Duplex unwinding and ATPase activities of the DEAD-box helicase eIF4A are coupled by eIF4G and eIF4B

Eukaryotic initiation factor (eIF) 4A is a DEAD-box helicase that stimulates translation initiation by unwinding mRNA secondary structure. The accessory proteins eIF4G, eIF4B, and eIF4H enhance the duplex unwinding activity of eIF4A, but the extent to which they modulate eIF4A activity is poorly understood. Here, we use real-time fluorescence assays to determine the kinetic parameters of duplex unwinding and ATP hydrolysis by these initiation factors. To ensure efficient duplex unwinding, eIF4B and eIF4G cooperatively activate the duplex unwinding activity of eIF4A. Our data reveal that eIF4H is much less efficient at stimulating eIF4A unwinding activity than eIF4B, implying that eIF4H is not able to completely substitute for eIF4B in duplex unwinding. By monitoring unwinding and ATPase assays under identical conditions, we demonstrate that eIF4B couples the ATP hydrolysis cycle of eIF4A with strand separation, thereby minimizing nonproductive unwinding events. Using duplex substrates with altered GC contents but similar predicted thermal stabilities, we further show that the rate of formation of productive unwinding complexes is strongly influenced by the local stability per base pair, in addition to the stability of the entire duplex. This finding explains how a change in the GC content of a hairpin is able to influence translation initiation while maintaining the overall predicted thermal stability.     

Mechanism of nucleic acid unwinding by SARS-CoV helicase

The non-structural protein 13 (nsp13) of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is a helicase that separates double-stranded RNA (dsRNA) or DNA (dsDNA) with a 5' → 3' polarity, using the energy of nucleotide hydrolysis. We determined the minimal mechanism of helicase function by nsp13. We showed a clear unwinding lag with increasing length of the double-stranded region of the nucleic acid, suggesting the presence of intermediates in the unwinding process. To elucidate the nature of the intermediates we carried out transient kinetic analysis of the nsp13 helicase activity. We demonstrated that the enzyme unwinds nucleic acid in discrete steps of 9.3 base-pairs (bp) each, with a catalytic rate of 30 steps per second. Therefore the net unwinding rate is ~280 base-pairs per second. We also showed that nsp12, the SARS-CoV RNA-dependent RNA polymerase (RdRp), enhances (2-fold) the catalytic efficiency of nsp13 by increasing the step size of nucleic acid (RNA/RNA or DNA/DNA) unwinding. This effect is specific for SARS-CoV nsp12, as no change in nsp13 activity was observed when foot-and-mouth-disease virus RdRp was used in place of nsp12. Our data provide experimental evidence that nsp13 and nsp12 can function in a concerted manner to improve the efficiency of viral replication and enhance our understanding of nsp13 function during SARS-CoV RNA synthesis.     

Formation of DNA triple helices inhibits DNA unwinding by the SV40 large T-antigen helicase.

Previous studies have indicated that d(TC)n.d(GA)n microsatellites may serve as arrest signals for mammalian DNA replication through the ability of such sequences to form DNA triple helices and thereby inhibit replication enzymes. To further test this hypothesis, we examined the ability of d(TC)i.d(GA)i.d(TC)i triplexes to inhibit DNA unwinding in vitro by a model eukaryotic DNA helicase, the SV40 large T-antigen. DNA substrates that were able to form triplexes, and non-triplex-forming control substrates, were tested. We found that the presence of DNA triplexes, as assayed by endonuclease S1 and osmium tetroxide footprinting, significantly inhibited DNA unwinding by T-antigen. Strong inhibition was observed not only at acidic pH values, in which the triplexes were most stable, but also at physiological pH values in the range 6.9-7.2. Little or no inhibition was detected at pH 8.7. Based on these results, and on previous studies of DNA polymerases, we suggest that DNA triplexes may form in vivo and cause replication arrest through a dual inhibition of duplex unwinding by DNA helicases and of nascent strand synthesis by DNA polymerases. DNA triplexes also have the potential to inhibit recombination and repair processes in which helicases and polymerases are involved.     

