Helicase Reaction Research Articles

Measuring the impact of cofactors on RNA helicase activities.

RNA helicases are the largest class of enzymes in eukaryotic RNA metabolism. In cells, protein cofactors regulate RNA helicase functions and impact biochemical helicase activities. Understanding how cofactors affect enzymatic activities of RNA helicases is thus critical for delineating physical roles and regulation of RNA helicases in cells. Here, we discuss approaches and conceptual considerations for the design of experiments to interrogate cofactor effects on RNA helicase activities in vitro. We outline the mechanistic frame for helicase reactions, discuss optimization of experimental setup and reaction parameters for measuring cofactor effects on RNA helicase activities, and provide basic guides to data analysis and interpretation. The described approaches are also instructive for determining the impact of small molecule inhibitors of RNA helicases.

Characterization of the MCM homohexamer from the thermoacidophilic euryarchaeon Picrophilus torridus.

The typical archaeal MCM exhibits helicase activity independently in vitro. This study characterizes MCM from the euryarchaeon Picrophilus torridus. While PtMCM hydrolyzes ATP in DNA-independent manner, it displays very poor ability to unwind DNA independently, and then too only under acidic conditions. The protein exists stably in complex with PtGINS in whole cell lysates, interacting directly with PtGINS under neutral and acidic conditions. GINS strongly activates MCM helicase activity, but only at low pH. In consonance with this, PtGINS activates PtMCM-mediated ATP hydrolysis only at low pH, with the amount of ATP hydrolyzed during the helicase reaction increasing more than fifty-fold in the presence of GINS. While the stimulation of MCM-mediated helicase activity by GINS has been reported in MCMs from P.furiosus, T.kodakarensis, and very recently, T.acidophilum, to the best of our knowledge, this is the first report of an MCM helicase demonstrating DNA unwinding activity only at such acidic pH, across all archaea and eukaryotes. PtGINS may induce/stabilize a conducive conformation of PtMCM under acidic conditions, favouring PtMCM-mediated DNA unwinding coupled to ATP hydrolysis. Our findings underscore the existence of divergent modes of replication regulation among archaea and the importance of investigating replication events in more archaeal organisms.

Identification of the PcrA DNA helicase reaction pathway by applying advanced targeted molecular dynamic simulations

PcrA DNA helicase uses the free energy of hydrolysis and binding of ATP to unwind double-stranded DNA (ds-DNA). There are two states of PcrA, termed the substrate and product complexes and, through the conformational changes between these two states, PcrA moves along ds-DNA and separates the two strands. In this study, two different methods, namely chain minimisation (CM, less reliable method) and auto targeted molecular dynamic (TMD) simulation (more reliable), were performed to generate two different initial reaction pathways between these two states, and then fixed root mean square distance (RMSD) TMD simulation was performed to optimise these two initial pathways. In general, the two optimised pathways share very similar major conformational changes, but are different in the minor motions. The potential energy profiles of the two improved pathways are generally similar, but the one generated by the improved TMD path is slightly lower. Considering the poor reliability of the initial path generated by CM and insignificant improvements of the auto-TMD path, our study suggests that fixed RMSD TMD simulation can generate reliable reaction pathways, but the different initial paths still have some influence on the detailed conformational analysis.

Plasmodium falciparum RuvB2 translocates in 5′–3′ direction, relocalizes during schizont stage and its enzymatic activities are up regulated by RuvB3 of the same complex

Two similar proteins RuvB like1 (Rvb1/Pontin) and RuvB like2 (Rvb2/Reptin) of AAA+ family of enzymes are present in yeast to human and are well known to be involved in diverse cellular activities. The human malaria parasite Plasmodium falciparum contains three different RuvB like proteins. Thus it has been of interest to explore why P. falciparum requires three RuvB like proteins and how these enzymes are biochemically regulated. In this study, we present the detailed biochemical characterization of PfRuvB2. The complex of PfRuvB3 was immunopurified and the presence of PfRuvB2 was confirmed. The in vitro interaction study shows that PfRuvB2 interacts only with PfRuvB3 but not with PfRuvB1. The recombinant as well as endogenous PfRuvB2 contains ATPase as well as weak DNA helicase activities. The presence of PfRuvB3 in the helicase reaction of PfRuvB2 increases the helicase activity significantly. Interestingly PfRuvB2/PfRuvB3 complex preferentially translocates and unwinds DNA in the 5′–3′ direction. In vivo studies showed that PfRuvB2 is expressed in all the asexual intraerythrocytic developmental stages and localizes mainly in the nucleus during merozoite, ring and trophozoite stages while during schizont stage it relocalizes partially in the nucleus and partially towards cytoplasm. As PfRuvB3 is specific to intraerythrocytic mitosis so we interpret that PfPuvB3 interacts with PfRuvB2 during schizont/intraerythrocytic mitosis and acts as its modulator mainly for the appreciable helicase activity.

