Cooperative translocation enhances the unwinding of duplex DNA by SARS coronavirus helicase nsP13 Na-Ra Lee 1 , Hyun-Mi Kwon 2 , Kkothanahreum Park 2 , Sangtaek Oh 3 , Yong-Joo Jeong 1, * and Dong-Eun Kim 2, * 1 Department of Bio and Nanochemistry, Kookmin University, Seoul 136-702, 2 Department of Bioscience and Biotechnology, and WCU program, Konkuk University, Seoul 143-701 and 3 PharmcoGenomic Research Center, Inje University, Busan 633-165, Republic of Korea Received April 1, 2010; Revised July 6, 2010; Accepted July 8, 2010 ABSTRACT SARS coronavirus encodes non-structural protein 13 (nsP13), a nucleic acid helicase/NTPase belong- ing to superfamily 1 helicase, which efficiently unwinds both partial-duplex RNA and DNA. In this study, unwinding of DNA substrates that had differ- ent duplex lengths and 5 0 -overhangs was examined under single-turnover reaction conditions in the presence of excess enzyme. The amount of DNA unwound decreased significantly as the length of the duplex increased, indicating a poor in vitro processivity. However, the quantity of duplex DNA unwound increased as the length of the single- stranded 5 0 -tail increased for the 50-bp duplex. This enhanced processivity was also observed for duplex DNA that had a longer single-stranded gap in between. These results demonstrate that nsP13 requires the presence of a long 5 0 -overhang to unwind longer DNA duplexes. In addition, enhanced DNA unwinding was observed for gapped DNA substrates that had a 5 0 -overhang, indicating that the translocated nsP13 molecules pile up and the preceding helicase facilitate DNA unwinding. Together with the propensity of oligomer formation of nsP13 molecules, we propose that the cooperative translocation by the functionally interacting oligomers of the helicase molecules loaded onto the 5 0 -overhang account for the observed enhanced processivity of DNA unwinding. INTRODUCTION Severe acute respiratory syndrome (SARS) that claimed almost 800 deaths in southern China within a few months between 2002 and 2003 was caused by a novel coronavirus, SARS coronavirus (SCV) (1). SARS con- tinues to be a serious concern as long as there is no vaccine or effective drug. SCV is a single-stranded (ss) RNA positive-strand virus with a genome of 29 727 nt (2,3). A single 21.2-kb replicase gene at the 5 0 -end region of the SCV genome is translated into two large replicative polyproteins, pp1ab (790 kDa) and pp1a (490 kDa), which is generated with and without ribosomal frame- shifting (1), respectively (4,5). These two translational products are subsequently processed by the viral main protease termed M pro or 3CL pro , generating a number of non-structural proteins (nsPs). These nsPs include the RNA-dependent RNA polymerase (nsP12) and the NTPase/helicase (nsP13), which primarily constitute the membrane-bound viral replicase complex (6,7). The viral replicase synthesizes the entire viral genome (replica- tion) as well as eight subgenomic mRNAs (transcription) (8,9). Since the viral helicase has been identified as a potential target for therapy in other viruses due to its indispensabil- ity in viral genome replication (10–12), SCV NTPase/ Helicase (nsP13), which was recently purified and characterized, was suggested as an attractive target for the development of anti-SCV agents (7). Thus, a lot of efforts have been made to identify and test small molecule inhibitors of the SCV helicase as drug candidates (13–16). RNA and DNA aptamers against SCV helicase were also reported to have an inhibitory effect against nucleic acids unwinding (17,18). Although the tertiary structure of the SCV NTPase/Helicase has not been ex- perimentally verified, the structure prediction of the protein was recently reported (19,20). SCV nsP13 has been shown to contain two separate domains, i.e. the helicase domain (Hel) and a metal-binding domain (MBD), which consists of several conserved Cys/His residues at the N-terminal (6,19). The SCV helicase Hel domain has been categorized into the Superfamily I helicase based on the conserved sequence motifs as shown in the Escherichia coli Rep helicase (21). *To whom correspondence should be addressed. Tel: +82 2 2049 6062; Fax: +82 2 3436 6062; Email: [email protected]Correspondence may also be addressed to Yong-Joo Jeong. Tel: +82 2 910 5454; Fax: +82 2 910 4415; Email: [email protected]7626–7636 Nucleic Acids Research, 2010, Vol. 38, No. 21 Published online 29 July 2010 doi:10.1093/nar/gkq647 ß The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. by guest on June 4, 2015 http://nar.oxfordjournals.org/ Downloaded from
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Cooperative translocation enhances the unwindingof duplex DNA by SARS coronavirus helicase nsP13Na-Ra Lee1, Hyun-Mi Kwon2, Kkothanahreum Park2, Sangtaek Oh3, Yong-Joo Jeong1,*
and Dong-Eun Kim2,*
1Department of Bio and Nanochemistry, Kookmin University, Seoul 136-702, 2Department of Bioscience andBiotechnology, and WCU program, Konkuk University, Seoul 143-701 and 3PharmcoGenomic Research Center,Inje University, Busan 633-165, Republic of Korea
Received April 1, 2010; Revised July 6, 2010; Accepted July 8, 2010
ABSTRACT
SARS coronavirus encodes non-structural protein13 (nsP13), a nucleic acid helicase/NTPase belong-ing to superfamily 1 helicase, which efficientlyunwinds both partial-duplex RNA and DNA. In thisstudy, unwinding of DNA substrates that had differ-ent duplex lengths and 50-overhangs was examinedunder single-turnover reaction conditions in thepresence of excess enzyme. The amount of DNAunwound decreased significantly as the length ofthe duplex increased, indicating a poor in vitroprocessivity. However, the quantity of duplex DNAunwound increased as the length of the single-stranded 50-tail increased for the 50-bp duplex.This enhanced processivity was also observed forduplex DNA that had a longer single-stranded gapin between. These results demonstrate that nsP13requires the presence of a long 50-overhang tounwind longer DNA duplexes. In addition,enhanced DNA unwinding was observed forgapped DNA substrates that had a 50-overhang,indicating that the translocated nsP13 moleculespile up and the preceding helicase facilitate DNAunwinding. Together with the propensity ofoligomer formation of nsP13 molecules, wepropose that the cooperative translocation by thefunctionally interacting oligomers of the helicasemolecules loaded onto the 50-overhang accountfor the observed enhanced processivity of DNAunwinding.
