JBC Papers in Press. Published on July 7, 2006 as Manuscript M605518200 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M605518200 THE REPLICATIVE HELICASES OF BACTERIA, ARCHAEA AND EUKARYA CAN UNWIND RNA-DNA HYBRID SUBSTRATES* Jae-Ho Shin and Zvi Kelman From the University of Maryland Biotechnology Institute, Center for Advanced Research in Biotechnology, 9600 Gudelsky Drive, Rockville, MD 20850 Running title: Unwinding of RNA by DNA helicases Address correspondence to: Zvi Kelman, University of Maryland Biotechnology Institute, Center for Advanced Research in Biotechnology, 9600 Gudelsky Drive, Rockville, MD 20850, Tel: (240) 314-6294, Fax: (240) 314-6255, E-mail: [email protected] Replicative helicases are replicative helicases of bacteria, archaea and hexameric enzymes that unwind DNA eukarya suggest that these enzymes form during chromosomal replication. They ring-shaped structures that encircle and use energy from nucleoside triphosphate translocate along DNA while utilizing the hydrolysis to translocate along one strand energy derived from NTP hydrolysis to of the duplex DNA and displace the separate duplex DNA at the front of the Do w complementary strand. Here, the ability replication fork (1). nlo a of a replicative helicase from each of the The bacterial DnaB helicase is a de d three domains - bacteria, archaea and homo-hexamer that binds and translocates fro m eukarya - to unwind RNA-containing along single-stranded (ss)1 and double- h ttp substrate was determined. It is shown stranded (ds) DNA and possesses a 5’→3’ ://w that all three helicases can unwind DNA- helicase activity and DNA-dependent w w RNA hybrids while translocating along ATPase activity (2). The eukaryotic .jb c .o the single stranded DNA. No unwinding minichromosome maintenance (MCM) rg could be observed when the helicases complex is a family of six related by/ g were provided with a single-stranded polypeptides (Mcm2–7), each of which is ue s RNA overhang. Using DNA, RNA and essential for cell viability. Biochemical t on A DNA-RNA chimeric oligonucleotides it studies have shown that a dimeric complex p was found that while the enzymes can of the Mcm4,6,7 heterotrimer contains ril 1 5 bind both DNA and RNA, they could 3’→5’ DNA helicase activity, ssDNA , 20 1 9 translocate only along DNA, and only binding, and DNA-dependent ATPase DNA stimulates the enzymes’ ATPase activity, and is capable of translocating activity. Recent observations suggest that along ss and dsDNA. In vitro, the Mcm2 helicases may interact with enzymes and Mcm3,5 complexes were shown to participating in RNA metabolism, and inhibit helicase activity (3,4). Although it that RNA-DNA hybrids may be present has not yet been shown, the MCM complex on the chromosomes. Thus, the results is thought to function as the eukaryotic presented here may suggest a new role for replicative helicase. In most archaeal the replicative helicases during species studied, a single MCM homologue chromosomal replication or in other has been identified. The structure of the cellular processes. protein is not yet clear; hexamers, heptamers, dodecamers and filaments have INTRODUCTION been reported (5). Biochemical studies revealed that the archaeal enzyme possesses Biochemical studies with the an ATP-dependent 3’→5’ helicase activity, 1 Copyright 2006 by The American Society for Biochemistry and Molecular Biology, Inc. DNA dependent ATPase activity, and can Biotin-dT and 2-o-methyl NTPs were bind and translocate along ss and dsDNA obtained from Glen Research. mtMCM was (5,6). purified as previously described (10), The eukaryotic MCM complex was spMCM was kindly provided by Dr. Jerard shown to interact with RNA polymerase Hurwitz (Memorial Sloan–Kettering Cancer (7,8) and was found in a complex with Yph1 Center) and Dr. Joon-Kyu Lee (Seoul (9), a protein needed for 60S ribosomal National University), and ecDnaB was biogenesis which may also participate in kindly provided by Dr. Mike O’Donnell polysome translation. Thus it is possible (The Rockefeller University). that some replicative helicases participate in cellular processes involving RNA in Methods addition to their role in chromosomal DNA Preparation of substrates for helicase assays replication. To date, it has not been Oligonucleotides used for the established whether the MCM complex is preparation of the different helicase capable of translocating and/or unwinding substrates are listed in Supplementary Table RNA or DNA-RNA hybrids. 