Methods for detecting ATP hydrolysis and nucleic acid unwinding of Japanese encephalitis virus NS3 helicase

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic pathogen that is prevalent in south-east Asia. Because there is no specific antiviral agent, JEV still causes a high rate of neurologic sequelae and mortality in humans. The helicase encoded by the NS3 gene of JEV has emerged recently as a novel antiviral target for treatment. In this study, a soluble recombinant JEV helicase protein was expressed and purified. Methods for detecting the ATP hydrolysis and nucleic acid unwinding activity were developed by luminescence and fluorescence resonance energy transfer (FRET). The concentrations of enzyme, substrate, capture strand, ATP, and divalent ions were optimised in the ATPase and helicase reactions. The feasibility of using these two methods for high-throughput screening of NS3 helicase inhibitors is discussed.

Abstract 2747: EWS-FLI1 reduces RNA Helicase A activity.

Abstract Background: EWS-FLI1 requires the protein binding of RNA Helicase A (RHA) to enable oncogenic transformation. RHA is a DExH box RNA helicase family member. RHA has critical roles modulating transcription, splicing and translation. RHA also functions as a scaffolding protein in multi-protein complexes including BRCA1, DICER, EGFR, POL2R, TOPO2. Our previous work demonstrated that YK-4-279 inhibited the protein-protein interaction of RHA, with EWS-FLI1 and led to cellular apoptosis. The biochemical mechanism remained cryptic, so we investigated the effect of EWS-FLI1 upon the helicase activity of RHA. Methods: Full-length purified recombinant RHA from insect cells. Infrared-labeled double stranded RNA was used as a substrate to measure helicase activity of RHA. We included full-length recombinant EWS-FLI1 in to the helicase reaction to test the effect of EWS-FLI1. Results: RHA unwinds double stranded RNA in the presence of ATP with Km value of 5.322 nM. We also showed that RHA manifests reannealing activity of single stranded RNA to double stranded RNA in the absence of ATP. Unlike ADP, ATP and additionally the non-hyrolysable gamma-S-ATP prevent reannealing activity of RHA. A molecular modeling of RHA structure predicted the surface exposure of the region (between 823-832aa), which EWS-FLI1 interacts with. The function of this region is not well described but presumed to support helicase activity. EWS-FLI1 interferes RNA helicase activity of RHA in a dose dependent manner. Moreover, the reduction of RHA protein level exerted cytotoxic effect on Ewing Sarcoma cells. Decreased RHA protein level changed EWS-FLI1 and its target gene expression profile. IGFBP3, GRK5, p21, and PRR3 gene expression levels increased massively. RPLA1 and NR0B1 mRNA levels were also increased by moderately in these cells. The change in the profile may contribute to apoptosis. Conclusions: The purified recombinant RHA is very active in vitro helicase assays. We demonstrated that RHA's helicase activity might depend on ATP binding but not ATP hydrolysis. The protein partnering as well as posttranslational modification of RHA might modulate the helicase and/or reannealing activity of RHA, which may augment oncogenesis of EWS-FLI1. This is the first time that a protein partner reduces the helicase activity of RHA. Further experiments are ongoing that investigate whether YK-4-279 blocking of EWS-FLI1 binding to RHA will restore the helicase activity of RHA as part of it contribution to cell death. Citation Format: Hayriye Verda Erkizan, Kamal Sajwan, Maksymilian Chruszcz, Jeffrey Schneider, Sarah E. Gamble, Aykut Uren, Wladek Minor, Radhakrishnan Padmanabhan, Jeffrey Toretsky. EWS-FLI1 reduces RNA Helicase A activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2747. doi:10.1158/1538-7445.AM2013-2747

Biogenic polyamines spermine and spermidine activate RNA polymerase and inhibit RNA helicase of hepatitis C virus

Influence of the biogenic polyamines spermine, spermidine, and putrescine as well as their derivatives on the replication enzymes of hepatitis C virus (HCV) was investigated. It was found that spermine and spermidine activate HCV RNA-dependent RNA polymerase (NS5B protein). This effect was not caused by the stabilization of the enzyme or by competition with template-primer complex, but rather it was due to achievement of true maximum velocity V(max). Natural polyamines and their derivatives effectively inhibited the helicase reaction catalyzed by another enzyme of HCV replication - helicase/NTPase (NS3 protein). However, these compounds affected neither the NTPase reaction nor its activation by polynucleotides. Activation of the HCV RNA polymerase and inhibition of the viral helicase were shown at physiological concentrations of the polyamines. These data suggest that biogenic polyamines may cause differently directed effects on the replication of the HCV genome in an infected cell.