INTRODUCTION
Severe acute respiratory syndrome (SARS) that claimedalmost 800 deaths in southern China within a few monthsbetween 2002 and 2003 was caused by a novel
coronavirus, SARS coronavirus (SCV) (1). SARS con-tinues to be a serious concern as long as there is novaccine or effective drug. SCV is a single-stranded (ss)RNA positive-strand virus with a genome of 29 727 nt(2,3). A single 21.2-kb replicase gene at the 50-end regionof the SCV genome is translated into two large replicativepolyproteins, pp1ab (�790 kDa) and pp1a (�490 kDa),which is generated with and without ribosomal frame-shifting (�1), respectively (4,5). These two translationalproducts are subsequently processed by the viral mainprotease termed Mpro or 3CLpro, generating a numberof non-structural proteins (nsPs). These nsPs includethe RNA-dependent RNA polymerase (nsP12) and theNTPase/helicase (nsP13), which primarily constitutethe membrane-bound viral replicase complex (6,7). Theviral replicase synthesizes the entire viral genome (replica-tion) as well as eight subgenomic mRNAs (transcription)(8,9).
Since the viral helicase has been identified as a potentialtarget for therapy in other viruses due to its indispensabil-ity in viral genome replication (10–12), SCV NTPase/Helicase (nsP13), which was recently purified andcharacterized, was suggested as an attractive target forthe development of anti-SCV agents (7). Thus, a lot ofefforts have been made to identify and test smallmolecule inhibitors of the SCV helicase as drug candidates(13–16). RNA and DNA aptamers against SCV helicasewere also reported to have an inhibitory effect againstnucleic acids unwinding (17,18). Although the tertiarystructure of the SCV NTPase/Helicase has not been ex-perimentally verified, the structure prediction of theprotein was recently reported (19,20). SCV nsP13 hasbeen shown to contain two separate domains, i.e. thehelicase domain (Hel) and a metal-binding domain(MBD), which consists of several conserved Cys/Hisresidues at the N-terminal (6,19). The SCV helicase Heldomain has been categorized into the Superfamily Ihelicase based on the conserved sequence motifs asshown in the Escherichia coli Rep helicase (21).
*To whom correspondence should be addressed. Tel: +82 2 2049 6062; Fax: +82 2 3436 6062; Email: [email protected] may also be addressed to Yong-Joo Jeong. Tel: +82 2 910 5454; Fax: +82 2 910 4415; Email: [email protected]
7626–7636 Nucleic Acids Research, 2010, Vol. 38, No. 21 Published online 29 July 2010doi:10.1093/nar/gkq647
� The Author(s) 2010. Published by Oxford University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
by guest on June 4, 2015http://nar.oxfordjournals.org/
Helicases are molecular motor proteins that translocatealong the nucleic acid and separate double-stranded (ds)nucleic acid into two ss nucleic acids using the energygenerated from nucleoside triphosphate (NTP) hydrolysis(22–24). In choosing duplex nucleic acids as substrates forunwinding, most known helicases unwind only one type ofnucleic acid, either the DNA or RNA duplex. However,SCV helicase nsP13 has been shown to display activity ondifferent types of nucleic acid substrates. The helicasensP13 can unwind both RNA and DNA duplexes with a50- to 30- polarity, in which the presence of 50 ss overhang isabsolutely required to unwind DNA duplexes (6,7).Furthermore, it has been observed that both ss RNAand DNA stimulated the ATPase activity of the helicaseto the same degree (7,25). Thus, it is highly important tounderstand the duplex nucleic acid unwinding mechanismof the nsP13 helicase with respect to putative controllingfactor(s) in nucleic acid unwinding.
In this study, the DNA duplex was chosen as a modelsubstrate, and the unwinding of DNA substrates with dif-ferent duplex lengths and 50-overhang by this helicase waskinetically investigated. We demonstrated that the lengthof the 50-overhang, which was used as the loading strandfor helicase oligomers, was an important factor dictatingthe processivity of the DNA unwinding process. Thus, wepropose that ‘functional interaction’ of nsP13 helicaseoligomers loaded onto the 50-overhang accounts for theobserved cooperative translocation of the helicase inDNA unwinding and processivity, which is similar to themechanism previously suggested for hepatitis C virus NS3helicase (26).
MATERIALS AND METHODS
Protein and nucleic acids
SCV helicase nsP13 was overexpressed in E. coli andpurified as described earlier (18). DNA substrates werepurchased from Integrated DNA Technologies(Coralville, IA, USA), and purified by a denaturing (8Murea) polyacrylamide gel electrophoresis (PAGE). TheDNA concentration was determined by 260 nm absorb-ance and its extinction coefficient. To produce duplex ortriplex DNA substrates, the 32P-labeled DNA strand wasmixed with a 2-fold excess of complementary DNA andthen annealed by heating to 95�C followed by slowlycooling. The DNA substrates used in this study arelisted in Table 1.