1. The helicase substrates were made as D A study was therefore initiated to follows. Five pmol of DNA or RNA o w n determine the ability of replicative helicases oligonucleotides (as indicated in figure lo a d from the three domains - bacteria, archaea legends) were labeled at the 5’ end using e d and eukarya - to unwind RNA-containing 16.7 pmol of [γ-32P]ATP (3,000 Ci/mmol) fro m substrates. It is shown here that the and T4 polynucleotide kinase. Labeling h ttp Escherichia coli DnaB protein (ecDnaB), reactions were stopped by adding EDTA to ://w the Methanothermobacter a final concentration of 25 mM. The labeled ww thermautotrophicus MCM protein oligonucleotide was hybridized to the .jbc .o (mtMCM), and the Schizosaccharomyces complementary oligonucleotide(s) at a 1:2 rg b/ pombe Mcm4,6,7 complex (spMCM) cannot molar ratio in a buffer containing 40 mM y g u unwind duplex RNA substrates. All can, Hepes-NaOH (pH 7.5) and 50 mM NaCl by es t o however, unwind a DNA-RNA hybrid heating to 100°C for 3 min followed by slow n A ssusDbsNtrAat e swtrahnedn, trbaunts locnaotitn g whaleonn g onthlye cDoNolAin glo atdoi n2g5 ˚bCu.f feAr ft(e0r. 1h%y bxriydlieznaet iocny,a n5oXl, pril 15 , 2 overhanging ssRNA was provided. It is also 0.1% bromophenol blue and 50% glycerol) 0 1 9 found that although all three enzymes can was added to a final concentration of 1X and bind RNA, RNA cannot stimulate the the mixture was electrophoresed through an enzymes’ ATPase activity. 8% native polyacrylamide gel for 1h at 100V in 0.5X TBE (45 mM Tris, 4.5 mM EXPERIMENTAL PROCEDURES boric acid, 0.5 mM EDTA) to remove unincorporated [γ-32P]ATP and free Materials oligonucleotides. The substrates were ATP and [γ-32P]ATP were obtained located by autoradiography and the products from GE Biosciences, and oligonucleotides were excised from the gel, which was sliced and biotinylated oligonucleotides were into small pieces and incubated at 37˚C for synthesized by the CARB DNA synthesis 16 hr in 3 volumes of an elution buffer facility. Biotin was obtained from Sigma, containing 0.5 M ammonium acetate, 10 Streptavidin from Rockland mM magnesium acetate, 1 mM EDTA, pH Immunochemicals, and nitroavidin 8.0. After centrifugation, the supernatants (CaptAvidinTM) from Molecular Probes. were collected and the substrates were 2 ethanol precipitated and resuspended in TE 100mM EDTA, 0.1% xylene cyanol, 0.1% (10 mM Tris-HCl (pH 7.5), 1 mM EDTA). bromophenol blue and 50% glycerol, and Due to low recovery of duplex RNA chilled on ice. Aliquots were loaded onto an substrates, 10 fold more RNA 8% native polyacrylamide gel in 0.5X TBE oligonucleotide (50 pmol) was 5’ end and electrophoresed for 1.5 h at 200 V at labeled with the same amount of [γ-32P]ATP 4°C. Gels were visualized and quantitated (16.7 pmol, 3,000mCi/mmol) and T4 by phosphorimaging. All helicase polynucleotide kinase. The specific activity experiments were repeated at least three of the DNA substrates was approximately times and their averages with standard 3,000 cpm/fmol, and that of duplex RNA deviations are shown in the figures together approximately 500 cpm/fmol. with representative gels. DNA helicase assay Filter binding assay DNA helicase activity of the ecDnaB Oligonucleotides for DNA and RNA was measured in reaction mixtures (15 µl) filter binding assays were prepared by containing 20 mM