Modulation of UvrD Helicase Activity by Covalent DNA-Protein Cross-links

UvrD (DNA helicase II) has been implicated in DNA replication, DNA recombination, nucleotide excision repair, and methyl-directed mismatch repair. The enzymatic function of UvrD is to translocate along a DNA strand in a 3' to 5' direction and unwind duplex DNA utilizing a DNA-dependent ATPase activity. In addition, UvrD interacts with many other proteins involved in the above processes and is hypothesized to facilitate protein turnover, thus promoting further DNA processing. Although UvrD interactions with proteins bound to DNA have significant biological implications, the effects of covalent DNA-protein cross-links on UvrD helicase activity have not been characterized. Herein, we demonstrate that UvrD-catalyzed strand separation was inhibited on a DNA strand to which a 16-kDa protein was covalently bound. Our sequestration studies suggest that the inhibition of UvrD activity is most likely due to a translocation block and not helicase sequestration on the cross-link-containing DNA substrate. In contrast, no inhibition of UvrD-catalyzed strand separation was apparent when the protein was linked to the complementary strand. The latter result is surprising given the earlier observations that the DNA in this covalent complex is severely bent ( approximately 70 degrees ), with both DNA strands making multiple contacts with the cross-linked protein. In addition, UvrD was shown to be required for replication of plasmid DNAs containing covalent DNA-protein complexes. Combined, these data suggest a critical role for UvrD in the processing of DNA-protein cross-links.

Synthesis and biological properties of pyrimidine 4′-fluoronucleosides and 4′-fluorouridine 5′-O-triphosphate

4'- Fluoro-2',3'-O-isopropylidenecytidine was synthesized via interaction of 5'-O-acetyl-4'-fluoro-2',3'-O-isopropylideneuridine with triazole and 4-chlorophenyl dichlorophosphate followed by ammonolysis. Treatment of 5'-O-acetyl-4'-fluoro-2',3'-O-isopropylidenecytidine with hydroxylamine resulted in 5'-O-acetyl-4'-fluoro-2',3'-O-isopropylidene-N(4)-hydroxycytidine. Subsequent removal of 2',3'-O-isopropylidene groups gave 5'-O-acetyl derivatives of 4'-fluorouridine, 4'-fluorocytidine and 4'-fluoro-N(4)-hydroxycytidine. 5'-O-Triphosphate of 4'-fluorouridine was obtained in three steps starting from 4'-fluoro-2',3'-O-isopropylideneuridine. The 4'-fluoro uridine 5'-O-triphospate was found to be an effective inhibitor of HCV RNA-dependent RNA polymerase, substrate for NTPase reaction, catalyzed by protein NS3 HCV (a rate of the analogue hydrolysis was similar to that of ATP) and an activator for helicase reaction (with an efficacy only three fold lower than that of ATP).

Structural Basis for DNA Strand Separation by the Unconventional Winged-Helix Domain of RecQ Helicase WRN

The RecQ family of DNA helicases including WRN (Werner syndrome protein) and BLM (Bloom syndrome protein) protects the genome against deleterious changes. Here we report the cocrystal structure of the RecQ C-terminal (RQC) domain of human WRN bound to a DNA duplex. In the complex, the RQC domain specifically interacted with a blunt end of the duplex and, surprisingly, unpaired a Watson-Crick base pair in the absence of an ATPase domain. The beta wing, an extended hairpin motif that is characteristic of winged-helix motifs, was used as a "separating knife" to wedge between the first and second base pairs, whereas the recognition helix, a principal component of helix-turn-helix motifs that are usually embedded within DNA grooves, was unprecedentedly excluded from the interaction. Our results demonstrate a function of the winged-helix motif central to the helicase reaction, establishing the first structural paradigm concerning the DNA structure-specific activities of the RecQ helicases.

