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Effect of heliquinomycin on the activity of humanminichromosome maintenance 4/6/7 helicaseYukio Ishimi1,2, Takafumi Sugiyama1, Ryou Nakaya1, Makoto Kanamori3, Toshiyuki Kohno2,Takemi Enomoto3and Makoto Chino41 College of Science*, Ibaraki University, Japan2 Macromolecular Structure Research Group*, Mitsubishi Kagaku Institute of Life Sciences, Tokyo, Japan3 Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Miyagi, Japan4 Pharmaceuticals Group, Nippon Kayaku Co. Ltd., Tokyo, JapanMinichromosome maintenance (MCM) proteins areessential factors for the prevention of the loss of extra-chromosomal DNA in Saccharomyces cerevisiae [1–3].A heterohexameric MCM2–7 protein complex has beenidentified as a component of the DNA replicationlicensing system that ensures a single round of DNAreplication per cell cycle [4–7]. This complex functionsas a replicative DNA helicase that drives the unwindingof the DNA duplex prior to semiconservative DNAsynthesis at the replication forks. This notion is sup-ported by the following findings. First, all of theMCM2–7 proteins possess DNA-dependent ATPasemotifs that are common features of DNA helicases [8].Second, the MCM4/6/7 subcomplex forms the core ofthe MCM2–7 hexamer and exhibits intrinsic DNAhelicase activity in vitro [9–12]. Third, in S. cerevisiae,MCM2–7 proteins play an essential role in both the ini-tiation and elongation of DNA replication [13], andthese proteins migrate on the genome together with thereplication forks [14,15]. One of the intricacies relatedto the function of the MCM2–7 complex is that an iso-lated MCM2–7 complex does not exhibit definite DNAhelicase activity in vitro, but the MCM4/6/7 hexamerdoes. Further, the interaction between the MCM2protein and the MCM4/6/7 hexamer, or between theMCM3/5 proteins and the MCM4/6/7 hexamer,Keywordsanticancer drug; DNA replication; MCM4/6/7 helicaseCorrespondenceY. Ishimi, Ibaraki University, 2-1-1 Bunkyo,Mito, Ibaraki 310-8512, JapanFax: +81 29 228 8439Tel: +81 29 228 8439E-mail: ishimi@mx.ibaraki.ac.jp(Received 9 March 2009, revised 13 April2009, accepted 16 April 2009)doi:10.1111/j.1742-4658.2009.07064.xThe antibiotic heliquinomycin, which inhibits cellular DNA replication at ahalf-maximal inhibitory concentration (IC50) of 1.4–4 lm, was found to inhi-bit the DNA helicase activity of the human minichromosome maintenance(MCM) 4/6/7 complex at an IC50value of 2.4 lm. In contrast, 14 lm heliqui-nomycin did not inhibit significantly either the DNA helicase activity of theSV40 T antigen and Werner protein or the oligonucleotide displacementactivity of human replication protein A. At IC50values of 25 and 6.5 lm,heliquinomycin inhibited the RNA priming and DNA polymerization activi-ties, respectively, of human DNA polymerase-a/primase. Thus, of theenzymes studied, the MCM4/6/7 complex was the most sensitive to heliqui-nomycin; this suggests that MCM helicase is one of the main targets ofheliquinomycin in vivo. It was observed that heliquinomycin did not inhibit theATPase activity of the MCM4/6/7 complex to a great extent in the absenceof single-stranded DNA. In contrast, heliquinomycin at an IC50value of5.2 lm inhibited the ATPase activity of the MCM4/6/7 complex in the pres-ence of single-stranded DNA. This suggests that heliquinomycin interfereswith the interaction of the MCM4/6/7 complex with single-stranded DNA.AbbreviationsBrdU, bromodeoxyuridine; FITC, fluorescein isothiocyanate; IC50,half-maximal inhibitory concentration; MCM, minichromosomemaintenance; RPA, replication protein A.