DNA unwinding assays
The nsP13 (200 nM) and 32P -labeled DNA substrates(5 nM) were mixed in buffer MixA [50mM Tris–Cl(pH 6.8), 50mM NaCl, 2mM ATP, 5mM EDTA and10% glycerol]. The mixture was preincubated for 5minat 22�C and the unwinding reactions were initiated byadding an equal volume of MixB [2mM ATP, 13mMMgCl2 and 3 mM trap DNA (unlabeled bottom strand)].After various times, the reactions were quenched byadding an equal volume of the quenching solution(100mM EDTA, 0.4% SDS, 20% glycerol and 0.1%
bromophenol blue). The ds and ssDNAs were resolvedby native (urea-free) PAGE. Size markers for theunwound products were produced by heating the duplexsubstrates with 600-fold trap DNA at 95�C. The radio-activity was quantified using a Cyclone (PerkinElmer)and analyzed by OptiQuant/Cyclone software (PackardInstrument Company). The ratio of unwound productswas calculated as described earlier (27) and the datawere fit to a single-exponential equation [Equation (1)].
FðtÞ ¼ A � ð1� expð�k1�tÞÞ ð1Þ
Where F(t) is fraction unwound at time t, A is the amp-litude, k1 is the observed rate constant of the burst phase.
Chemical cross-linking
The chemical cross-linking reagent dimethylsuberimidate(DMS) was used to study the oligomerization of nsP13. A300mg/ml DMS stock solution was prepared immediatelybefore use in ice-cold triethanolamine (TEA) buffer (TEA/HCl, 0.15M, pH 8.2) and the pH was readjusted to 8.2with 1N NaOH. The nsP13 (4mM) was preincubated in50mM TEA buffer (pH 8.2) containing 50mM NaCl and5mM EDTA for 10min at 22�C and cross-linking wasinitiated by adding DMS (10mg/ml). After incubationfor 1min at 22�C, the cross-linking reactions werequenched by adding an equal volume of 1M glycine.The quenched samples were analyzed by SDS–PAGE(4–12% gradient) and proteins were detected byCoomassie Brilliant Blue R-250 staining.
RESULTS AND DISCUSSION
SCV helicase nsP13 has been shown to have dsDNA un-winding activity and was shown to belong to the family ofSF1 helicases (28). Like many SF1 helicases, the SCVhelicase requires an exposed ss DNA or RNA forbinding to the nucleic acid complex and the helicase trans-locates along the nucleic acids by hydrolyzing ATP. Wehave previously shown that ss DNA or RNA stimulatesthe ATP hydrolysis activity, but dsDNA does not (25).Although the crystal structures of a few typical SF1helicases complexed with DNA were revealed (29,30),e.g. Rep and PcrA, only a computational 3D model hasbeen proposed for the SCV helicase nsP13 (20). Therefore,details about the nucleic acid unwinding mechanism ofSCV helicase nsP13 have to be deduced from biochemicalstudies only. Hence, we investigated how the mechanisticefficiency of dsDNA unwinding by nsP13 is affected bythe structural features of the dsDNAs, such as duplexlength, ssDNA tail length and artificial ss gap betweenthe two DNA duplexes.
Single-turnover kinetics of duplex DNA unwindingby nsP13
Because nsP13 has been shown to unwind both DNA andRNA duplexes that have a 50-tail (6,7), we designed DNAduplexes with a 50-tail at one side as the unwinding sub-strates to investigate the single-turnover kinetics ofDNA unwinding by the helicase. Previously, analysis ofnucleic acids unwinding by the coronavirus helicase was
performed using a multiple turnover kinetics experiment,in which the helicase may rebind to the substrates aftercompletion of a cycle of unwinding (6,7). In contrast, ourexperimental setup of the single-turnover conditionenabled us to obtain quantitative aspects of the unwindingkinetics, especially the processivity of unwinding by thehelicase. The reaction scheme is shown in Figure 1A, inwhich the helicase is initially allowed to bind to the sub-strate without ATP hydrolysis. In the absence of ATPhydrolysis, unwinding was not observed (data not
shown). However, the addition of magnesium ions to thereaction allowed the helicase to unwind the DNA duplexes(Figure 1A).
The duplex DNA unwinding reaction was initiated byadding a solution of MgCl2 (13mM) and a large excess(3.0 mM) of trap oligonucleotides (unlabeled bottomstrand) to a mixture of the pre-incubated reaction contain-ing duplex DNA substrates (5 nM) and helicase (200 nM).The trap oligonucleotides served to prevent there-initiation of unwinding by trapping free helicases and
Table 1. DNA substrates for unwinding by nsP13 helicase
any helicases that dissociated from the substrates duringunwinding (Supplementary Data). The trap oligonucleo-tides also prevented reannealing of the displaced bottomstrand (Supplementary Data). Under the single-turnoverreaction condition, the nsP13 helicase unwinded thedsDNA substrates and generated ssDNA products thatwere resolved by non-denaturing PAGE (Figure 1B).The kinetic time-course of ssDNA accumulation wasplotted and fitted to an exponential function to ob-tain the reaction amplitudes and unwinding rates of onlythe helicases that were initially bound to the DNAsubstrates.