Coupling translocation with nucleic acid unwinding by NS3 helicase

We present a semiquantitative model for translocation and unwinding activities of monomeric nonstructural protein 3 (NS3) helicase. The model is based on structural, biochemical, and single-molecule measurements. The model predicts that the NS3 helicase actively unwinds duplex by reducing more than 50% the free energy that stabilizes base pairing/stacking. The unwinding activity slows the movement of the helicase in a sequence-dependent manner, lowering the average unwinding efficiency to less than 1 bp per ATP cycle. When bound with ATP, the NS3 helicase can display significant translocational diffusion. This increases displacement fluctuations of the helicase, decreases the average unwinding efficiency, and enhances the sequence dependence. Also, interactions between the helicase and the duplex stabilize the helicase at the junction, facilitating the helicase's unwinding activity while preventing it from dissociating. In the presence of translocational diffusion during active unwinding, the dissociation rate of the helicase also exhibits sequence dependence. Based on unwinding velocity fluctuations measured from single-molecule experiments, we estimate the diffusion rate to be on the order of 10 s^− 1 . The generic features of coupling single-stranded nucleic acid translocation with duplex unwinding presented in this work may apply generally to a class of helicases.     

G-quadruplex formation at the 3' end of telomere DNA inhibits its extension by telomerase, polymerase and unwinding by helicase

Telomere G-quadruplex is emerging as a promising anti-cancer target due to its inhibition to telomerase, an enzyme expressed in more than 85% tumors. Telomerase-mediated telomere extension and some other reactions require a free 3' telomere end in single-stranded form. G-quadruplex formation near the 3' end of telomere DNA can leave a 3' single-stranded tail of various sizes. How these terminal structures affect reactions at telomere end is not clear. In this work, we studied the 3' tail size-dependence of telomere extension by either telomerase or the alternative lengthening of telomere (ALT) mechanism as well as telomere G-quadruplex unwinding. We show that these reactions require a minimal tail of 8, 12 and 6 nt, respectively. Since we have shown that G-quadruplex tends to form at the farthest 3' distal end of telomere DNA leaving a tail of no more than 5 nt, these results imply that G-quadruplex formation may play a role in regulating reactions at the telomere ends and, as a result, serve as effective drug target for intervening telomere function.     

Human mitochondrial DNA helicase TWINKLE is both an unwinding and annealing helicase

TWINKLE is a nucleus-encoded human mitochondrial (mt)DNA helicase. Point mutations in TWINKLE are associated with heritable neuromuscular diseases characterized by deletions in the mtDNA. To understand the biochemical basis of these diseases, it is important to define the roles of TWINKLE in mtDNA metabolism by studying its enzymatic activities. To this end, we purified native TWINKLE from Escherichia coli. The recombinant TWINKLE assembles into hexamers and higher oligomers, and addition of MgUTP stabilizes hexamers over higher oligomers. Probing into the DNA unwinding activity, we discovered that the efficiency of unwinding is greatly enhanced in the presence of a heterologous single strand-binding protein or a single-stranded (ss) DNA that is complementary to the unwound strand. We show that TWINKLE, although a helicase, has an antagonistic activity of annealing two complementary ssDNAs that interferes with unwinding in the absence of gp2.5 or ssDNA trap. Furthermore, only ssDNA and not double-stranded (ds)DNA competitively inhibits the annealing activity, although both DNAs bind with high affinities. This implies that dsDNA binds to a site that is distinct from the ssDNA-binding site that promotes annealing. Fluorescence anisotropy competition binding experiments suggest that TWINKLE has more than one ssDNA-binding sites, and we speculate that a surface-exposed ssDNA-specific site is involved in catalyzing DNA annealing. We propose that the strand annealing activity of TWINKLE may play a role in recombination-mediated replication initiation found in the mitochondria of mammalian brain and heart or in replication fork regression during repair of damaged DNA replication forks.     

Conserved helicase domain of human RecQ4 is required for strand annealing-independent DNA unwinding

Humans have five members of the well conserved RecQ helicase family: RecQ1, Bloom syndrome protein (BLM), Werner syndrome protein (WRN), RecQ4, and RecQ5, which are all known for their roles in maintaining genome stability. BLM, WRN, and RecQ4 are associated with premature aging and cancer predisposition. Of the three, RecQ4's biological and cellular roles have been least thoroughly characterized. Here we tested the helicase activity of purified human RecQ4 on various substrates. Consistent with recent results, we detected ATP-dependent RecQ4 unwinding of forked duplexes. However, our results provide the first evidence that human RecQ4's unwinding is independent of strand annealing, and that it does not require the presence of excess ssDNA. Moreover, we demonstrate that a point mutation of the conserved lysine in the Walker A motif abolished helicase activity, implying that not the N-terminal portion, but the helicase domain is solely responsible for the enzyme's unwinding activity. In addition, we demonstrate a novel stimulation of RecQ4's helicase activity by replication protein A, similar to that of RecQ1, BLM, WRN, and RecQ5. Together, these data indicate that specific biochemical activities and protein partners of RecQ4 are conserved with those of the other RecQ helicases.     