*[Corrections added on 18 May 2009 after first online publication: in affiliation 1, ‘Macromolecular Structure Research Group’ has beenreplaced by ‘College of Science’, and in affiliation 2 ‘Macromolecular Structure Research Group’ has been inserted.]3382 FEBS Journal 276 (2009) 3382–3391 ª 2009 The Authors Journal compilation ª 2009 FEBSinhibits helicase activity [16,17]. On the basis of theseprevious reports, we propose that a structural changein the MCM2–7 complex may generate the MCM4/6/7hexamer, which, in turn, exhibits helicase activity.Another possibility is that the DNA helicase activity ofthe MCM2–7 complex may be attributed to the inter-action of this complex with other proteins. It has beenreported that the CMG complex, which consists of theCdc45 protein, MCM2–7 hexamer and the GINS com-plex purified from Drosophila embryo extracts, exhibitsDNA helicase activity in vitro [18]. Furthermore, it hasbeen demonstrated recently that the MCM2–7 complexprepared from S. cerevisiae exhibits DNA helicaseactivity in the presence of potassium acetate or gluta-mate. These results suggest that the MCM2–7 complexfunctions as a replicative DNA helicase in vivo [19].Heliquinomycin, which is an antibiotic [20,21], inhib-its cellular DNA replication and RNA synthesis. Toelucidate its cellular targets for the inhibition of DNAsynthesis, we examined the effects of heliquinomycinon the DNA helicase activities of the MCM4/6/7 com-plex, SV40 T antigen and Werner protein, on the oligo-nucleotide displacement activity of replication proteinA (RPA), and on the RNA priming and DNA poly-merization activities of the DNA polymerase-a/primasecomplex. The results indicated that, among all theenzymes examined, the MCM4/6/7 helicase was themost sensitive to heliquinomycin. It was observed that,in the absence of single-stranded DNA, heliquinomycindid not inhibit the ATPase activity of the MCM4/6/7complex to a great extent; in contrast, in the presenceof DNA, this antibiotic inhibited the ATPase activity.This result suggests that heliquinomycin inhibits thehelicase activity of MCM4/6/7 by interfering with theinteraction of this complex with single-stranded DNA.ResultsSensitivity of cellular DNA replication toheliquinomycinHeliquinomycin with a relative molecular mass of698 Da was isolated from Streptomyces sp. as an anti-biotic (Fig. S1) [20]. It has been shown that heliquino-mycin inhibits DNA replication in various transformedcells at a half-maximal inhibitory concentration (IC50)of 1.4–4 lm [21]. In these experiments, DNA synthesiswas measured by the incorporation of labelled thymi-dine into DNA. To confirm this, human HeLa cellswere pulse labelled with bromodeoxyuridine (BrdU) inthe presence of increasing concentrations of heliquino-mycin (Fig. 1A). BrdU incorporated into DNA wasdetected by staining the cells with anti-BrdU Ig andABFig. 1. Effect of heliquinomycin (HQ) on the incorporation of BrdUinto DNA in HeLa cells. (A) Logarithmically growing HeLa cells wereincubated with the indicated concentrations of heliquinomycin for1 h and then pulse labelled with BrdU for 20 min. The incorporatedBrdU in the cells was detected by incubation of the cells with anti-BrdU Ig, followed by FITC-labelled anti-rat Ig. MCM7 was detectedby incubation of the cells with anti-MCM7 Ig, followed by Cy3-labelled anti-mouse Ig. (B) One hundred Cy3-stained cells wereselected, and the fluorescence intensity of FITC in the cells wasquantified. The average level of intensity in the cells cultured in thepresence of heliquinomycin was expressed in comparison with thatin the cells cultured in the absence of heliquinomycin.Y. Ishimi et al. Inhibition of MCM4/6/7 helicase with heliquinomycinFEBS Journal 276 (2009) 3382–3391 ª 2009 The Authors Journal compilation ª 2009 FEBS 3383then with fluorescein isothiocyanate (FITC)-labelledsecond Ig. The cells were also stained with specific Igto detect MCM7 protein in the nucleus. In the absenceof heliquinomycin, approximately 27% of the cellswere stained with anti-BrdU Ig. As the concentrationof heliquinomycin was increased, the proportion ofBrdU-positive cells and the intensity of the signal grad-ually decreased. Almost no BrdU-positive cells weredetected in the presence of 14 lm heliquinomycin. Incontrast, staining with anti-MCM7 Ig was not changedby the presence of heliquinomycin. The fluorescencederived from the incorporated BrdU was quantified inthese experiments, and the IC50value was determinedto be 2.3 lm (Fig. 1B), which is similar to the valuereported previously [21].Sensitivity of MCM4/6/7 helicase toheliquinomycinHeliquinomycin at an IC50value of 7–14 lm inhibitsthe activity of the cellular DNA helicase called DNAhelicase I [22], but scarcely affects the activities oftopoisomerases and the replication of the