helicase activity). In the accompanying paper, we report the altered properties of a proteolytic fragment of the 41 protein helicase and its effect on in vitro DNA synthesis in the T4 multienzyme replication system.     

Recognition of single-stranded DNA by the bacteriophage T4-induced type II topoisomerase

The bacteriophage T4-induced type II DNA topoisomerase has been shown previously to make a reversible double strand break in DNA double helices. In addition, this enzyme is shown here to bind tightly and to cleave single-stranded DNA molecules. The evidence that the single-stranded DNA cleavage activity is intrinsic to the topoisomerase includes: 1) protein linkage to the 5' ends of the newly cleaved DNA; 2) coelution of essentially homogeneous topoisomerase and the DNA cleavage activity; 3) inhibition of both single-stranded DNA cleavage and double-stranded DNA relaxation by oxolinic acid; and 4) inhibition of duplex DNA relaxation by single-stranded DNA. The major cleavage sites on phi X174 viral DNA substrates have been mapped, and several cleavage sites analyzed to determine the exact nucleotide position of cleavage. Major cleavage sites are found very near the base of predicted hairpin helices in the single-stranded DNA substrates, suggesting that DNA secondary structure recognition is important in the cleavage reaction. On the other hand, there are also many weaker cleavage sites with no obvious sequence requirements. Many of the properties of the single-stranded DNA cleavage reaction examined here differ from those of the oxolinic acid-dependent, double-stranded DNA cleavage reaction catalyzed by the same enzyme.     

Displacement of a DNA binding protein by Dda helicase

Bacteriophage T4 Dda helicase has recently been shown to be active as a monomer for unwinding of short duplex oligonucleotides and for displacing streptavidin from 3′-biotinylated oligonucleotides. However, its activity for streptavidin displacement and DNA unwinding has been shown to increase as the number of Dda molecules bound to the substrate molecule increases. A substrate was designed to address the ability of Dda to displace DNA binding proteins. A DNA binding site for the Escherichia coli trp repressor was introduced into an oligonucleotide substrate for Dda helicase containing single-stranded overhang. Here we show that a Dda monomer is insufficient to displace the E.coli trp repressor from dsDNA under single turnover conditions, although the substrate is unwound and the repressor displaced when the single-stranded overhang is long enough to accommodate two Dda molecules. The quantity of product formed increases when the substrate is able to accommodate more than two Dda molecules. These results indicate that multiple Dda molecules act to displace DNA binding proteins in a manner that correlates with the DNA unwinding activity and streptavidin displacement activity. We suggest a cooperative inchworm model to describe the activities of Dda helicase.     

Mutations of bacteriophage T4 59 helicase loader defective in binding fork DNA and in interactions with T4 32 single-stranded DNA-binding protein

Bacteriophage T4 gene 59 protein greatly stimulates the loading of the T4 gene 41 helicase in vitro and is required for recombination and recombination-dependent DNA replication in vivo. 59 protein binds preferentially to forked DNA and interacts directly with the T4 41 helicase and gene 32 single-stranded DNA-binding protein. The helicase loader is an almost completely alpha-helical, two-domain protein, whose N-terminal domain has strong structural similarity to the DNA-binding domains of high mobility group proteins. We have previously speculated that this high mobility group-like region may bind the duplex ahead of the fork, with the C-terminal domain providing separate binding sites for the fork arms and at least part of the docking area for the helicase and 32 protein. Here, we characterize several mutants of 59 protein in an initial effort to test this model. We find that the I87A mutation, at the position where the fork arms would separate in the model, is defective in binding fork DNA. As a consequence, it is defective in stimulating both unwinding by the helicase and replication by the T4 system. 59 protein with a deletion of the two C-terminal residues, Lys(216) and Tyr(217), binds fork DNA normally. In contrast to the wild type, the deletion protein fails to promote binding of 32 protein on short fork DNA. However, it binds 32 protein in the absence of DNA. The deletion is also somewhat defective in stimulating unwinding of fork DNA by the helicase and replication by the T4 system. We suggest that the absence of the two terminal residues may alter the configuration of the lagging strand fork arm on the surface of the C-terminal domain, so that it is a poorer docking site for the helicase and 32 protein.     

DNA helicase requirements for DNA replication during bacteriophage T4 infection.

The lytic bacteriophage T4 uses multiple mechanisms to initiate the replication of its DNA. Initiation occurs predominantly at replication origins at early times of infection, but there is a switch to genetic recombination-dependent initiation at late times of infection. The T4 insertion-substitution system was used to create a deletion in the T4 dda gene, which encodes a 5'-3' DNA helicase that stimulates both DNA replication and recombination reactions in vitro. The deletion caused a delay in T4 DNA synthesis at early times of infection, suggesting that the Dda protein is involved in the initiation of origin-dependent DNA synthesis. However, DNA synthesis eventually reached nearly wild-type levels, and the final number of phages produced per bacterium was similar to that of the wild type. When the dda mutant phage also contained a mutation in T4 gene 59 (a gene normally required only for recombination-dependent DNA replication), essentially no DNA was synthesized. Recent in vitro studies have shown that the gene 59 protein loads a component of the primosome, the T4 gene 41 DNA helicase, onto DNA. A molecular model for replication initiation is presented that is based on our genetic data.     