Tris-HCl (pH 7.5), 10 labeling the oligonucleotide using [γ- D mM MgCl2, 2 mM DTT, 100 µg/ml BSA, 5 32P]ATP and T4 polynucleotide kinase. own mM ATP, 10 fmol of 32P-labeled DNA Unincorporated [γ-32P]ATP was removed lo a d substrate and ecDnaB protein as indicated in from the single-stranded substrate using ed the figure legends. Mixtures were incubated sephadex G-50 column chromatography. fro m at 37°C for the time indicated in the legends Nitrocellulose filter binding assays http for figures 1, 3, 6 and supplementary figures were performed with 50 fmol of 32P-labeled ://w w 1 and 2. substrate. The binding reactions were w DNA helicase activity of the performed at 60˚C (mtMCM), 30˚C .jbc .o mtMCM was measured in reaction mixtures (spMCM) or 37˚C (ecDnaB) for 10 min in brg/ (15 µl) containing 20 mM Tris-HCl (pH 20µl reaction buffer containing 20mM y g u 8.5), 10 mM MgCl2, 2 mM DTT, 100 µg/ml Hepes-NaOH (pH 7.5), 10mM MgCl2, 2mM est o BSA, 5 mM ATP, 10 fmol of 32P-labeled DTT, and 100µg/ml BSA. After incubation, n A p DNA substrate and mtMCM proteins as the mixture was filtered through an alkaline- ril 1 indicated in the figure legends. Mixtures washed nitrocellulose filter (Millipore, HA 5, 2 were incubated at 60°C for the time 0.45 µm) (11), which was subsequently 01 9 indicated in the legends for figures 1, 3, 6 washed with 20mM Hepes-NaOH (pH 7.5). and supplementary figures 1 and 2. The radioactivity adsorbed to the filter was DNA helicase activity of the measured by liquid scintillation counting. spMCM complex was measured in reaction All DNA binding experiments were mixtures (15 µl) containing 25 mM Hepes- repeated three times and their averages with NaOH (pH 7.5), 25 mM potassium acetate, standard deviations are shown in Figure 4. 10 mM magnesium acetate, 5 mM ATP, 1 mM DTT, 100 µg/ml BSA, 10 fmol of 32P- Streptavidin/nitroavidin displacement assay labeled DNA substrate, and spMCM Biotinylated oligonucleotides were complex as indicated in the figure legends. 5’ end labeled using [γ-32P]ATP and T4 Mixtures were incubated at 30°C for the polynucleotide kinase and purified as time indicated in the legends for figures 1, 3, described above for the preparation of 6 and supplementary figures 1 and 2. substrates for filter binding assay. Ten fmol Reactions were stopped by adding of oligonucleotides (as indicated in figure 5µl of loading buffer containing 2% SDS, legends) were incubated with 750 fmol of 3 streptavidin for the experiments with the The replicative helicases of bacteria, archaea mtMCM or with 1.5 pmol of nitroavidin for and eukarya can unwind DNA-RNA hybrid the experiments with spMCM and ecDnaB It has not yet been determined in the respective helicase reaction mixtures whether the archaeal or eukaryal MCM (15 µl) at 30˚C for 10 min. Next, the proteins are capable of unwinding substrates helicase was added together with 10 pmol of containing RNA, and only limited studies free biotin for each reaction (to trap and have been performed with the bacterial sequester streptavidin or nitroavidin when DnaB protein (12). Thus the ability of a released from the oligonucleotides by the replicative helicase from bacteria, archaea helicase). After incubation for 1 hr at 55˚C and eukarya to unwind RNA-containing (mtMCM), 30˚C (spMCM) or 37˚C substrate was determined. (ecDnaB) 5 µl of 5X loading buffer (100mM It was shown previously that the EDTA, 0.1% xylene cyanol, 0.1% spMCM complex is unable to displace a bromophenol blue and 50% glycerol) was DNA substrate with a duplex region of 20 added to each sample, followed by bp or longer when provided with only a 3′ electrophoretic analysis on a native 8% ssDNA overhang ((13) and Supplementary D polyacrylamide gel in 0.5X TBE. The gels Figure 1). To unwind that substrate, the o w n were analyzed using phosphorimaging. All helicase required a forked DNA structure lo a d experiments were repeated 3 times with containing both 