Unwinding Rate Research Articles

DNA Unwinding By DnaB and the DnaB/TAU Complex

The replicative helicase for E. coli is DnaB, a hexameric, ring-shaped motor protein that encircles and translocates along ssDNA, denaturing dsDNA in advance of its motion by sterically occluding the complementary strand to the outside of the ring. Using multiplexed single-molecule measurements with magnetic tweezers, we investigate the translocation and unwinding activities of DnaB. We find that DnaB's interaction with the ss/dsDNA junction is dependent on the geometry of the DNA substrate and applied force, suggesting that the hexamer interacts with the occluded strand during unwinding. We have also found that the structure of the bound nucleotides within DnaB's central channel is highly compact relative to the contour length of ssDNA, consistent with crystal structures of related hexameric helicases. Finally, in all our experiments, we find high variance in the rates of unwinding as well as frequent pausing, indicating that individual hexamers fluctuate among different conformations with different activities. To investigate DnaB's variable nature, we test the effect on helicase activity of interactions with the tau subunit of the Pol III holoenzyme, which is thought to regulate DnaB's unwinding rate.

Stepping Mechanism of Bacteriophage T7 Helicase and Priming Loop Observed Using Single-Molecule Fret Methods

Bacteriophage T7 gp4, a hexameric ring-shaped helicase, serves as a model protein for replicative helicases. T7 helicase couples dTTP hydrolysis to directional movement and DNA strand separation. Previous studies have shown that its DNA unwinding rate and dTTPase rate are sensitive to base pair (bp) stability.We confirm that the unwinding rate of T7 helicase decreases with increasing base pair (bp) stability. For duplexes containing > 35% GC basepairs, we observed stochastic pauses during unwinding.

DNA Structure Specificity Conferred on a Replicative Helicase by Its Loader

Prokaryotic and eukaryotic replicative helicases can translocate along single-stranded and double-stranded DNA, with the central cavity of these multimeric ring helicases being able to accommodate both forms of DNA. Translocation by such helicases along single-stranded DNA results in the unwinding of forked DNA by steric exclusion and appears critical in unwinding of parental strands at the replication fork, whereas translocation over double-stranded DNA has no well-defined role. We have found that the accessory factor, DnaC, that promotes loading of the Escherichia coli replicative helicase DnaB onto single-stranded DNA may also act to confer DNA structure specificity on DnaB helicase. When present in excess, DnaC inhibits DnaB translocation over double-stranded DNA but not over single-stranded DNA. Inhibition of DnaB translocation over double-stranded DNA requires the ATP-bound form of DnaC, and this inhibition is relieved during translocation over single-stranded DNA indicating that stimulation of DnaC ATPase is responsible for this DNA structure specificity. These findings demonstrate that DnaC may provide the DNA structure specificity lacking in DnaB, limiting DnaB translocation to bona fide replication forks. The ability of other replicative helicases to translocate along single-stranded and double-stranded DNA raises the possibility that analogous regulatory mechanisms exist in other organisms.

NS4A regulates the ATPase activity of the NS3 helicase: a novel cofactor role of the non-structural protein NS4A from West Nile virus

Using constructs that encode the individual West Nile virus (WNV) NS3helicase (NS3hel) and NS3hel linked to the hydrophilic, N-terminal 1-50 sequence of NS4A, we demonstrated that the presence of NS4A allows NS3hel to conserve energy in the course of oligonucleotide substrate unwinding. Using NS4A mutants, we also determined that the C-terminal acidic EELPD/E motif of NS4A, which appears to be functionally similar to the acidic EFDEMEE motif of hepatitis C virus (HCV) NS4A, is essential for regulating the ATPase activity of NS3hel. We concluded that, similar to HCV NS4A, NS4A of WNV acts as a cofactor for NS3hel and allows helicase to sustain the unwinding rate of the viral RNA under conditions of ATP deficiency.