Real-time monitoring of RNA helicase activity using fluorescence resonance energy transfer in vitro

We have developed a continuous fluorescence assay based on fluorescence resonance energy transfer (FRET) for the monitoring of RNA helicase activity in vitro. The assay is tested using the hepatitis C virus (HCV) NS3 helicase as a model. We prepared a double-stranded RNA (dsRNA) substrate with a 5′ fluorophore-labeled strand hybridized to a 3′ quencher-labeled strand. When the dsRNA is unwound by helicase, the fluorescence of the fluorophore is emitted following the separation of the strands. Unlike in conventional gel-based assays, this new assay eliminates the complex and time-consuming steps, and can be used to simply measure the real-time kinetics in a single helicase reaction. Our results demonstrate that Alexa Fluor 488 and BHQ1 are an effective fluorophore–quencher pair, and this assay is suitable for the quantitative measurement of the RNA helicase activity of HCV NS3. Moreover, we found that several extracts of marine organisms exhibited different inhibitory effects on the RNA and DNA helicase activities of HCV NS3. We propose that this assay will be useful for monitoring the detailed kinetics of RNA unwinding mechanisms and screening RNA helicase inhibitors at high throughput.

Characterization of the Mycobacterial AdnAB DNA Motor Provides Insights into the Evolution of Bacterial Motor-Nuclease Machines

Mycobacterial AdnAB exemplifies a family of heterodimeric motor-nucleases involved in processing DNA double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal RecB-like nuclease module. Here we conducted a biochemical characterization of the AdnAB motor, using a nuclease-inactivated heterodimer. AdnAB is a vigorous single strand DNA (ssDNA)-dependent ATPase (k(cat) 415 s(-1)), and the affinity of the motor for the ssDNA cofactor increases 140-fold as DNA length is extended from 12 to 44 nucleotides. Using a streptavidin displacement assay, we demonstrate that AdnAB is a 3' --> 5' translocase on ssDNA. AdnAB binds stably to DSB ends. In the presence of ATP, the motor unwinds the DNA duplex without requiring an ssDNA loading strand. We integrate these findings into a model of DSB unwinding in which the "leading" AdnB and "lagging" AdnA motor domains track in tandem, 3' to 5', along the same DNA single strand. This contrasts with RecBCD, in which the RecB and RecD motors track in parallel along the two separated DNA single strands. The effects of 5' and 3' terminal obstacles on ssDNA cleavage by wild-type AdnAB suggest that the AdnA nuclease receives and processes the displaced 5' strand, while the AdnB nuclease cleaves the displaced 3' strand. We present evidence that the distinctive "molecular ruler" function of the ATP-dependent single strand DNase, whereby AdnAB measures the distance from the 5'-end to the sites of incision, reflects directional pumping of the ssDNA through the AdnAB motor into the AdnB nuclease. These and other findings suggest a scenario for the descent of the RecBCD- and AddAB-type DSB-processing machines from an ancestral AdnAB-like enzyme.

Hepatitis C virus helicase/NTPase: an efficient expression system and new inhibitors

A method has been developed for obtaining a full-length protein NS3 of hepatitis C virus with the yield of 6.5 mg/liter of cell culture, and conditions for measuring its NTPase and helicase activities have been optimized. The helicase reaction can proceed in two modes depending on the enzyme and substrate concentration ratio: it can be non-catalytic in the case of enzyme excess and catalytic in the case of tenfold substrate excess. In the latter case, helicase activity is coupled with NTPase and is stimulated by ATP. A number of NTP and inorganic pyrophosphate analogs were studied as substrates and/or inhibitors of NS3 NTPase activity, and it was found that the structure of nucleic base and ribose fragment of NTP molecule has a slight effect on its inhibitory (substrate) properties. Among the nucleotide derivatives, the most efficient inhibitor of NTPase activity is 2 -deoxythymidine 5 -phosphoryl-beta,gamma-hypophosphate, and among pyrophosphate analogs imidodiphosphate exhibited maximal inhibitory activity. These compounds were studied as inhibitors of the helicase reaction, and it was shown that imidodiphosphate efficiently inhibited the ATP-dependent helicase reaction and had almost no effect on the ATP-independent duplex unwinding. However, the inhibitory effect of 2 -deoxythymidine 5 -phosphoryl-beta,gamma-hypophosphate was insignificant in both cases, which is due to the possibility of helicase activation by this ATP analog.