SV40chromosome in vitro [21]. To understand the cellulartargets of this antibiotic during DNA replication, theeffects of this antibiotic on the helicase activities of thehuman MCM4/6/7 complex, SV40 T antigen andWerner protein were examined. In addition to thesethree proteins, the human DNA polymerase-a/primasecomplex and the human RPA complex were purifiedto near homogeneity (Fig. S2). Some unidentified pro-teins were found in the purified DNA polymerase-a/primase complex and the human RPA complex. It hasbeen reported that MCM3 interacts with DNApolymerase-a/primase [23]. We examined the presenceof MCM4, 5 and 6 proteins in the purified DNA+ MCM4/6/7AB+ Tag0(μM)0.431.44.31443HQ00.431.44.31443%17-mer17-mer/M130(μM) HQ0.43 1.4 4.31443TagMCM0(μM) HQ0.431201008060402001.44.31443%+ Werner0 0.431.44.3 14 43(μM)HQ17-mer17-mer/M13Fig. 2. Effect of heliquinomycin on DNA helicase activity. (A) Top:effects of increasing concentrations of heliquinomycin (HQ) on theDNA helicase activities of the MCM4/6/7 complex and the SV40 Tantigen. Dimethyl sulfoxide solution (0.4 lL) containing or lackingheliquinomycin was added to the reaction mixture. The final con-centrations of heliquinomycin added to the reaction mixture areindicated at the top. The DNA helicase activity was measured asthe activity that displaces 17-mer oligonucleotides annealed toM13mp18 single-stranded DNA. Bottom: the proportion of dis-placed 17-mer oligonucleotides in total DNA was considered to be100% in the control reaction mixture lacking heliquinomycin, andthe proportions in the mixtures containing heliquinomycin were cal-culated in relation to the control value. The horizontal line is dis-played on a logarithmic scale. Four independent experiments wereperformed for the MCM4/6/7 complex, and an average of the val-ues was plotted together with the standard deviations. Two inde-pendent experiments were performed for the T antigen and anaverage of the values was plotted. (B) Top: effects of increasingconcentrations of heliquinomycin on the DNA helicase activity ofthe Werner protein. Bottom: proportion of displaced 17-mer oligo-nucleotides. Two independent experiments were performed, andan average of the values was plotted together with the error bars.Inhibition of MCM4/6/7 helicase with heliquinomycin Y. Ishimi et al.3384 FEBS Journal 276 (2009) 3382–3391 ª 2009 The Authors Journal compilation ª 2009 FEBSpolymerase-a/primase complex (Fig. S3). Only smallamounts of MCM4 (0.1% of total protein) andMCM5 (0.9%) proteins were detected, and MCM6was not found.DNA helicases were added to the DNA helicasereaction mixtures at the minimum amounts required todisplace almost all of the 17-mer oligonucleotides.Heliquinomycin at a concentration of 14 lm did notinhibit the helicase activity of the SV40 T antigen to agreat extent, but inhibited that of the MCM4/6/7 com-plex at an IC50value of 2.4 lm (Fig. 2A). Heliquino-mycin (14 lm) did not inhibit the helicase activity ofthe Werner protein to a great extent (Fig. 2B). Itshould be noted that the mobility of displaced frag-ments in the absence or presence of heliquinomycinwas different. The RPA complex displaces oligonucleo-tides annealed to M13 single-stranded DNA withouttriggering ATP hydrolysis [24]. We found that heliqui-nomycin scarcely affected the oligonucleotide displace-ment activity of RPA (Fig. S4). We also examined theeffect of heliquinomycin on the reactions of RNApriming (Fig. 3A) and DNA polymerization activity(Fig. 3B) of the DNA polymerase-a/primase complex.When dT50was used as a template, an RNA primer ofapproximately 10 nucleotides was synthesized only inthe presence of the above complex. The synthesisof the RNA primer was inhibited in the presence ofheliquinomycin at an IC50value of 25 lm. The DNApolymerization activity of the DNA polymerase-a/primase complex was measured using activated DNAas a template and a primer. The observed reduction inthe level of the incorporated nucleotides indicates thatheliquinomycin at an IC50value of 6.5 lm inhibits theDNA polymerization activity of the complex. Theseresults indicate that, among the enzymes studied,MCM4/6/7 is the most sensitive to heliquinomycin andthe DNA polymerase-a/primase