Translesion DNA polymerases remodel the replisome and alter the speed of the replicative helicase

All cells contain specialized translesion DNA polymerases that replicate past sites of DNA damage. We find that Escherichia coli translesion DNA polymerase II (Pol II) and polymerase IV (Pol IV) function with DnaB helicase and regulate its rate of unwinding, slowing it to as little as 1 bp/s. Furthermore, Pol II and Pol IV freely exchange with the polymerase III (Pol III) replicase on the beta-clamp and function with DnaB helicase to form alternative replisomes, even before Pol III stalls at a lesion. DNA damage-induced levels of Pol II and Pol IV dominate the clamp, slowing the helicase and stably maintaining the architecture of the replication machinery while keeping the fork moving. We propose that these dynamic actions provide additional time for normal excision repair of lesions before the replication fork reaches them and also enable the appropriate translesion polymerase to sample each lesion as it is encountered.

Fuel Specificity of the Hepatitis C Virus NS3 Helicase

The hepatitis C virus (HCV) NS3 protein is a helicase capable of unwinding duplex RNA or DNA. This study uses a newly developed molecular-beacon-based helicase assay (MBHA) to investigate how nucleoside triphosphates (NTPs) fuel HCV helicase-catalyzed DNA unwinding. The MBHA monitors the irreversible helicase-catalyzed displacement of an oligonucleotide-bound molecular beacon so that rates of helicase translocation can be directly measured in real time. The MBHA reveals that HCV helicase unwinds DNA at different rates depending on the nature and concentration of NTPs in solution, such that the fastest reactions are observed in the presence of CTP followed by ATP, UTP, and GTP. 3′-Deoxy-NTPs generally support faster DNA unwinding, with dTTP supporting faster rates than any other canonical (d)NTP. The presence of an intact NS3 protease domain makes HCV helicase somewhat less specific than truncated NS3 bearing only its helicase region (NS3h). Various NTPs bind NS3h with similar affinities, but each NTP supports a different unwinding rate and processivity. Studies with NTP analogs reveal that specificity is determined by the nature of the Watson–Crick base-pairing region of the NTP base and the nature of the functional groups attached to the 2′ and 3′ carbons of the NTP sugar. The divalent metal bridging the NTP to NS3h also influences observed unwinding rates, with Mn 2+ supporting about 10 times faster unwinding than Mg 2+. Unlike Mg 2+, Mn 2+ does not support HCV helicase-catalyzed ATP hydrolysis in the absence of stimulating nucleic acids. Results are discussed in relation to models for how ATP might fuel the unwinding reaction.

Studying RecBCD Helicase Translocation Along χ-DNA Using Tethered Particle Motion with a Stretching Force

Escherichia coli RecBCD helicase unwinds blunt-end duplex DNA to repair damaged DNA molecules in the homologous recombination pathway. Previous single-molecule experiments showed that RecBCD recognizes an 8 nt DNA sequence, χ, and lowers its unwinding rate afterward under saturating ATP condition. We have developed a single-molecule force-tethered particle motion (FTPM) method, which is modified from the conventional TPM method, and applied it to study RecBCD motion in detail. In the FTPM experiment, a stretching force is applied to the DNA-bead complex that suppresses the bead's Brownian motion, resulting in an improved spatial resolution at long DNA substrates. Based on the equipartition theorem, the mean-square displacement of the bead's Brownian motion measured by FTPM correlates linearly to DNA extension length with a predicted slope, circumventing the difficulties of conventional TPM experiments, such as nonlinearity and low resolution of long DNA substrates. The FTPM method offers the best resolution in the presence of only a small stretching force (0.20 pN). We used the FTPM method to investigate RecBCD helicase motion along 4.1 kb long χ-containing duplex DNA molecules, and observed that the translocation rate of RecBCD changes after the χ sequence under limited ATP concentrations. This suggests that χ recognition by RecBCD does not require saturating ATP conditions, contrary to what was previously reported.

Studying RecBCD Helicase Translocation along Chi-DNA Using Tethered Particle Motion with a Stretching Force