3’ and 5’ overhanging ed almost identical results. Representative gels ssDNA (13,14). Similar observations have fro m are shown in Figure 2. been made with the bacterial DnaB protein http (Supplementary Figure 1). Therefore, all ://w w ATPase assay substrates used in the first set of experiments w ATPase activity was measured in (Fig. 1) contained duplex regions of 25 bp in .jbc .o reaction mixtures (15 µl) containing 25 mM length and forked structures (containing rg b/ Hepes-NaOH (pH 7.5), 5 mM MgCl2, 1 mM both 3’ and 5’ overhang single stranded y gu dithiothreitol, 100 µg/ml bovine serum regions) as shown schematically at the top es t o albumin, 1.5 nmol of ATP containing 0.495 of each panel in Figure 1. n A pAmmoelr shaomf B[γi-o3s2Pci]eAnTcePs ), (a3n0d0 09 0 Cfim/moml oolf; enzymeFs irstto, asu naw cionndt rodlu, pthleex abDiliNtyA o f wthaes pril 15, 2 mtMCM (as hexamer) or 270 fmol of determined. All three helicases can unwind 01 9 spMCM and ecDnaB (as hexamer) in the such substrates with comparable efficiencies presence or absence of 2 pmol 25-base DNA to those previously reported for the enzymes or RNA oligonucleotide as indicated in the [Fig. 1 panels A-C lanes 3-5 (I), see also legend of Figure 5. After incubation at 60°C panels D-F]. Next, the ability of the helicase (mtMCM), 30°C (spMCM) or 37°C to unwind duplex RNA substrates was (ecDnaB) for 60 min, a 1µl aliquot was determined. None of the enzymes, mtMCM, spotted onto a polyethyleneimine cellulose spMCM or ecDnaB, could unwind these thin layer plate, and ATP and Pi were substrates [Fig. 1 panels A-C lanes 18-20 separated by chromatography in 1 M formic (IV), see also panels D-F]. acid and 0.5 M LiCl. The extent of ATP Although the enzymes cannot hydrolysis was quantitated by unwind duplex RNA, they may have the ability to unwind substrates containing phosphorimager analysis. RNA-DNA hybrids. There are two types of RESULTS such hybrid substrates. One contains 3’- overhanging DNA and 5’-overhanging 4 RNA, while the other has the reverse, 3’- cannot unwind RNA substrates they can overhanging RNA and 5’-overhanging separate DNA-RNA hybrids if provided DNA. The ability of the enzyme to unwind with a ssDNA region to move on. However, both types of substrates was evaluated. although the enzymes can unwind RNA- As shown in Figure 1A-C all three DNA hybrid substrates, the rate of helicases are capable of unwinding hybrid unwinding a duplex DNA may be different DNA-RNA substrates when translocating than that for a RNA-DNA hybrid. Thus, the along the DNA strand [Fig. 1A-C, lanes 8- experiments described in Figure 1 were 10 (II), see also panels D-E; recall that the repeated, varying the reaction time instead eukaryotic and archaeal MCM have a 3’→5’ of helicase concentration (Supplementary polarity on ssDNA while DnaB moves in the Fig. 2). Similar to the results of Figure 1, 5’→3’ direction]. No unwinding could be both the eukaryal and archaeal MCM observed, however, when the M. proteins are not as efficient at unwinding thermautotrophicus and S. pombe MCM hybrid substrates in comparison to their proteins were provided with a 3’ RNA activity on DNA substrates, but no clear overhang [Fig. 1A and B lanes 13-15 (III), difference in the rate of unwinding could be D see also panels D and E] or when the observed. o w n ecDnaB was provided with 5’ RNA lo a d overhang [Fig. 1C lanes 13-15 (III), see also The replicative helicases cannot translocate ed panel F]. along RNA fro m Interestingly, the E. coli enzyme The results shown in Figure 1 and http unwinds hybrid RNA-DNA duplex more Supplementary Figure 2 suggest that the ://w efficiently than duplex DNA [Fig. 1C helicases cannot move along RNA. To ww compare lanes 8-10 (II) to lanes 3-5 (I), see determine whether the enzyme can .jbc .o also panel F] while the MCM helicases are translocate along RNA, another, more direct, rg b/ slightly inhibited (Fig. 1A and B