DNA damage-induced translocation of the Werner helicase is regulated by acetylation

Werner syndrome is a rare autosomal recessive disorder involving the premature appearance of features reminiscent of human aging. Werner syndrome occurs by mutation of the WRN gene, encoding a DNA helicase. WRN contributes to the induction of the p53 tumor suppressor protein by various DNA damaging agents. Here we show that UV exposure leads to extensive translocation of WRN from the nucleolus to nucleoplasmic foci in a dose-dependent manner. Ionizing radiation also induces WRN translocation, albeit milder, partially through activation of the ATM kinase. The nucleoplasmic foci to which WRN is recruited display partial colocalization with PML nuclear bodies. The translocation of WRN into nucleoplasmic foci is significantly enhanced by the protein deacetylase inhibitor, Trichostatin A. Moreover, Trichostatin A delays the re-entry of WRN into the nucleolus at late times after irradiation. WRN is acetylated in vivo, and this is markedly stimulated by the acetyltransferase p300. Importantly, p300 augments the translocation of WRN into nucleoplasmic foci. These findings support the notion that WRN plays a role in the cellular response to DNA damage and suggest that the activity of WRN is modulated by DNA damage-induced post-translational modifications of WRN and possibly WRN-interacting proteins.     

Mechanism of translocation and kinetics of DNA unwinding by the helicase RecG

RecG is a DNA helicase involved in the repair of damage at a replication fork and catalyzes the reversal of the fork to a point beyond the damage in the template strand. It unwinds duplex DNA in reactions that are coupled to ATP hydrolysis. The kinetic mechanism of duplex DNA unwinding by RecG was analyzed using a quantitative fluorescence assay based on the process of contact quenching between Cy3 and Dabcyl groups attached to synthetic three-way DNA junctions. The data show that the protein moves at a rate of 26 bp s(-1) along the duplex DNA during the unwinding process. RecG ATPase activity during translocation indicates a constant rate of 7.6 s(-1), measured using a fluorescent phosphate sensor, MDCC-PBP. These two rates imply a movement of approximately 3 bp per ATP hydrolyzed. We demonstrate in several trapping experiments that RecG remains attached to DNA after translocation to the end of the arm of the synthetic DNA junction. ATPase activity continues after translocation is complete. Dissociation of RecG from the product DNA occurs only very slowly, suggesting strong interactions between them. The data support the idea that interactions of the duplex template arm with the protein are the major sites of binding and production of translocation.     

Bent out of shape: RNA unwinding by the DEAD-box helicase Vasa

RNA helicases of the DEAD-box family are involved in essentially all RNA-dependent cellular processes. In this issue of Cell, Sengoku et al. (2006) solve the structure of the DEAD-box protein Vasa in the presence of RNA and a nonhydrolyzable ATP analog and provide important insights into how this family of helicases unwinds RNA.     

Increasing the length of the single-stranded overhang enhances unwinding of duplex DNA by bacteriophage T4 Dda helicase

Dda has been shown previously to be active as a monomer for DNA unwinding [Nanduri et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 14722] and streptavidin displacement [Byrd and Raney (2004) Nat. Struct. Mol. Biol. 11, 531]. However, its activity for streptavidin displacement increased as a function of the length of single-stranded DNA. We investigated whether Dda exhibited enhanced DNA unwinding of partially duplex DNA substrates as a function of increasing the length of the single-stranded overhangs. DNA substrates were prepared containing 16 base pairs and single-stranded overhangs of 4, 6, 8, 12, 16, 20, and 24 nucleotides. Under single turnover conditions in the presence of excess enzyme, the quantity of DNA unwound increased significantly as the length of the single strand overhang increased. Increased processivity was observed when the DNA substrate contained longer single-stranded overhangs. Equilibrium binding studies indicated that Dda bound to the substrates containing the longer overhangs significantly better than the shorter overhangs. To determine whether the increased processivity for unwinding was due to multiple molecules of Dda or due to the increased binding affinity to the longer overhangs, DNA unwinding was conducted under pre-steady-state conditions, which favor binding of monomeric Dda. Under pre-steady-state conditions, the quantity of product decreased somewhat as the single-stranded length increased, from 12 to 24 nucleotides. Thus, when monomeric Dda is required to translocate longer distances prior to unwinding, processivity is lowered. Taken together, these results indicate that enhanced binding to the longer single-stranded overhangs was not responsible for enhanced processivity under conditions of excess enzyme. Rather, multiple molecules of Dda bound to the same substrate exhibit greater processivity for DNA unwinding.     