complex is also rela-tively sensitive to this antibiotic (Table 1).Sensitivity of MCM4/6/7 helicase toheliquinomycinTo understand the mechanism by which heliquino-mycin inhibits the activity of MCM4/6/7 helicase, we0 0.43 1.4 4.3 14 43HQ (μM)%0(μM)0.431.44.31443HQ171050nt+ Polα-primaseAB0(μM) HQ0.43 1.4 4.3 14 43%120100806040200100806040200Fig. 3. Effect of heliquinomycin on the RNA priming activity of theDNA polymerase-a/primase complex. (A) Top: effect of increasingconcentrations of heliquinomycin (HQ) on the RNA priming actionof the DNA polymerase-a/primase. RNA priming activity was mea-sured by the analysis of oligoA synthesis when dT50was used asthe template. The products were electrophoresed under denaturingconditions. The three oligonucleotides of A10, 17-mer and dT50were labelled at their 5¢ ends and electrophoresed to determine thesize of the synthesized oligoA fragment. The arrow indicates theposition of the RNA primer synthesized by DNA polymerase-a/prim-ase. Bottom: radioactivity of the synthesized RNA primer. Theradioactivity recorded for RNA in the control reaction mixture lack-ing heliquinomycin was considered to be 100%, and that recordedfor the reaction mixtures containing heliquinomycin was presentedin relation to this control value. Two independent experiments wereperformed, and an average of the values was plotted together witherror bars. (B) Effect of increasing concentrations of heliquinomycinon the DNA polymerization activity of the DNA polymerase-a/prim-ase. The reaction was performed using activated DNA as a primerand template. The acid-insoluble radioactive material trapped on theglass fibre filter was measured. The radioactivity recorded in thecase of the control reaction mixture lacking heliquinomycin wasconsidered to be 100%, and that recorded for the reaction mixturecontaining heliquinomycin was presented in relation to this controlvalue. Two independent experiments were performed, and an aver-age of the data was plotted together with the error bars.Y. Ishimi et al. Inhibition of MCM4/6/7 helicase with heliquinomycinFEBS Journal 276 (2009) 3382–3391 ª 2009 The Authors Journal compilation ª 2009 FEBS 3385examined the effect of heliquinomycin on the formationof the MCM4/6/7 complex (Fig. 4). In the absence ofheliquinomycin, the MCM4/6/7 complex, as detectedusing anti-MCM4 IgG, exhibits a trimeric or hexamericstructure, depending on its mobility in the gel. Weobserved that the hexamer–trimer proportion increasedslightly with an increase in the heliquinomycin concen-tration. The hexameric form of the MCM4/6/7 complexwas dominant in the presence of 14 lm of heliquino-mycin, and larger complexes were detected in the pres-ence of 43 lm of heliquinomycin. Thus, it appears thathigher concentrations of heliquinomycin affect signifi-cantly the formation of the MCM4/6/7 complex. Wealso examined the sensitivity of the ATPase activitiesof the MCM4/6/7 complex and the SV40 T antigento heliquinomycin in the absence of single-strandedDNA (Fig. 5). Heliquinomycin inhibited the ATPase0 (μM) HQ0.43 1.4 4.3 14 43 669 440 kDa (4/6/7)2 (4/6/7) Fig. 4. Effect of increasing concentrations of heliquinomycin (HQ)on the formation of the MCM4/6/7 complex. The MCM4/6/7 com-plex was incubated in the presence or absence of heliquinomycinand subsequently electrophoresed on a native polyacrylamide gel.The proteins in the gel were transferred onto a filter and theMCM4 protein was detected by incubating the filter with rabbitanti-MCM4 IgG, followed by horseradish peroxidase-conjugatedanti-rabbit IgG. Finally, the bound antibodies were examined forchemiluminescence using West Pico chemiluminescent substrate(Thermo Scientific, Rockford, IL, USA). The positions to whichthyroglobulin (669 kDa) and ferritin (440 kDa) migrated in the gelare indicated.TagMCM0(μM) HQ0.43 1.4 4.3 14 430(μM)0.431.44.31443HQ00.431.44.31443+ MCM4/6/7+ TagPiATP1201008060%40200Fig. 5. Top: effect of increasing concentrations of heliquinomycin(HQ) on the ATPase activities of the MCM4/6/7 complex (340 ng)and the SV40 T antigen (200 ng) in the absence of single-strandedDNA. After incubation under these conditions, an aliquot of the mix-ture was