compare approach was used. y g u lanes 8-10 (II) to lanes 3-5 (I), see also It was previously shown that the es t o panels D and E]. The reason for the mtMCM is capable of displacing n A ddiufef eretnoc e tihse nobt acslee ar.c o Hmopwoseivtieorn, it omfa y thbee sotlriegpotnauvcidleino tides whfirloem m oving abloiontgin DylNatAed, pril 15 , 2 substrates. It is well established that the and that ATP is required for this activity 01 9 stability of a DNA-RNA hybrid depends on (16). These experiments were performed which strand is the DNA and which is the using biotinylated oligonucleotides that were RNA (e.g. (15). Thus, although the same pre-bound by streptavidin. The mtMCM oligonucleotide sequences were used to was incubated with the substrate in the make the substrate for DnaB and the MCM presence of a large excess of biotin. The helicases, due to the different directionality biotin in solution served as a trap to bind of the DnaB and MCM (5’→3’ and 3’→5’, streptavidin upon its displacement from the respectively), the DNA and RNA strands are DNA by the helicase. “reversed” in the substrates used for ecDnaB A similar approach was used to in comparison to the two MCM proteins it determine whether the mtMCM is able to may be that the substrate used for the translocate along RNA. As shown in Figure ecDnaB is less stable compared to that used 2A, the enzyme can displace streptavidin for the MCM proteins. from biotinylated DNA (Fig. 2A lanes 3-6) The results presented in Figure 1 and ATP is required for the activity (Fig. 2A demonstrate that although the helicases lane 7). No displacement could be observed 5 when biotinylated RNA was used (Fig. 2A dashed line) was determined. On these lanes 10-13). substrates, the helicase would either It was previously shown that the assemble on the DNA and then move to the spMCM and ecDnaB could not displace RNA portion of the substrate, or perform the streptavidin from biotinylated reverse; assemble on the RNA and then oligonucleotides (16,17). Streptavidin binds move to the DNA. As shown in Figure 3, biotin very tightly (K ~10-15M-1 (18)), which each helicase is unable to unwind these d may explain the inability of the eukaryal and chimeric substrates regardless of whether it bacterial helicases to remove it from first assembles on the RNA (Fig. 3A-C lanes biotinylated oligonucleotides. An avidin 3-5) or DNA (Fig. 3A-C lanes 8-10) portion derivative, nitroavidin, binds biotin with of the substrate. These data demonstrate much lower affinity (K >10-9M-1 (19)). that assembly on DNA is not sufficient to d Therefore, the ability of the ecDnaB and promote RNA translocation and substrate spMCM complex to displace nitroavidin unwinding by the helicases. from biotinylated oligonucleotide was analyzed in an experiment similar to that All three replicative helicases can bind RNA D performed with streptavidin. It was found If the helicases cannot interact with o w n that both the spMCM (Fig. 2B lanes 3-5) RNA it would explain why the helicases lo a d and ecDnaB (Fig. 2C lanes 3-5) protein are cannot move along it. Therefore, the ability e d capable of displacing nitroavidin from of the ecDnaB, mtMCM and spMCM fro m biotinylated DNA. Neither enzyme could proteins to bind RNA was determined using h ttp do so if ATP was omitted from the reaction 50-mer oligonucleotide in a filter binding ://w (Fig. 2B and C lane 6), demonstrating that assay. As shown in Figure 4, all three ww active movement by the helicase is required. enzymes bind RNA, though not as well as .jbc .o In contrast, when biotinylated RNA was DNA. Nevertheless, the data suggest that rg b/ used instead of DNA, no nitroavidin lack of RNA binding is not the reason for y g u displacement could be observed (Fig. 2B the inability of the enzymes to move along e s t o and C lanes 9-11) supporting the conclusion it. n A dFriagwurne f2ro mth atF igthuer e en1z yamnde s Sucapnpnleomt emntoavrye