ATPase cycle and DNA unwinding kinetics of RecG helicase

The superfamily 2 bacterial helicase, RecG, is a monomeric enzyme with a role in DNA repair by reversing stalled replication forks. The helicase must act specifically and rapidly to prevent replication fork collapse. We have shown that RecG binds tightly and rapidly to four-strand oligonucleotide junctions, which mimic a stalled replication fork. The helicase unwinds such DNA junctions with a step-size of approximately four bases per ATP hydrolyzed. To gain an insight into this mechanism, we used fluorescent stopped-flow and quenched-flow to measure individual steps within the ATPase cycle of RecG, when bound to a DNA junction. The fluorescent ATP analogue, mantATP, was used throughout to determine the rate limiting steps, effects due to DNA and the main states in the cycle. Measurements, when possible, were also performed with unlabeled ATP to confirm the mechanism. The data show that the chemical step of hydrolysis is the rate limiting step in the cycle and that this step is greatly accelerated by bound DNA. The ADP release rate is similar to the cleavage rate, so that bound ATP and ADP would be the main states during the ATP cycle. Evidence is provided that the main structural rearrangements, which bring about DNA unwinding, are linked to these states.     

WRN helicase expression in Werner syndrome cell lines

Mutations in the chromosome 8p WRN gene cause Werner syndrome (WRN), a human autosomal recessive disease that mimics premature aging and is associated with genetic instability and an increased risk of cancer. All of the WRN mutations identified in WRN patients are predicted to truncate the WRN protein with loss of a C-terminal nuclear localization signal. However, many of these truncated proteins would retain WRN helicase and/or nuclease functional domains. We have used a combination of immune blot and immune precipitation assays to quantify WRN protein and its associated 3′→5′ helicase activity in genetically characterized WRN patient cell lines. None of the cell lines from patients harboring four different WRN mutations contained detectable WRN protein or immune-precipitable WRN helicase activity. Cell lines from WRN heterozygous individuals contained reduced amounts of both WRN protein and helicase activity. Quantitative immune blot analyses indicate that both lymphoblastoid cell lines and fibroblasts contain ~6 × 10⁴ WRN molecules/cell. Our results indicate that most WRN mutations result in functionally equivalent null alleles, that WRN heterozygote effects may result from haploinsufficiency and that successful modeling of WRN pathogenesis in the mouse or in other model systems will require the use of WRN mutations that eliminate WRN protein expression.     

Rapid unwinding of triplet repeat hairpins by Srs2 helicase of Saccharomyces cerevisiae

Expansions of trinucleotide repeats cause at least 15 heritable human diseases. Single-stranded triplet repeat DNA in vitro forms stable hairpins in a sequence-dependent manner that correlates with expansion risk in vivo. Hairpins are therefore considered likely intermediates during the expansion process. Unwinding of a hairpin by a DNA helicase would help protect against expansions. Yeast Srs2, but not the RecQ homolog Sgs1, blocks expansions in vivo in a manner largely dependent on its helicase function. The current study tested the idea that Srs2 would be faster at unwinding DNA substrates with an extrahelical triplet repeat hairpin embedded in a duplex context. These substrates should mimic the relevant intermediate structure thought to occur in vivo. Srs2 was faster than Sgs1 at unwinding several substrates containing triplet repeat hairpins or another structured loop. In contrast, control substrates with an unstructured loop or a Watson–Crick duplex were unwound equally well by both enzymes. Results with a fluorescently labeled, three-way junction showed that Srs2 unwinding proceeds unabated through extrahelical triplet repeats. In summary, Srs2 maintains its facile unwinding of triplet repeat hairpins embedded within duplex DNA, supporting the genetic evidence that Srs2 is a key helicase in Saccharomyces cerevisiae for preventing expansions.     

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.     

Phosphate release contributes to the rate-limiting step for unwinding by an RNA helicase

RNA helicases function in numerous aspects of RNA biology. These enzymes are RNA-stimulated ATPases that translocate on RNA and unwind or remodel structured RNA in an ATP-dependent fashion. How ATP and the ATPase cycle fuel the work performed by helicases is not completely clear. The hepatitis C virus RNA helicase, NS3, is an important model system for this class of enzymes. NS3 binding to a single-/double-strand RNA or DNA junction leads to ATP-independent melting of the duplex and formation of a complex capable of ATP-dependent unwinding by using a spring-loaded mechanism. We have established an RNA substrate for NS3 that can be unwound in a single sub-step. Our studies are consistent with a model in which a single ATP binding and/or hydrolysis event sets the unwinding spring and phosphate dissociation contributes to release of the spring, thereby driving the power stroke used for unwinding.