Processivity of duplex DNA unwinding
The helicase may dissociate from the duplex DNA beforeit is completely unwound. Therefore, to investigate theprocessivity of nsP13, kinetic studies of nsP13-catalyzedDNA unwinding were performed with DNA substratescontaining different duplex lengths (shown in Table 1)under the single-turnover reaction condition.Measurements of the reaction amplitude of the fractionof dsDNA molecules unwound allows one to calculatethe processivity of the helicase (31,32). As shown inFigure 2, the amplitude decreased as the length of theDNA duplex increased from 20 to 90 bp. This decrease
Figure 1. Single-turnover reaction of DNA unwinding by nsP13. (A) Schematic drawing of the unwinding of duplex DNA by nsP13. (B) Shown isthe representative native gel shift assay of nsP13 unwinding in the presence of the 15T30bp dsDNA substrate. Unwinding products were resolved onan 8% native-PAGE and visualized using a phosphorimager Cyclone.
in amplitude with an increase in duplex length may haveoccurred partly due to the reannealing of DNA strandsbehind the helicase and to the helicase dissociation fromthe DNA during unwinding. Nevertheless, the large excessof trap DNA that is included in the reaction rapidlyanneals to the strand that is displaced from the duplexsubstrates during unwinding. A decrease in the amplitudeof DNA unwound was observed in the absence of trapDNA at a given duplex DNA length (data not shown),indicating that the trap DNA prevented reannealing ofdisplaced strands after completion of unwinding.A plot of the final reaction amplitude versus duplex
length showed that processivity of the nsP13 helicase inunwinding of DNA with a 50-tail of 20 nt is very low(Figure 2B) compared with other related helicases, suchas NPH-II RNA helicase and HCV NS3 DNA helicase(33,34). The processivity of a helicase is defined as the
probability of unwinding a base pair versus dissociationof the helicase from the DNA at that position (24). Atpresent, it is hard to calculate the exact processivity ofthe nsP13 helicase due to the absence of ‘kinetic stepsize’ information of active nsP13 (31,35). The monomericform of most SF1 and SF2 helicases shows that they arerapid and processive at translocating along ssDNA, butnot processive in terms of dsDNA unwinding (36–38).This raises the possibility that nsP13 may function as anoligomer in its active form like other SF1 and SF2helicases, despite the fact that the helicase is non-processive by itself. Considering the hexameric ring-shaped helicases such as T7 bacteriophage helicase, theformation of ring was expected to result in highprocessivity due to DNA passage through a centralchannel. However, single-turnover kinetic analysisshowed that the T7 helicase by itself has a low processivityfor unwinding dsDNA (�15 bp), but had a 9-fold higherhelicase activity (�130 bp) in the presence of polymeraseand thioredoxin (31,39). The fact that SARS coronavirushas a polymerase and many non-structural proteins ofunknown functions led to the hypothesis that additionalproteins may be involved in the helicase activity ofnsP13 (40).
An alternative but not exclusive explanation for the lowprocessivity of nsP13 is that the DNA may not be theproper substrate for nsP13. Despite the fact that theSARS nsP13 helicase is a RNA helicase, little is knownabout its interaction with RNA. Although we have previ-ously investigated the ATP hydrolysis activity of nsP13 inthe presence of ssDNA or ssRNA (25), more rigorousexperiments to monitor its affinities toward DNA orRNA are required to explain this low processivity.Whether nsP13 has different affinities for various typesof DNAs or RNAs might be a determining factor forthe processivity of nsP13 in duplex unwinding.
Enhancement of duplex DNA unwinding by increasingthe length of 50-overhang
Partial duplex DNA substrates containing 50 bp with a50-ssDNA overhang of varying lengths (Tn50bp inTable 1) were designed in order to investigate whetherDNA unwinding is dependent on the length of theloading strand. Although nsP13 has been shown to havestimulated ATPase activity in the presence of polynucleo-tides, detailed binding affinity experiments have not beenconducted. More importantly, the extent of stimulation ofATPase activity with ss nucleic acids was previouslyshown to be proportional to the length of ssDNA orssRNA at a given concentration (25). These observationsmay indicate that nsP13 binds to the ss nucleic acids with adefined binding site size. Thus, we can speculate that asthe length of the loading strand increases, more nsP13helicases would be bound to the substrate DNA underthe single-turnover reaction condition, in which thehelicase outnumbers the amount of substrate DNAs.
Time-courses of duplex DNA unwinding by 100 nMnsP13 with 2.5 nM of each duplex DNA substrate withvarious 50-tail lengths (0, 5, 10, 15, 20, 30 and 40 nt) areshown in Figure 3. The quantity of unwound duplex DNA
Figure 2. Unwinding of duplex DNA substrates with varying length.(A) Single-turnover unwinding of duplex DNA substrates with differentlengths by nsP13. dsDNA substrates contained 20 T tail and 20 bp(open square), 30 bp (filled circle), 40 bp (open circle), 50 bp (filledinverted triangle), 60 bp (open inverted triangle), 90 bp (filled square).The amplitudes are as follows: 20 bp=0.98±0.0077; 30 bp=0.91±0.020; 40 bp=0.37±0.016; 50 bp=0.21±0.0082; 60 bp=0.062±0.0045 and 90 bp=0.028±0.0074. (B) Amplitudes of nP13are shown in the bar graph against duplex length and amplitudes ofNPH (filled triangle) and NS3(filled circle) are marked on the samegraph for comparison.