RNA cannot stimulate the ATPase activity pril 15 , 2 along RNA. of the replicative helicases 0 1 9 Taken together, the data presented The data presented in Figure 4 above demonstrate that the bacterial, demonstrate that the enzymes can bind archaeal and eukaryal helicases cannot RNA. However, to unwind the substrate the translocate along RNA or unwind RNA enzymes not only need to interact with the duplexes. They all can, however, displace substrate but also to move along it. It is well RNA from a RNA-DNA hybrid while established that the movement of ecDnaB, moving along the DNA strand. mtMCM and spMCM along ssDNA It is possible that the helicase cannot stimulates their ATPase activity. initiate translocation from RNA but could Stimulation of ATPase activity is therefore move along RNA if movement starts on indirect evidence for helicase movement DNA. Thus the ability of the helicases to along nucleic acids. Thus, an ATPase assay unwind substrates containing RNA-DNA was used to determine whether the helicases chimeric oligonucleotides (schematically are capable of translocation along RNA. shown at the top of each panel of Fig. 3; As expected, the ATPase activity of DNA strands, solid line; RNA strands, all three helicases is stimulated by ssDNA 6 (Fig. 5A-C). RNA, on the other hand, does displacement of the right duplex region [Fig. not stimulate ATPase activity; only the basal 6A-C lanes 8-10 (II), see also panel D-F]. level of activity was observed with mtMCM Translocation along the hybrid duplex is and spMCM (Fig. 5A and B). The ecDnaB indistinguishable from translocation along protein exhibits a different behavior. Its duplex DNA [Fig. 5A-C compare lanes 8-10 ATPase activity is inhibited in the presence (II) to lanes 3-5 (I), see also panel D-F]. It of RNA (Fig. 5C). The reason for this is not is possible, however, that the bacterial and clear. As the MCM and DnaB proteins have eukaryal enzymes displace the RNA prior to different structures it is possible that upon the displacement of the downstream duplex. binding to RNA the ecDnaB structure is This is not the case, however, as neither of locked in a conformation that prevents the the enzymes can unwind DNA or RNA intrinsic ATPase activity, the access of the duplexes of 25 nucleotides if not provided nucleoside to the active site, or dissociation with a forked DNA substrate of the ADP after hydrolysis. (Supplementary Fig. 1B and C). In addition, all three enzymes have the ability to displace The replicative helicases can translocate streptavidin (mtMCM) or nitroavidin D along DNA-RNA duplexes (spMCM and ecDnaB) from DNA-RNA o w n The replicative helicases of bacteria, hybrids (data not shown). lo a d archaea and eukarya are all capable of These results show that, in contrast e d translocating along duplex DNA (16,17,20). to RNA translocation, when the RNA is a fro m The substrates required for dsDNA part of a RNA-DNA hybrid duplex the h ttp translocation, however, are different for the helicase can move along it without major ://w three enzymes. While the archaeal MCM difficulty. ww can slide onto duplex DNA (16), the .jbc .o eukaryotic MCM requires a 3’ ssDNA DISCUSSION rg b/ overhang (16,20) and DnaB requires a 5’ y g u ssDNA overhang (17). The study described here e s t o After establishing that the helicases demonstrates that the three replicative n A caabnilnitoyt tmo omveo vael oanlogn Rg Na AR N(FAig-Ds. N1A-3 )d, utphleeixr hdeolmicaaisness ofs tluifdei,e da,r e rceappraebsleen toinf gu nwthien ditnhgre ea pril 15 , 2 was determined using substrates similar to DNA-RNA hybrid. What might the in vivo 0 1 9 those previously used to demonstrate role of such activity be? Does the dsDNA translocation by the enzymes replicative helicase encounter RNA-DNA (16,20). It was previously shown that while hybrids during chromosomal DNA DnaB and the eukaryotic MCM require a replication? A growing number of studies