E. coli RecBCD helicase unwinds blunt-end duplex DNA to repair damaged DNA molecules in the homologous recombination pathway. Previous single-molecule experiments show that RecBCD recognizes an 8 nt DNA sequence, chi, and lowers its unwinding rate afterwards under saturating ATP condition. We have developed a single-molecule Force Tethered Particle Motion (FTPM) method, which is modified from the conventional TPM method, and applied it to study RecBCD motion in details. In the FTPM experiment, a stretching force is applied to the DNA-bead complex, and suppressed bead's Brownian motion, resulting in an improved spatial resolution at long DNA substrates. Based on the equipartition theorem, the mean square displacement (MSD) of the bead Brownian motion measured by FTPM correlates linearly to DNA extension length with a predicted slope, circumventing the difficulties, such as non-linearality and low resolution of long DNA substrates in conventional TPM experiments. The FTPM method offers the best resolution (56 bp at 433 bp long DNA) in the presence of only a small stretching force (0.20 pN). We have used the FTPM method to investigate the RecBCD helicase motion along 4.1 kb long chi-containing duplex DNA molecules, and observed that translocation rate of RecBCD changes after chi sequence under limited ATP concentrations. This suggests that chi recognition by RecBCD does not require saturating ATP conditions, contrary to what have been previously reported.

NS3 Helicase from the Hepatitis C Virus Can Function as a Monomer or Oligomer Depending on Enzyme and Substrate Concentrations

Hepatitis C virus NS3 helicase can unwind double-stranded DNA and RNA and has been proposed to form oligomeric structures. Here we examine the DNA unwinding activity of monomeric NS3. Oligomerization was measured by preparing a fluorescently labeled form of NS3, which was titrated with unlabeled NS3, resulting in a hyperbolic increase in fluorescence anisotropy and providing an apparent equilibrium dissociation constant of 236 nm. To evaluate the DNA binding activity of individual subunits within NS3 oligomers, two oligonucleotides were labeled with fluorescent donor or acceptor molecules and then titrated with NS3. Upon the addition of increasing concentrations of NS3, fluorescence energy transfer was observed, which reached a plateau at a 1:1 ratio of NS3 to oligonucleotides, indicating that each subunit within the oligomeric form of NS3 binds to DNA. DNA unwinding was measured under multiple turnover conditions with increasing concentrations of NS3; however, no increase in specific activity was observed, even at enzyme concentrations greater than the apparent dissociation constant for oligomerization. An ATPase-deficient form of NS3, NS3(D290A), was prepared to explore the functional consequences of oligomerization. Under single turnover conditions in the presence of excess concentration of NS3 relative to DNA, NS3(D290A) exhibited a dominant negative effect. However, under multiple turnover conditions in which DNA concentration was in excess to enzyme concentration, NS3(D290A) did not exhibit a dominant negative effect. Taken together, these data support a model in which monomeric forms of NS3 are active. Oligomerization of NS3 occurs, but subunits can function independently or cooperatively, dependent upon the relative concentration of the DNA.

Replisome Dynamics And Polymerase Exchanges Enable Forks To Cope With Various Obsticals

Replication only occurs once in the life of a cell, but the DNA genome is very large and many obstacles are encountered along the way. For example, RNA polymerases are slow and will sometimes be encountered by the replication fork. We find that the E. coli replisome successfully bypasses an in-line RNA polymerase by displacing it and using the transcript as a primer for continued leading strand synthesis. Encounter of the replisome with a head-on RNA polymerase transcribing the lagging strand will also be discussed. Encounter of the replisome with DNA lesions are often avoided during normal conditions though their efficient repair before the replication fork reaches them. But when damage to DNA is extensive, replisome encounters with DNA lesions are likely to be frequent, eventually leading to strand breaks and cell death. We find that the damage induced polymerases, Pol II and Pol IV, form “alternative” replisomes with DnaB helicase and the beta sliding clamp - even in the complete absence of Pol III. Pol II and Pol IV are much slower than Pol III, and we find that these polymerases control the DnaB helicase, coordinating the rate of unwinding to match their speed of synthesis (1-5 ntd/s). These forks are highly stabile, over 10 min, implying that these polymerases stabilize DnaB and preserve the fork, preventing it from collapse. We also find that Pol II, Pol III and Pol IV are highly fluid in their exchange among one another on the clamp at a moving replisome. Overall, these results imply that alternative polymerases may slow the fork during times of extensive DNA damage, thereby acting as a mechanical checkpoint to give more time for lesions to be repaired.