increased as the length of the ss 50-tail increased. Theseresults suggest that more ssDNA products were formedfrom duplex DNA substrates that could potentially bindmore than one helicase molecule on the 50-tail, implyingthat multiple nsP13 molecules might exhibit a higherprocessivity. Alternatively, a tighter binding affinity ofthe helicase monomer or oligomers to longer ss tailsmight also explain this increase in DNA unwinding.Based on these two explanations, we hypothesized thatthe helicase may occupy the defined length of thessDNA overhang as a minimal binding site size and thestronger affinity is caused by cooperative binding ofmultiple helicases on a longer ssDNA. A tail length of20 nt enabled nsP13 to marginally unwind 50 bp duplexDNA substrates, and a tail length of 40 nt allowednsP13 to maximally unwind more duplex DNA (>95%)
that was 50 bp in length. We postulate that the functionalnsP13 monomer or oligomer requires a ssDNA loadingstrand that is at least 20 nt to translocate and unwindduplex DNA, which is analogous to a putative bindingsite size.To further examine the effect of the length of the
loading stand on nsP13 DNA duplex unwindingkinetics, we designed a series of duplex DNA substratescontaining a DNA gap on the bottom strand (Table 1 andFigure 4). The gapped DNA duplex substrates did nothave a 50-tail at either end, which is available as loadingstrand for the helicase. Instead, the nsP13 helicase mayrecognize the internal ssDNA portion in the gappedDNA as the loading strand. The gap size was variedfrom 0 to 20 nt and unwinding at the bottom of the
Figure 4. Unwinding of gapped DNA substrates by nsP13.(A) Unwinding of DNA substrates containing gaps of varying length:20 bases gap (filled circle), 15 bases gap (open circle), 10 bases gap(filled inverted triangle), and 5 bases gap (open inverted triangle).The substrates consisted of two ds regions in the 50-side (30 bp) and30 side (30 bp) of the upper strand. The gap was located between thetwo ds regions. Reaction amplitudes of gapped DNA substrates were asfollows: 20 bases gap=0.17±0.0088, 15 bases gap=0.10±0.0063,10 bases gap=0.071±0.0027 and 5 bases gap=0.016±0.0008.(B) The bar graph shows the amplitudes of nsP13-catalyzed unwindingwith triple-stranded DNA substrates containing gaps of differentlengths.
Figure 3. Unwinding of DNA substrates with varying length of50-overhang by nsP13. (A) Single-turnover unwinding of 50 bp duplexDNA substrates with 50-overhangs of varying lengths by 0T (filledcircle), 5 T (open circle), 10T (filled inverted triangle), 15T (openinverted triangle), 20T (filled square), 30T (open square) and 40T(filled diamond). Data were fit to a single-exponential. The amplitudeswere as follows: 0 T=0.0035±0.0013, 5T=0.038±0.017, 10T=0.042±0.0028, 15T=0.074±0.0040, 20T=0.18±0.0031, 30T=0.55±0.013 and 40T=0.95±0.0039. (B) The bar graph shows thereaction amplitudes of nsP13-catalyzed dsDNA unwinding as thelength of the 50-overhang increased from 0 to 40 nt.
DNA strand (30 nt) annealed to the 30-side of the topstrand was monitored. Because entry of the helicase isnot available at both blunt ends of the gapped DNA,the only available entry point of the helicase would bethe ssDNA gap. In these experiments, the bottom strand(30 nt) annealed to the 50-side of the top strand wasdisplaced only when an overhang was present at the50-end of the gapped DNA duplex (data not shown).When blunt ended duplex DNA with an internal gap of
0 nt (nicked DNA) was reacted with the nsP13 helicase, nossDNA product was observed, suggesting that thepresence of ssDNA of a certain length is required for un-winding by nsP13. Increasing the gap size allowed nsP13to bind to ssDNA and to unwind the bottom strand DNAannealed to the 30 side of the top strand. When gaps of 5or 10 nt were present in the bottom strand, the nsP13helicase unwound the substrate with a very lowprocessivity; <10% of substrates were unwound(Figure 4B). However, more ssDNA was formed whensubstrates contained gaps of 15 or 20 nt in the middle ofthe duplex DNA (Figure 4B). This result indicates that thelonger loading strand facilitated more binding of nsP13molecules to the ssDNA gap, resulting in a higherprocessivity in duplex (30 bp) unwinding. An increase inthe amplitude of ssDNA product formation for DNA sub-strates containing 5–20 nt gaps was observed, indicatingthat DNA unwinding processivity was enhanced by anincrease in the ssDNA gap. Interestingly, the amplitudeobtained for 30-bp duplex DNA substrates containing a20-nt gap was not as high as when the substrate containeda 20-nt 50-tail overhang (>0.9 in Figure 2). This resultsuggests that the state of the nsP13 helicases bound tothe 20-nt ssDNA gap were different from the one boundto the 20-nt 50-tail.The results of this study clearly show that increasing the
ssDNA tail length enhances the unwinding amplitude.This result was also previously observed for SF1 andSF2 helicase (26,41); longer ssDNA tails provide morechances for helicase molecules to bind to ssDNA andmultiple binding leads to an enhanced unwinding effi-ciency. In the present experimental setup, where theenzyme concentration was greater than the substrate con-centration, all ssDNA tails were assumed to be initiallybound by nsP13. The stacked oligomers and forwardmovement of nsP13 caused by the binding of multiplensP13s on the ssDNA tail are believed to be the mainreasons for the enhanced unwinding on substrates with alonger ssDNA loading strand. Taken together, whennsP13 concentration was in excess, the amplitude ofssDNA formation increased as the length of either thessDNA overhang or gap increased. These results suggestthat multiple nsP13 molecules may bind to the ssDNAloading strand and unwind longer duplex DNA with ahigher processivity.
Cooperative translocation by oligomer of nsP13 helicasesin duplex unwinding
To further investigate whether multiple nsP13s can trackthrough the loading strand and participate in the unwind-ing of the DNA duplex, we prepared gapped DNA
substrates with or without a 50-ssDNA tail (Table 1).Gapped substrates with a 50-ssDNA tail, which containeda ssDNA gap that was 10 nt in length between the 30 bp ofthe lead duplex and 20 bp of the trailing duplex(20T30bp10G20bp in Table 1), was readily unwound bynsP13, resulting in displacement of the two duplexes(Figure 5A). However, for the gapped substrate that didnot have the 50-ssDNA tail (0T30bp10G20bp in Table 1),nsP13 was able to unwind only the 20 bp of the trailingduplex and not the 30 bp of the leading duplex(Figure 5A). This result indicates that the two gappedDNA substrates are recognized differently by nsP13 andthe 50-ssDNA loading strand is of primary importance forrecognition and unwinding of the DNA duplex. Notably,a 10-nt gap was sufficient for the internal initiation site tocompletely unwind the 20 bp of the trailing DNA duplex,which was not observed for the unwinding of the longer30-bp DNA duplex with a 10 nt gap (substrate ‘10 Gap’ inFigure 4). This indicates that nsP13 requires the presenceof a longer 50-ssDNA track to unwind a longer duplexDNA.