in single-stranded overhang region (5’ for bacteria and eukarya suggest that such DnaB and 3’ for MCM) the archaeal enzyme RNA-DNA hybrids (R-loops) do exist on can initiate duplex translocation from a blunt DNA. Therefore, if these structures are not end (16,20). Three oligonucleotides were removed from the DNA ahead of the constructed and hybridized to produce the replication fork the replicative helicase has desired single-stranded and double-stranded to remove or bypass them. regions of DNA or RNA (schematically Studies conducted with bacteria and shown at the top of each panel in Fig. 6). As eukarya demonstrated that under some shown in Figure 6, all three enzymes are circumstances RNA-DNA hybrids form as capable of translocating along the RNA- the result of transcription in cells harboring DNA duplex, as is evident from the mutations in certain genes. For example, 7 transcription in E. coli cells harboring a formation of a R-loop containing RNA- mutation in topoisomerase I results in stable DNA hybrids on the chromosome R-loop RNA-DNA hybrids (21,22). (27,29,30). Similarly, studies in Saccharomyces Thus, either as a result of mutation or cerevisiae Hpr1 mutant cells show that due to normal cell growth, the replicative RNA-DNA hybrid structures are formed helicase may encounter RNA along its path during transcription (23). And the depletion during chromosomal replication. As the of the splicing factor ASF/SF2 from chicken helicase would be translocating along DNA cells also results in the formation of RNA- following assembly at the origin, the data DNA hybrids (24). presented here suggest that such RNA on Although these and other examples DNA would not inhibit DNA replication, as demonstrate that mutations in different it likely would be removed by the helicase. genes can result in transcription-mediated The replicative helicases, at least the RNA-DNA hybrid formation, the base eukaryotic MCM complex, may also play a composition of the gene transcribed may direct role during transcription elongation. dictate whether R-loops are formed. It was MCM was shown to interact with the D shown that when transcription results in long eukaryotic RNA polymerase (7,8). The o w n purine-rich stretches of RNA it promotes the MCM complex also interacts with the lo a d formation of RNA-DNA hybrids (25,26). transcription activator Stat1 (31-33), and e d RNA-DNA hybrids may also play interaction with MCM is required for Stat1- fro m important roles during normal cell growth. mediated transcription (33). It was also h ttp It is well established that dsRNA can found that MCM moves along with RNA ://w regulate gene expression post- polymerase during transcription and it was ww transcriptionally via RNA interference. suggested that the MCM may unwind the .jbc .o Recently, evidence suggests that short RNA DNA ahead of the RNA polymerase (33). It rg b/ molecules may also directly regulate is tempting to speculate that the bacterial y g u transcription by regulating cytosine and archaeal helicases may also have roles e s t o methylation at promoter regions and by in transcription. In addition, in light of the n A crehfreormenactiens thmeroedinif)i.c a tiAonlt hou((g2h7 ,2th8e) exaancdt duantdae pr recseerntateind hceirrceu, mit sitsa anlcseos pothsesi btlrea nthscart,i pift pril 15 , 2 mechanism by which the RNA regulates reannealed to the template DNA, the 0 1 9 these cellular process is unknown, it was helicase could unwind it behind the RNA suggested that it may be mediated by the polymerase. REFERENCE 1. Patel, S. S., and Picha, K. M. (2000) Annu Rev Biochem 69, 651-697 2. Kornberg, A., and Baker, T. A. (1992) DNA replication, 2nd / Ed., W.H. Freeman, New York 3. Lei, M., and Tye, B. K. (2001) J Cell Sci 114, 1447-1454. 4. Forsburg, S. L. (2004) Microbiol Mol Biol Rev 68, 109-131 5. Kelman, Z., and White, M. F. 