subjected to thin layer chromatography. The radioactivity atthe sites to which Piand ATP migrated was measured, and the ratioof the released Pito ATP was calculated. The ratio obtained in thecase of the reaction performed without the enzymes was subtractedfrom that obtained in the reactions performed with the enzymes.Bottom: the ratio obtained in the case of the control reaction mixturewhich lacked heliquinomycin was considered to be 100%, and thatrecorded for the reaction mixture that contained heliquinomycin waspresented in relation to this control value. Two independent experi-ments were performed for the MCM4/6/7 complex, and an averageof the values was plotted together with error bars.Table 1. Sensitivity of the enzymes to heliquinomycin. The IC50values indicated are those calculated in the present study as wellas in a previous investigation [21]. Those determined in the pre-vious investigation are marked by an asterisk.IC50(lM)Human cellular DNA synthesis* 1.4–4HeLa DNA synthesis 2.5SV40 chromosome replication in vitro*>72T antigen helicase 43HeLa helicase I* 7–14hMCM4/6/7 helicase 2.4Werner helicase > 43Human RPA > 43DNA polymerase a 6.5DNA primase 25Topoisomerase I* 145Topoisomerase II* 43Inhibition of MCM4/6/7 helicase with heliquinomycin Y. Ishimi et al.3386 FEBS Journal 276 (2009) 3382–3391 ª 2009 The Authors Journal compilation ª 2009 FEBSactivities of these two helicases only slightly. We alsoexamined its effect on these activities in the presence ofheat-denatured, single-stranded DNA (Fig. 6); underthese conditions, the ATPase activity of the MCM4/6/7complex increased manifold [9]. Heliquinomycin scar-cely inhibited the ATPase activity of the T antigen, butinhibited that of the MCM4/6/7 helicase at an IC50value of 5.2 lm. The ATPase activity of MCM4/6/7was also stimulated in the presence of M13mp18 single-stranded DNA in place of heat-denatured DNA, andthe stimulated activity was also inhibited in the pres-ence of heliquinomycin (Fig. S5). These results suggestthat heliquinomycin interferes with the interaction ofthe MCM4/6/7 complex with single-stranded DNA,which is required to increase the ATPase activity of thiscomplex.DiscussionHeliquinomycin was first characterized as a compoundthat inhibits bacterial cell growth, and was found toinhibit DNA synthesis in several cancer cells at anIC50value of 1.4–4 lm [21]. In the present study, wefound that heliquinomycin inhibits BrdU incorporationinto DNA at an IC50value of 2.3 lm; this result isconsistent with the previous findings. The reportedstudy also indicated that the cell cycle progression ofHeLa cells was retarded during the S phase and thecells were arrested in the G2 phase in the presence ofheliquinomycin [21]. Heliquinomycin inhibits thecellular DNA helicase, helicase I, at an IC50value of7–14 lm, but does not inhibit the activity of topoi-somerases. Our study indicates that heliquinomycininhibits the activity of human MCM4/6/7 helicase atan IC50value of 2.4 lm, but scarcely inhibits the DNAhelicase activity of the SV40 T antigen and the Wernerprotein, or the oligonucleotide displacement activity ofhuman RPA. Further, it inhibits the RNA primingand DNA polymerization activities of the human DNApolymerase-a/primase at IC50values of 25 and 6.5 lm,respectively. We also examined the effect of heliquino-mycin on the DNA helicase activity of human REC-QL4 protein. Heliquinomycin inhibited this activity atan IC50value of 14 lm (data not presented). Thus,among the enzymes studied, MCM4/6/7 helicase wasfound to be the most sensitive to heliquinomycin.These results suggest that MCM helicase and DNApolymerases may be the critical targets of heliquinomy-cin during cellular DNA replication. Further, weobserved that the checkpoint system that is induced bythe inhibition of DNA polymerases during DNA repli-cation is not induced in HeLa cells treated with 4.3 lmheliquinomycin (data not presented). This suggests thatthe MCM helicase, rather than the DNA polymerases,is the main target of heliquinomycin in vivo.Heliquinomycin not only inhibited the helicase activ-ity of the MCM4/6/7 complex, but also inhibited thesingle-stranded DNA-dependent ATPase activity ofthe