Establishing a Mechanistic Basis for the Large Kinetic Steps of the NS3 Helicase

The NS3 helicase from hepatitis C virus is a prototypical DEx(H/D) RNA helicase. NS3 has been shown to unwind RNA in a discontinuous manner, pausing after long apparent steps of unwinding. We systematically examined the effects of duplex stability and ionic conditions on the periodicity of the NS3 unwinding cycle. The kinetic step size for NS3 unwinding was examined on diverse substrate sequences. The kinetic step size (16 bp/step) was found to be independent of RNA duplex stability and composition, but it exhibited strong dependence on monovalent salt concentration, decreasing to approximately 11 bp/step at low [NaCl]. We addressed this behavior by analyzing the oligomeric state of NS3 at various salt concentrations. Whereas only NS3 oligomers are capable of processive unwinding, we found that monomeric NS3 is an active helicase that unwinds with low processivity. We demonstrate that low salt conditions enhance unwinding by monomeric NS3, which is likely to account for the reduction in apparent step size under low salt conditions. Based on results reported here, as well as available structural and single molecule data, we present an unwinding mechanism that addresses the apparent periodicity of NS3 unwinding, the magnitude of the step size, and that integrates the various stepwise motions observed for NS3. We propose that the large kinetic step size of NS3 unwinding reflects a delayed, periodic release of the separated RNA product strand from a secondary binding site that is located in the NTPase domain (Domain II) of NS3. These findings suggest that the mechanism of product release represents an important and unexplored feature of helicase mechanism.

Impediment of E. coli UvrD by DNA-destabilizing force reveals a strained-inchworm mechanism of DNA unwinding

Escherichia coli UvrD is a non-ring-shaped model helicase, displaying a 3'-5' polarity in DNA unwinding. Using a transverse magnetic tweezer and DNA hairpins, we measured the unwinding kinetics of UvrD at various DNA-destabilizing forces. The multiform patterns of unwinding bursts and the distributions of the off-times favour the mechanism that UvrD unwinds DNA as a dimer. The two subunits of the dimer coordinate to unwind DNA processively. They can jointly switch strands and translocate backwards on the other strand to allow slow (approximately 40 bp/s) rewinding, or unbind simultaneously to allow quick rehybridization. Partial dissociation of the dimer results in pauses in the middle of the unwinding or increases the translocation rate from approximately 40 to approximately 150 nt/s in the middle of the rewinding. Moreover, the unwinding rate was surprisingly found to decrease from approximately 45 to approximately 10 bp/s when the force is increased from 2 to 12 pN. The results lead to a strained-inchworm mechanism in which a conformational change that bends and tenses the ssDNA is required to activate the dimer.

Influence of DNA End Structure on the Mechanism of Initiation of DNA Unwinding by the Escherichiacoli RecBCD and RecBC Helicases

Escherichia coli RecBCD is a bipolar DNA helicase possessing two motor subunits (RecB, a 3′-to-5′ translocase, and RecD, a 5′-to-3′ translocase) that is involved in the major pathway of recombinational repair. Previous studies indicated that the minimal kinetic mechanism needed to describe the ATP-dependent unwinding of blunt-ended DNA by RecBCD in vitro is a sequential n-step mechanism with two to three additional kinetic steps prior to initiating DNA unwinding. Since RecBCD can “melt out” ∼ 6 bp upon binding to the end of a blunt-ended DNA duplex in a Mg 2+-dependent but ATP-independent reaction, we investigated the effects of noncomplementary single-stranded (ss) DNA tails [3′-(dT) 6 and 5′-(dT) 6 or 5′-(dT) 10] on the mechanism of RecBCD and RecBC unwinding of duplex DNA using rapid kinetic methods. As with blunt-ended DNA, RecBCD unwinding of DNA possessing 3′-(dT) 6 and 5′-(dT) 6 noncomplementary ssDNA tails is well described by a sequential n-step mechanism with the same unwinding rate ( mk U = 774 ± 16 bp s − 1 ) and kinetic step size ( m = 3.3 ± 1.3 bp), yet two to three additional kinetic steps are still required prior to initiation of DNA unwinding ( k C = 45 ± 2 s − 1 ). However, when the noncomplementary 5′ ssDNA tail is extended to 10 nt [5′-(dT) 10 and 3′-(dT) 6], the DNA end structure for which RecBCD displays optimal binding affinity, the additional kinetic steps are no longer needed, although a slightly slower unwinding rate ( mk U = 538 ± 24 bp s − 1 ) is observed with a similar kinetic step size ( m = 3.9 ± 0.5 bp). The RecBC DNA helicase (without the RecD subunit) does not initiate unwinding efficiently from a blunt DNA end. However, RecBC does initiate well from a DNA end possessing noncomplementary twin 5′-(dT) 6 and 3′-(dT) 6 tails, and unwinding can be described by a simple uniform n-step sequential scheme, without the need for the additional k C initiation steps, with a similar kinetic step size ( m = 4.4 ± 1.7 bp) and unwinding rate ( mk obs = 396 ± 15 bp s − 1 ). These results suggest that the additional kinetic steps with rate constant k C required for RecBCD to initiate unwinding of blunt-ended and twin (dT) 6-tailed DNA reflect processes needed to engage the RecD motor with the 5′ ssDNA.