The unwinding of duplex regions in DNA by the simian virus 40 large tumor antigen-associated DNA helicase activity

The DNA helicase activity associated with purified simian virus 40 (SV40) large tumor (T) antigen has been examined. A variety of DNA substrates were used to characterize this ATP-dependent activity. Linear single-stranded M13 DNA containing short duplex regions at both ends was used to show that SV40 T antigen helicase displaced the short, annealed fragment by unwinding in a 3' to 5' direction. Three different partial duplex structures consisting of 71-, 343-, and 851-nucleotide long fragments annealed to M13 single-stranded circular DNA were used to show that SV40 T antigen can readily unwind short and long duplex regions with almost equal facility. ATP and MgCl2 were required for this reaction. With the exception of GTP, dGTP, and CTP, the other common nucleoside triphosphates substituted for ATP with varied efficiency, while adenosine 5'-O-(thiotriphosphate) was inactive. The T antigen helicase activity was also examined using completely duplex DNA fragments (approximately 300 base pairs) with or without the SV40 origin sequence as substrates. In reactions containing small amounts (0.6 ng) of DNA, the ATP-dependent unwinding of duplex DNA fragments occurred with no dependence on the origin sequence. This reaction was stimulated 5- to 6-fold by the addition of the Escherichia coli single-stranded DNA-binding protein. When competitor DNA was added so that the ratio of SV40 T antigen to DNA was reduced 1000-fold, only DNA fragments containing a functional SV40 origin of replication were unwound. This reaction was dependent on ATP, MgCl2, and a DNA-binding protein, and was stimulated by inorganic phosphate or creatine phosphate. The origin sequence requirements for the unwinding reaction were the same as those for replication (the 64-base pair sequence present at T antigen binding site 2). Thus, under specified conditions, only duplex DNA fragments containing an intact SV40 core origin were unwound. In contrast, unwinding of partially duplex segments of DNA flanked by single-stranded regions can occur with no sequence specificity.     

Single-molecule studies reveal dynamics of DNA unwinding by the ring-shaped T7 helicase

Helicases are molecular motors that separate DNA strands for efficient replication of genomes. We probed the kinetics of individual ring-shaped T7 helicase molecules as they unwound double-stranded DNA (dsDNA) or translocated on single-stranded DNA (ssDNA). A distinctive DNA sequence dependence was observed in the unwinding rate that correlated with the local DNA unzipping energy landscape. The unwinding rate increased approximately 10-fold (approaching the ssDNA translocation rate) when a destabilizing force on the DNA fork junction was increased from 5 to 11 pN. These observations reveal a fundamental difference between the mechanisms of ring-shaped and nonring-shaped helicases. The observed force-velocity and sequence dependence are not consistent with a simple passive unwinding model. However, an active unwinding model fully supports the data even though the helicase on its own does not unwind at its optimal rate. This work offers insights into possible ways helicase activity is enhanced by associated proteins.     

DNA helicase mutants of bacteriophage T4 that are defective in DNA recombination

We previously isolated a plasmid-borne, recombination-deficient mutant derivative of the bacteriophage T4 DNA helicase gene 41. We have now transferred this 41rrh1 mutation into the phage genome in order to characterize its mutational effects further. The mutation impairs a recombination pathway that is distinct from the pathway involving uvsX, which is essential for strand transfer, and it also eliminates most homologous recombination between a plasmid and the T4 genome. Although 41rrh1 does not affect T4 DNA replication from some origins, it does inactivate plasmid replication that is dependent on ori(uvsY) and ori(34), as well as recombination-dependent DNA replication. Combination of 41rrh1 with some uvsX alleles is lethal. Based on these results, we propose that gene 41 contributes to DNA recombination through its role in DNA replication. Received: 3 February 1999 / Accepted: 20 July 1999     

Evidence for a functional monomeric form of the bacteriophage T4 DdA helicase. Dda does not form stable oligomeric structures

The active form of many helicases is oligomeric, possibly because oligomerization provides multiple DNA binding sites needed for unwinding of DNA. In order to understand the mechanism of the bacteriophage T4 Dda helicase, the potential requirement for oligomerization was investigated. Chemical cross-linking and high pressure gel filtration chromatography provided little evidence for the formation of an oligomeric species. The specific activity for ssDNA stimulated ATPase activity was independent of Dda concentration. Dda was mutated to produce an ATPase-deficient protein (K38A Dda) by altering a residue within a conserved, nucleotide binding loop. The helicase activity of K38A Dda was inactivated, although DNA binding properties were similar to Dda. In the presence of limiting DNA substrate, the rate of unwinding by Dda was not changed; however, the amplitude of product formation was reduced in the presence of increasing concentrations of K38A Dda. The reduction was between that expected for a monomeric or dimeric helicase based on simple competition for substrate binding. When unwinding of DNA was measured in the presence of excess DNA substrate, addition of K38A Dda caused no reduction in the observed rate for strand separation. Taken together, these results indicate that oligomerization of Dda is not required for DNA unwinding.