It was of interest to determine whether nsP13 mighttraverse from the 50-ssDNA tail to the internal gap andfacilitate unwinding of the incoming duplex DNA, like atrain on a railroad track pushing another train in the lead.To this end, we prepared DNA substrates with a longertrailing duplex (30 bp) and 10-nt gap (substrates inFigure 5B). As stated earlier, the 10-nt gap was not suffi-cient for nsP13 to completely unwind the 30-bp DNAduplex on the 30-side, which was unwound when thegapped DNA substrate without the 50-ssDNA overhangwas used (0T20bp10G30bp in Figure 5B). Note that ablunt-ended DNA substrate (the lead 20-bp duplex of0T20bp10G30bp) slightly unwound under this experimen-tal setup. A portion of the blunt end duplex might have a50-ssDNA at the end due to breathing of the DNA duplex,which supports unwinding of the 20-bp DNA duplex.However, this effect was not sufficient for the unwindingof the longer 30-bp DNA duplex (square symbols inFigure 5A). Most importantly, nsP13 was able to com-pletely unwind the 30 bp of the trailing duplex in the10-nt gapped DNA substrate containing a 50-ssDNA tail(20T20bp10G30bp in Figure 5B). This result suggests thatthe translocated nsP13 molecules pile up and facilitate un-winding of the 30-side of the DNA duplex by the helicasesthat are internally bound at the gap. This may be thereason why the 30-bp DNA duplex containing the 10-ntgap and 50-overhang (20T20bp10G30bp) unwound moreefficiently than the gapped DNA substrate containing thelonger lead DNA duplex (30 bp) with a blunt end(‘10 Gap’ shown in Figure 4). A portion of the nsP13helicases may stack up after the 50-side of the DNAduplex is unwound and may then unwind the 30-side ofthe DNA duplex. Therefore, the nsP13 helicase can trackthrough the loading strand and facilitate unwinding of theDNA duplex as long as the translocated helicases stay onthe track.
In order to investigate whether oligomerization of thensP13 monomer is facilitated in the presence of ssDNAand ATP, we used a chemical cross-linking assay toexamine the oligomeric state of nsP13 in the presence or
Figure 5. Unwinding of gapped DNA substrates by nsP13 with or without a 50-overhang. S indicates substrates that were incubated in the absenceof nsP13 for 3000 s. –E indicates the absence of the enzyme (nsP13). H indicates substrates that were heated in the presence of excess trap DNA.Each substrate is described in the graph. (A) The amplitudes of 0T30bp10G20bp were as follows: 30b (filled square)=0.031±0.011, 20b (filleddiamond)=0.96±0.025. The amplitudes of 20T30bp10G20bp were as follows: 30b (filled circle)=1.05±0.013, 20b (filled inverted tri-angle)=1.01±0.014. (B) The amplitudes of 0T20bp10G30bp were as follows: 30b (filled square)=0.69±0.22, 20b (filled diamond)=0.16±0.074.The amplitudes of 20T20bp10G30bp were as follows: 30b (filled circle)=0.98±0.012, 20b (filled inverted triangle)=1.05±0.0088.
absence of ligands. Chemical cross-linking with DMSshowed that the degree of cross-linking was slightlyaltered in the presence of ligands; a higher order oligomerswere observed in the presence of longer ssDNA(arrowheads in Figure 6). Interestingly, the nsP13protein itself readily formed a dimer and trimer in theabsence of any ligands. Thus, oligomerization of nsP13is an intrinsic property of the protein, which is facilitatedin the presence of long ssDNA and ATP. However, wecould not determine the definite oligomeric state of thehelicase in this study, suggesting that a dynamicoligomeric status of the helicase might exist in thepresence of ss DNA.Based on the results obtained when the length of the
50-ssDNA tail and internal ssDNA gap in duplex DNAwas varied, together with the observation that nsP13oligomerization occurs in the presence of ssDNA, we
believe that nsP13 more efficiently unwinds partial-duplexnucleic acid substrates containing ss regions at the 50-sidethat are long enough to allow the binding of multiplemonomers (Figure 7). The proposed model, schematicallyshown in Figure 7, is very similar to that proposed for theunwinding of DNA by the HCV NS3 helicase and T4 Ddahelicase (26,41). The proposed model for DNA unwindingby those helicases does not require protein oligomeriza-tion per se to obtain greater processivity. The increase inthe helicase activity was explained by functionalcooperativity rather than structural contacts betweenhelicase monomers. Although multiple nsP13 helicasesdo not necessarily need to form a functional oligomericform, multiple molecules of nsP13 are more efficient atunwinding longer substrates under single-turnover condi-tions. As depicted in Figure 7, multiple molecules of nsP13loaded on the leading strand are more likely to completethe unwinding process before leading helicase monomersdisassociate from the substrate. This cooperative trans-location is demonstrated by our results on the unwindingof the gapped duplex DNAs with or without the 50-ssDNAoverhang (Figure 5). Unwinding of the 30-side of the DNAduplex with a short gap size was impeded when fewernsP13 molecules translocated from the 50-side of theDNA, suggesting that multiple nsP13 monomers loadedon the same ssDNA track function together to producemore displaced ssDNA than a single nsP13 monomer cando. Taken together, the model presented in Figure 7 is asummary of how the non-processive nsP13 belonging toSF 1 helicase accomplishes substantial processivity in un-winding duplex nucleic acids in vitro. It should be notedthat nsP13 may interact with other hosts and/or viralproteins when unwinding its RNA genome in infectedcells and it may exhibit different properties during RNAunwinding.