Transcription Termination Factor Rho Can Displace Streptavidin from Biotinylated RNA

In Escherichia coli, binding of the hexameric Rho protein to naked C-rich Rut (Rho utilization) regions of nascent RNA transcripts initiates Rho-dependent termination of transcription. Although the ring-shaped Rho factor exhibits in vitro RNA-dependent ATPase and directional RNA-DNA helicase activities, the actual molecular mechanisms used by Rho to disrupt the intricate network of interactions that cement the ternary transcription complex remain elusive. Here, we show that Rho is a molecular motor that can apply significant disruptive forces on heterologous nucleoprotein assemblies such as streptavidin bound to biotinylated RNA molecules. ATP-dependent disruption of the biotin-streptavidin interaction demonstrates that Rho is not mechanistically limited to the melting of nucleic acid base pairs within molecular complexes and confirms that specific interactions with the roadblock target are not required for Rho to operate properly. We also show that Rho-induced streptavidin displacement depends significantly on the identity of the biotinylated transcript as well as on the position, nature, and length of the biotin link to the RNA chain. Altogether, our data are consistent with a "snow plough" type of mechanism of action whereby an early rearrangement of the Rho-substrate complex (activation) is rate-limiting, physical force (pulling) is exerted on the RNA chain by residues of the central Rho channel, and removal of structural obstacles from the RNA track stems from their nonspecific steric exclusion from the hexamer central hole. In this context, a simple model for the regulation of Rho-dependent termination based on the modulation of disruptive dynamic loading by secondary factors is proposed.

The Human Werner Syndrome Protein Stimulates Repair of Oxidative DNA Base Damage by the DNA Glycosylase NEIL1

The mammalian DNA glycosylase, NEIL1, specific for repair of oxidatively damaged bases in the genome via the base excision repair pathway, is activated by reactive oxygen species and prevents toxicity due to radiation. We show here that the Werner syndrome protein (WRN), a member of the RecQ family of DNA helicases, associates with NEIL1 in the early damage-sensing step of base excision repair. WRN stimulates NEIL1 in excision of oxidative lesions from bubble DNA substrates. The binary interaction between NEIL1 and WRN (K(D) = 60 nM) involves C-terminal residues 288-349 of NEIL1 and the RecQ C-terminal (RQC) region of WRN, and is independent of the helicase activity WRN. Exposure to oxidative stress enhances the NEIL-WRN association concomitant with their strong nuclear co-localization. WRN-depleted cells accumulate some prototypical oxidized bases (e.g. 8-oxoguanine, FapyG, and FapyA) indicating a physiological function of WRN in oxidative damage repair in mammalian genomes. Interestingly, WRN deficiency does not have an additive effect on in vivo damage accumulation in NEIL1 knockdown cells suggesting that WRN participates in the same repair pathway as NEIL1.

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.

Nucleic Acid Unwinding by Hepatitis C Virus and Bacteriophage T7 Helicases Is Sensitive to Base Pair Stability

Helicases are motor enzymes that convert the chemical energy of NTP hydrolysis into mechanical force for motion and nucleic acid strand separation. Within the cell, helicases process a range of nucleic acid sequences. It is not known whether this composite rate of moving and opening the strands of nucleic acids depends on the base sequence. Our presteady state kinetic studies of helicases from two classes, the ring-shaped T7 helicase and two forms of non-ring-shaped hepatitis C virus (HCV) helicase, show that both the unwinding rate and processivity depend on the sequence and decrease as the nucleic acid stability increases. The DNA unwinding activity of T7 helicase and the RNA unwinding activity of HCV helicases decrease steeply with increasing base pair stability. On the other hand, the DNA unwinding activity of HCV helicases is less sensitive to base pair stability. These results predict that helicases will fall into a spectrum of modest to high sensitivity to base pair stability depending on their biological role in the cell. Modeling of the dependence provided the degree of the active involvement of helicase in base pair destabilization during the unwinding process and distinguished between passive and active mechanisms of unwinding.

DNA Unwinding by Escherichia coli DNA Helicase I (TraI) Provides Evidence for a Processive Monomeric Molecular Motor

The F plasmid TraI protein (DNA helicase I) plays an essential role in conjugative DNA transfer as both a transesterase and a helicase. Previous work has shown that the 192-kDa TraI protein is a highly processive helicase, catalytically separating >850 bp under steady-state conditions. In this report, we examine the kinetic mechanism describing DNA unwinding of TraI. The kinetic step size of TraI was measured under both single turnover and pre-steady-state conditions. The resulting kinetic step-size estimate was approximately 6-8 bp step(-1). TraI can separate double-stranded DNA at a rate of approximately 1100 bp s(-1), similar to the measured unwinding rate of the RecBCD helicase, and appears to dissociate very slowly from the 3' terminus following translocation and strand-separation events. Analyses of pre-steady-state burst amplitudes indicate that TraI can function as a monomer, similar to the bacteriophage T4 helicase, Dda. However, unlike Dda, TraI is a highly processive monomeric helicase, making it unique among the DNA helicases characterized thus far.