Coupling of DNA unwinding to nucleotide hydrolysis in a ring-shaped helicase

The ring-shaped T7 helicase uses the energy of dTTP hydrolysis to perform the mechanical work of translocation and base pair (bp) separation. We have shown that the unwinding rate of T7 helicase decreases with increasing DNA stability. Here, we show that the dTTPase rate also decreases with increasing DNA stability, which indicates close linkage between chemical transition steps and translocation steps of unwinding. We find that the force-producing step during unwinding is not associated with dTTP binding, but dTTP hydrolysis or P(i) release. We determine that T7 helicase extracts approximately 3.7 kcal/mol energy from dTTPase to carry out the work of strand separation. Using this energy, T7 helicase unwinds approximately 4 bp of AT-rich DNA or 1-2 bp of GC-rich DNA. T7 helicase therefore adjusts both its speed and coupling ratio (bp/dTTP) to match the work of DNA unwinding. We discuss the mechanistic implications of the variable bp/dTTP that indicates T7 helicase either undergoes backward movements/futile hydrolysis or unwinds DNA with a variable bp-step size; 'long and fast' steps on AT-rich and 'short and slow' steps on GC-rich DNA.

Effects of Mutagenic and Chain-Terminating Nucleotide Analogs on Enzymes Isolated from Hepatitis C Virus Strains of Various Genotypes

The development of effective therapies for hepatitis C virus (HCV) must take into account genetic variation among HCV strains. Response rates to interferon-based treatments, including the current preferred treatment of pegylated alpha interferon administered with ribavirin, are genotype specific. Of the numerous HCV inhibitors currently in development as antiviral drugs, nucleoside analogs that target the conserved NS5B active site seem to be quite effective against diverse HCV strains. To test this hypothesis, we examined the effects of a panel of nucleotide analogs, including ribavirin triphosphate (RTP) and several chain-terminating nucleoside triphosphates, on the activities of purified HCV NS5B polymerases derived from genotype 1a, 1b, and 2a strains. Unlike the genotype-specific effects on NS5B activity reported previously for nonnucleoside inhibitors (F. Pauwels, W. Mostmans, L. M. Quirynen, L. van der Helm, C. W. Boutton, A. S. Rueff, E. Cleiren, P. Raboisson, D. Surleraux, O. Nyanguile, and K. A. Simmen, J. Virol. 81:6909-6919, 2007), only minor differences in inhibition were observed among the various genotypes; thus, nucleoside analogs that are current drug candidates may be more promising for treatment of a broader variety of HCV strains. We also examined the effects of RTP on the HCV NS3 helicase/ATPase. As with the polymerase, only minor differences were observed among 1a-, 1b-, and 2a-derived enzymes. RTP did not inhibit the rate of NS3 helicase-catalyzed DNA unwinding but served instead as a substrate to fuel unwinding. NS3 added to RNA synthesis reactions relieved inhibition of the polymerase by RTP, presumably due to RTP hydrolysis. These results suggest that NS3 can limit the incorporation of ribavirin into viral RNA, thus reducing its inhibitory or mutagenic effects.