Figure 7. Proposed mechanism of DNA unwinding by the nsP13 helicase with different processivity.
Figure 6. Chemical cross-linking using DMS indicates nsP13 oligomer-ization. Coomassie blue staining of a SDS–PAGE (4–12%) gel showingcross-linking of nsP13 either in the absence or presence of ATP (2mM)and ssDNA (125 nM; 10, 30, 50, 70, 90, 110 nt). See the text for detailedexperiment.
The Korean Government, National Research Foundationof Korea (KRF-2008-313-C00531) and a grant (A090410)of the Korea Healthcare technology R&D Project,Ministry for Health, Welfare & Family Affairs, Republicof Korea (to Y.-J.J.); National Research Foundation ofKorea through the WCU Project (R33-10128) and a grant(20080401034026) from the BioGreen 21 Program, RuralDevelopment Administration, Republic of Korea (toD.-E.K.). Funding for open access charge: Grant (2008-0062074) from the Ministry of Education, Science andTechnology, the Korean government.
Conflict of interest statement. None declared.
REFERENCES
1. World Health Organization report. (2003) Cumulative Number ofReported Probable Cases of Severe Acute Respiratory Syndrome(SARS), http://www.who.int/csr/sars/country/en (1 July 2010, datelast accessed).
2. Marra,M.A., Jones,S.J., Astell,C.R., Holt,R.A., Brooks-Wilson,A.,Butterfield,Y.S., Khattra,J., Asano,J.K., Barber,S.A., Chan,S.Y.et al. (2003) The genome sequence of the SARS-associatedcoronavirus. Science, 300, 1399–1404.
3. Rota,P.A., Oberste,M.S., Monroe,S.S., Nix,W.A., Campagnoli,R.,Icenogle,J.P., Penaranda,S., Bankamp,B., Maher,K., Chen,M.H.et al. (2003) Characterization of a novel coronavirus associatedwith severe acute respiratory syndrome. Science, 300, 1394–1399.
4. Ziebuhr,J., Snijder,E.J. and Gorbalenya,A.E. (2000) Virus-encodedproteinases and proteolytic processing in the Nidovirales.J. Gen. Virol., 81, 853–879.
5. Lai,M.M. and Cavanagh,D. (1997) The molecular biology ofcoronaviruses. Adv. Virus Res., 48, 1–100.
6. Ivanov,K.A., Thiel,V., Dobbe,J.C., van der Meer,Y., Snijder,E.J.and Ziebuhr,J. (2004) Multiple enzymatic activities associatedwith severe acute respiratory syndrome coronavirus helicase.J. Virol., 78, 5619–5632.
7. Tanner,J.A., Watt,R.M., Chai,Y.B., Lu,L.Y., Lin,M.C.,Peiris,J.S., Poon,L.L., Kung,H.F. and Huang,J.D. (2003) Thesevere acute respiratory syndrome (SARS) coronavirus NTPase/helicase belongs to a distinct class of 50 to 30 viral helicases.J. Biol. Chem., 278, 39578–39582.
8. Thiel,V., Ivanov,K.A., Putics,A., Hertzig,T., Schelle,B., Bayer,S.,Weissbrich,B., Snijder,E.J., Rabenau,H., Doerr,H.W. et al. (2003)Mechanisms and enzymes involved in SARS coronavirus genomeexpression. J. Gen. Virol., 84, 2305–2315.
9. Snijder,E.J., Bredenbeek,P.J., Dobbe,J.C., Thiel,V., Ziebuhr,J.,Poon,L.L., Guan,Y., Rozanov,M., Spaan,W.J. andGorbalenya,A.E. (2003) Unique and conserved features of genomeand proteome of SARS-coronavirus, an early split-off from thecoronavirus group 2 lineage. J. Mol. Biol., 331, 991–1004.
10. Borowski,P., Schalinski,S. and Schmitz,H. (2002) Nucleotidetriphosphatase/helicase of hepatitis C virus as a target forantiviral therapy. Antiviral Res., 55, 397–412.
11. Crute,J.J., Grygon,C.A., Hargrave,K.D., Simoneau,B.,Faucher,A.M., Bolger,G., Kibler,P., Liuzzi,M. andCordingley,M.G. (2002) Herpes simplex virus helicase-primaseinhibitors are active in animal models of human disease.Nat. Med., 8, 386–391.
12. Kleymann,G., Fischer,R., Betz,U.A., Hendrix,M., Bender,W.,Schneider,U., Handke,G., Eckenberg,P., Hewlett,G., Pevzner,V.et al. (2002) New helicase-primase inhibitors as drug candidatesfor the treatment of herpes simplex disease. Nat. Med., 8,392–398.
13. Tanner,J.A., Zheng,B.J., Zhou,J., Watt,R.M., Jiang,J.Q.,Wong,K.L., Lin,Y.P., Lu,L.Y., He,M.L., Kung,H.F. et al. (2005)The adamantane-derived bananins are potent inhibitors of thehelicase activities and replication of SARS coronavirus.Chem. Biol., 12, 303–311.
17. Shum,K.T. and Tanner,J.A. (2008) Differential inhibitoryactivities and stabilisation of DNA aptamers against the SARScoronavirus helicase. Chembiochem ., 9, 3037–3045.