Inhibition of Werner Syndrome Helicase Activity by Benzo[a]pyrene Diol Epoxide Adducts Can Be Overcome by Replication Protein A

RecQ helicases are believed to function in repairing replication forks stalled by DNA damage and may also play a role in the intra-S-phase checkpoint, which delays the replication of damaged DNA, thus permitting repair to occur. Since little is known regarding the effects of DNA damage on RecQ helicases, and because the replication and recombination defects in Werner syndrome cells may reflect abnormal processing of damaged DNA associated with the replication fork, we examined the effects of specific bulky, covalent adducts at N(6) of deoxyadenosine (dA) or N(2) of deoxyguanosine (dG) on Werner (WRN) syndrome helicase activity. The adducts are derived from the optically active 7,8-diol 9,10-epoxide (DE) metabolites of the carcinogen benzo[a]pyrene (BaP). The results demonstrate that WRN helicase activity is inhibited in a strand-specific manner by BaP DE-dG adducts only when on the translocating strand. These adducts either occupy the minor groove without significant perturbation of DNA structure (trans adducts) or cause base displacement at the adduct site (cis adducts). In contrast, helicase activity is only mildly affected by intercalating BaP DE-dA adducts that locally perturb DNA double helical structure. This differs from our previous observation that intercalating dA adducts derived from benzo[c]phenanthrene (BcPh) DEs inhibit WRN activity in a strand- and stereospecific manner. Partial unwinding of the DNA helix at BaP DE-dA adduct sites may make such adducted DNAs more susceptible to the action of helicase than DNA containing the corresponding BcPh DE-dA adducts, which cause little or no destabilization of duplex DNA. The single-stranded DNA binding protein RPA, an auxiliary factor for WRN helicase, enabled the DNA unwinding enzyme to overcome inhibition by either the trans-R or cis-R BaP DE-dG adduct, suggesting that WRN and RPA may function together to unwind duplex DNA harboring specific covalent adducts that otherwise block WRN helicase acting alone.

Competition between the DNA unwinding and strand pairing activities of the Werner and Bloom syndrome proteins

BackgroundThe premature aging and cancer-prone Werner and Bloom syndromes are caused by defects in the RecQ helicase enzymes WRN and BLM, respectively. Recently, both WRN and BLM (as well as several other RecQ members) have been shown to possess a strand annealing activity in addition to the requisite DNA unwinding activity. Since an annealing function would appear to directly oppose the action of a helicase, we have examined in this study the dynamic equilibrium between unwinding and annealing mediated by either WRN or BLM.ResultsOur investigation into the competition between annealing and unwinding demonstrates that, under standard reaction conditions, WRN- or BLM-mediated annealing can partially or completely mask unwinding as measured in standard helicase assays. Several strategies were employed to suppress the annealing activity so that the actual strength of WRN- or BLM-dependent unwinding could be more accurately assessed. Interestingly, if a DNA oligomer complementary to one strand of the DNA substrate to be unwound is added during the helicase reaction, both WRN and BLM unwinding is enhanced, presumably by preventing protein-mediated re-annealing. This strategy allowed measurement of WRN-catalyzed unwinding of long (80 base pair) duplex regions and fully complementary, blunt-ended duplexes, both of which were otherwise quite refractory to the helicase activity of WRN. Similarly, the addition of trap strand stimulated the ability of BLM to unwind long and blunt-ended duplexes. The stimulatory effect of the human replication protein A (hRPA, the eukaryotic single-stranded DNA binding protein) on both WRN- and BLM-dependent unwinding was also re-examined in light of its possible role in preventing re-annealing. Our results show that hRPA influences the outcome of WRN and BLM helicase assays by both inhibiting re-annealing and directly promoting unwinding, with the larger contribution from the latter mechanism.ConclusionThese findings indicate that measurements of unwinding by WRN, BLM, and probably other RecQ helicases are complicated by their annealing properties. Thus, WRN- and BLM-dependent unwinding activities are significantly stronger than previously believed. Since this broadens the range of potential physiological substrates for WRN and BLM, our findings have relevance for understanding their functions in vitro and in vivo.