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This work was supported by a Research Scholar Grant from the American Cancer Society (RSG-04-050-01- GMC). 1The abbreviations used are: dsDNA, double-stranded DNA; ec, Escherichia coli; MCM, minichromosome maintenance; mt, Methanothermobacter thermautotrophicus; sp, 9 Schizosaccharomyces pombe; ssDNA, single-stranded DNA FIGURE LEGENDS Figure 1. mtMCM, ecDnaB and spMCM can unwind DNA-RNA hybrid substrates. DNA helicase assays were performed as described in "Experimental Procedures" using 10 fmol of substrate with increasing amounts of the mtMCM (A and D), spMCM (B and E) or ecDnaB (C and F) for 1 hr at 60, 30 and 37°C, respectively. Panels A-C are representative gels. 32P-labeled strands are marked by an asterisk. DNA strands are represented by a solid line and RNA strands by a dashed line. Panel A, lanes 1, 6, 11 and 16, boiled substrate; lanes 2, 7, 12 and 17, substrate only; lanes 3, 8, 13, and 18, 10 fmol of mtMCM as hexamer; lanes 4, 9, 14, and 19, 30 fmol of mtMCM as hexamer; lanes 5, 10, 15, and 20, 90 fmol of mtMCM as hexamer; lanes 4, 9, 14, and 19, 270 fmol of mtMCM as hexamer. Panels B and C, lanes 1, 6, 11 and 16, boiled substrate; lanes 2, 7, 12 and 17, substrate only; lanes 3, 8, 13, and 18, 30 fmol of spMCM (B) or ecDnaB (C) as hexamer; lanes 4, 9, 14, and 19, 90 fmol of spMCM (B) or ecDnaB (C) as hexamer; lanes 5, 10, 15, and 20, 270 fmol of spMCM (B) or ecDnaB (C) as hexamer; lanes 4, 9, D 14, and 19, 90 fmol of spMCM (B) or ecDnaB (C) as hexamer. Panels D-F, summary of three o w n independent experiments with standard deviation of the percent displacement by the mtMCM lo a d (D), spMCM (E) and ecDnaB (F). DNA/DNA substrate (I), closed circle; DNA/RNA substrate e d (II, translocation on the DNA strand), open circle; DNA/RNA substrate (III, translocation on the fro m RNA strand), closed triangle; RNA/RNA substrate (IV), open triangle. h ttp ://w Figure 2. mtMCM, ecDnaB and spMCM can displace streptavidin or nitroavidin from ww biotinylated DNA but not RNA. .jbc .o Biotinylated ssDNA or ssRNA oligonucleotide was pre-incubated with a 75-fold molar rg b/ excess of streptavidin (A) or a 150-fold molar excess of nitroavidin (B and C) at 30˚C for 10 y g u min. Assays were performed as described in “Experimental Procedures” using 10 fmol of e s t o substrate DNA or RNA with increasing amounts of mtMCM (A), spMCM (B) or ecDnaB (C) for n A 1D NhrA a ts u5b5s,t r3a0t ea; nlda n3e7s° 8C-,1 r4e sRpNecAti vseulbys, trinat eth. eL panreesse n1c aen odf 81,0 b piomtionly loaft ebdi ootliing. o nPuacnleelo tAid, el;a nlaense 1s -27 pril 15 , 2 and 9, biotinylated oligonucleotide bound to streptavidin; lanes 3 and 10, 10 fmol of mtMCM as 0 1 9 hexamer; lanes 4 and 11, 30 fmol of mtMCM as hexamer; lanes 5 and 12, 90 fmol of mtMCM as hexamer; lanes 6 and 13, 270 fmol of mtMCM as hexamer; lane 7 and 14, 270 fmol of mtMCM as hexamer but without ATP. Panels B and C, lanes 1-6 DNA substrate; lanes 7-12 RNA substrate. Lanes 1 and 7, biotinylated oligonucleotide; lanes 2 and 8, biotinylated oligonucleotide bound to nitroavidin; lanes 3 and 9, 30 fmol of spMCM (B) or ecDnaB (C) as hexamer; lanes 4 and 10, 90 fmol of spMCM (B) or ecDnaB (C) as hexamer; lanes 5 and 11, 270 fmol of spMCM (B) or ecDnaB (C) as hexamer; lanes 6 and 12, 270 fmol of spMCM (B) or ecDnaB (C) as hexamer but without ATP. Streptavidin or nitroavidin are marked by closed circle and 32P- labeled strands are marked by an asterisk. Figure 3. mtMCM, ecDnaB and spMCM are unable to unwind chimeric DNA-RNA substrates. DNA helicase assays were performed as described in "Experimental Procedures" using 10 fmol of substrates with increasing amounts of the mtMCM (A), spMCM (B) or ecDnaB (C) for 1 hr at 55, 30 and 37°C, respectively. 32P-labeled strands are marked by asterisks. DNA strands are represented by a solid line and RNA by a dashed line. Panel A, lanes 1 and 6, 10
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