18. Jang,K.J., Lee,N.R., Yeo,W.S., Jeong,Y.J. and Kim,D.E. (2008)Isolation of inhibitory RNA aptamers against severe acuterespiratory syndrome (SARS) coronavirus NTPase/Helicase.Biochem. Biophys. Res. Commun., 366, 738–744.
20. Hoffmann,M., Eitner,K., von Grotthuss,M., Rychlewski,L.,Banachowicz,E., Grabarkiewicz,T., Szkoda,T. and Kolinski,A.(2006) Three dimensional model of severe acute respiratorysyndrome coronavirus helicase ATPase catalytic domain andmolecular design of severe acute respiratory syndrome coronavirushelicase inhibitors. J. Comput. Aided Mol. Des., 20, 305–319.
21. Gorbalenya,A.E., Koonin,E.V., Donchenko,A.P. and Blinov,V.M.(1989) Coronavirus genome: prediction of putativefunctional domains in the non-structural polyprotein bycomparative amino acid sequence analysis. Nucleic Acids Res., 17,4847–4861.
22. Patel,S.S. and Picha,K.M. (2000) Structure and function ofhexameric helicases. Annu. Rev. Biochem, 69, 651–697.
23. Patel,S.S. and Donmez,I. (2006) Mechanisms of helicases.J. Biol. Chem., 281, 18265–18268.
24. Lohman,T.M. and Bjornson,K.P. (1996) Mechanisms ofhelicase-catalyzed DNA unwinding. Ann. Rev. Biochem., 65,169–214.
25. Lee,N.R., Lee,A.R., Lee,B., Kim,D.E. and Jeong,Y.J. (2009) ATPhydrolysis analysis of severe acute respiratory syndrome (SARS)coronavirus helicase. Bull. Kor. Chem. Soc., 30, 1724–1728.
26. Levin,M.K., Wang,Y.H. and Patel,S.S. (2004) The functionalinteraction of the hepatitis C virus helicase molecules isresponsible for unwinding processivity. J. Biol. Chem., 279,26005–26012.
27. Ahnert,P. and Patel,S.S. (1997) Asymmetric interactions ofhexameric bacteriophage T7 DNA helicase with the 50- and30-tails of the forked DNA substrate. J. Biol. Chem., 272,32267–32273.
28. Kwong,A.D., Rao,B.G. and Jeang,K.T. (2005) Viral and cellularRNA helicases as antiviral targets. Nat. Rev. Drug. Discov., 4,845–853.
29. Velankar,S.S., Soultanas,P., Dillingham,M.S., Subramanya,H.S.and Wigley,D.B. (1999) Crystal structures of complexes of PcrADNA helicase with a DNA substrate indicate an inchwormmechanism. Cell., 97, 75–84.
30. Korolev,S., Hsieh,J., Gauss,G.H., Lohman,T.M. and Waksman,G.(1997) Major domain swiveling revealed by the crystal structuresof complexes of E. coli Rep helicase bound to single-strandedDNA and ADP. Cell., 90, 635–647.
31. Jeong,Y.J., Levin,M.K. and Patel,S.S. (2004) TheDNA-unwinding mechanism of the ring helicase of bacteriophageT7. Proc. Natl Acad. Sci. USA, 101, 7264–7269.
32. Ali,J.A. and Lohman,T.M. (1997) Kinetic measurement of thestep size of DNA unwinding by Escherichia coli UvrD helicase.Science, 275, 377–380.
33. Jankowsky,E., Gross,C.H., Shuman,S. and Pyle,A.M. (2000) TheDExH protein NPH-II is a processive and directional motor forunwinding RNA. Nature., 403, 447–451.
34. Pang,P.S., Jankowsky,E., Planet,P.J. and Pyle,A.M. (2002) Thehepatitis C viral NS3 protein is a processive DNA helicase withcofactor enhanced RNA unwinding. EMBO J., 21, 1168–1176.
35. Tackett,A.J., Chen,Y., Cameron,C.E. and Raney,K.D. (2005)Multiple full-length NS3 molecules are required for optimalunwinding of oligonucleotide DNA in vitro. J. Biol. Chem., 280,10797–10806.
36. Cheng,W., Hsieh,J., Brendza,K.M. and Lohman,T.M. (2001) E.coli Rep oligomers are required to initiate DNA unwindingin vitro. J. Mol. Biol., 310, 327–350.
37. Fischer,C.J., Maluf,N.K. and Lohman,T.M. (2004) Mechanism ofATP-dependent translocation of E.coli UvrD monomers alongsingle-stranded DNA. J. Mol. Biol., 344, 1287–1309.
38. Niedziela-Majka,A., Chesnik,M.A., Tomko,E.J. andLohman,T.M. (2007) Bacillus stearothermophilus PcrA monomeris a single-stranded DNA translocase but not a processive helicasein vitro. J. Biol. Chem., 282, 27076–27085.
39. Stano,N.M., Jeong,Y.J., Donmez,I., Tummalapalli,P., Levin,M.K.and Patel,S.S. (2005) DNA synthesis provides the drivingforce to accelerate DNA unwinding by a helicase. Nature., 435,370–373.
40. Stadler,K., Masignani,V., Eickmann,M., Becker,S., Abrignani,S.,Klenk,H.D. and Rappuoli,R. (2003) SARS: beginning tounderstand a new virus. Nat. Rev. Microbiol., 1, 209–218.
41. Byrd,A.K. and Raney,K.D. (2005) Increasing the length ofthe single-stranded overhang enhances unwinding of duplexDNA by bacteriophage T4 Dda helicase. Biochemistry, 44,12990–12997.