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

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

RNA Unwinding Activity of the Hepatitis C Virus NS3 Helicase Is Modulated by the NS5B Polymerase

Hepatitis C virus (HCV) infects over 170 million persons worldwide. It is the leading cause of liver disease in the U.S. and is responsible for most liver transplants. Current treatments for this infectious disease are inadequate; therefore, new therapies must be developed. Several labs have obtained evidence for a protein complex that involves many of the nonstructural (NS) proteins encoded by the virus. NS3, NS4A, NS4B, NS5A, and NS5B appear to interact structurally and functionally. In this study, we investigated the interaction between the helicase, NS3, and the RNA polymerase, NS5B. Pull-down experiments and surface plasmon resonance data indicate a direct interaction between NS3 and NS5B that is primarily mediated through the protease domain of NS3. This interaction reduces the basal ATPase activity of NS3. However, NS5B stimulates product formation in RNA unwinding experiments under conditions of excess nucleic acid substrate. When the concentrations of NS3 and NS5B are in excess of nucleic acid substrate, NS5B reduces the rate of NS3-catalyzed unwinding. Under pre-steady-state conditions, in which NS3 and substrate concentrations are similar, product formation increased in the presence of NS5B. The increase was consistent with 1:1 complex formed between the two proteins. A fluorescently labeled form of NS3 was used to investigate this interaction through fluorescence polarization binding assays. Results from this assay support interactions that include a 1:1 complex formed between NS3 and NS5B. The modulation of NS3 by NS5B suggests that these proteins may function together during replication of the HCV genome.

Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism

Helicases are enzymes that couple ATP hydrolysis to the unwinding of double-stranded (ds) nucleic acids. The bacteriophage T4 helicase (gp41) is a hexameric helicase that promotes DNA replication within a highly coordinated protein complex termed the replisome. Despite recent progress, the gp41 unwinding mechanism and regulatory interactions within the replisome remain unclear. Here we use a single tethered DNA hairpin as a real-time reporter of gp41-mediated dsDNA unwinding and single-stranded (ss) DNA translocation with 3-base pair (bp) resolution. Although gp41 translocates on ssDNA as fast as the in vivo replication fork ( approximately 400 bp/s), its unwinding rate extrapolated to zero force is much slower ( approximately 30 bp/s). Together, our results have two implications: first, gp41 unwinds DNA through a passive mechanism; second, this weak helicase cannot efficiently unwind the T4 genome alone. Our results suggest that important regulations occur within the replisome to achieve rapid and processive replication.

Stimulation of the DNA unwinding activity of human DNA helicase II/Ku by phosphorylation

The Ku autoantigen is a heterodimeric protein of 70- and 83-kDa subunits, endowed with duplex DNA end-binding capacity and DNA helicase activity (Human DNA Helicase II, HDH II). HDH II/Ku is well established as the DNA binding component, the regulatory subunit as well as a substrate for the DNA-dependent protein kinase DNA-PK, a complex involved in the repair of DNA double-strand breaks and in V(D)J recombination in eukaryotes. The effects of phosphorylation by this kinase on the helicase activity of Escherichia coli-produced HDH II/Ku were studied. The rate of DNA unwinding by recombinant HDH II/Ku heterodimer is stimulated at least fivefold upon phosphorylation by DNA-PK cs. This stimulation is due to the effective transfer of phosphate residues to the helicase rather than the mere presence of the complex. In vitro dephosphorylation of HeLa cellular HDH II/Ku caused a significant decrease in the DNA helicase activity of this enzyme.

The yeast Pif1p DNA helicase preferentially unwinds RNA DNA substrates

Pif1p is the prototypical member of the PIF1 family of DNA helicases, a subfamily of SFI helicases conserved from yeast to humans. Baker's yeast Pif1p is involved in the maintenance of mitochondrial, ribosomal and telomeric DNA and may also have a general role in chromosomal replication by affecting Okazaki fragment maturation. Here we investigate the substrate preferences for Pif1p. The enzyme was preferentially active on RNA–DNA hybrids, as seen by faster unwinding rates on RNA–DNA hybrids compared to DNA–DNA hybrids. When using forked substrates, which have been shown previously to stimulate the enzyme, Pif1p demonstrated a preference for RNA–DNA hybrids. This preferential unwinding could not be correlated to preferential binding of Pif1p to the substrates that were the most readily unwound. Although the addition of the single-strand DNA-binding protein replication protein A (RPA) stimulated the helicase reaction on all substrates, it did not diminish the preference of Pif1p for RNA–DNA substrates. Thus, forked RNA–DNA substrates are the favored substrates for Pif1p in vitro. We discuss these findings in terms of the known biological roles of the enzyme.