complex. Heliquinomycin suppressed the ATPaseactivity of the complex in the absence of single-stranded DNA, but the enzymatic activity wassignificantly less sensitive to heliquinomycin. Thus,heliquinomycin may inhibit the ATPase activity andDNA helicase activity of the MCM4/6/7 complex byaffecting the ability of this complex to interact withsingle-stranded DNA. The finding that the activities0(μM)0.431.44.31443HQ00.431.44.31443+ MCM4/6/7AB+ TagPiATPTagMCM0(μM) HQ0.43 1.4 4.3 14 431201008060%40200Fig. 6. (A) Effect of increasing concentrations of heliquinomycin(HQ) on the ATPase activities of the MCM4/6/7 complex (120 ng)and SV40 T antigen (200 ng) in the presence of single-strandedDNA. The radioactivity at the sites to which Piand ATP migratedwas measured, and the ratio of the released Pito ATP was calcu-lated. The ratio in the case of the reaction performed without theenzyme was subtracted from that in the case of the reactions per-formed with these enzymes. (B) The ratio obtained in the case ofthe control reaction mixture which lacked heliquinomycin was con-sidered to be 100%, and that recorded for the reaction mixture thatcontained heliquinomycin was presented in relation to this controlvalue. Two independent experiments were performed for theMCM4/6/7 complex, and an average of the values was plottedtogether with error bars.Y. Ishimi et al. Inhibition of MCM4/6/7 helicase with heliquinomycinFEBS Journal 276 (2009) 3382–3391 ª 2009 The Authors Journal compilation ª 2009 FEBS 3387of DNA polymerase-a/primase are also inhibited athigher concentrations of heliquinomycin may suggestthat heliquinomycin interacts with single-strandedDNA to interfere with the activities. However, there isno evidence for the interaction of heliquinomycin withsingle-stranded DNA. In contrast, the formation of theMCM4/6/7 complex was inhibited by heliquinomycinat higher concentrations. Although these concentra-tions are higher than those that inhibit MCM4/6/7helicase activity, it is possible that heliquinomycininteracts directly with the MCM4/6/7 complex to inhi-bit the interaction of this complex with single-strandedDNA, even at low concentrations.MCM proteins are considered to be one of themost sensitive diagnostic markers for the detectionof cancer cells in human tissues [25]. The expressionof MCM proteins appears to be critical for thedevelopment of cancer cells, as this expression showsa strong correlation with the malignant transforma-tion of cells. The finding that MCM2–7 proteins areoverexpressed in transformed cancer cells [26]suggests that the upregulation of MCM proteinexpression may play a role in the development ofcancer cells. Consistent with this notion, it hasrecently been reported that deregulated expression ofthe MCM7 protein accelerates the transformation ofcells [27]. Thus, MCM proteins are among the mostcritical targets for achieving the inhibition of cancercell growth. Furthermore, heliquinomycin mayhave useful applications in the development ofMCM-specific anticancer drugs.Materials and methodsBrdU labelling of HeLa cellsHeLa cells were cultured in Dulbecco’s modified Eagle’smedium supplemented with 7% fetal calf serum. Cells cul-tured on coverslips were incubated with dimethyl sulfoxideor increasing concentrations of heliquinomycin for 1 h andthen pulse labelled with 20 lm BrdU for 20 min. After beingwashed with NaCl/Pi, the cells were fixed by incubation with4% paraformaldehyde in NaCl/Pifor 5 min at room tem-perature. The cells were washed with NaCl/Pi, and thenpermeabilized and blocked by incubation with 0.1% TritonX-100, 0.02% SDS and 2% nonfat dried milk in NaCl/Pifor 1 h at 37 °C. The cells were incubated overnight withanti-MCM7 mouse Ig (sc-9966; Santa Cruz Biotechnology,Santa Cruz, CA, USA) at 4 °C in the above-mentionedblocking solution. The cells were washed with the samesolution and then incubated with cyanine-3 (Cy3)-conju-gated anti-rabbit IgG (Jackson ImmunoResearch, WestGrove, PA, USA) for 1.5 h at 37 °C in the blocking solu-tion. They were then re-fixed, treated with 4 m HCl for30 min at room temperature and incubated with rat anti-BrdU Ig (clone BU1/75; Harlan Sera Laboratory, Belton,Leicestershire, UK), followed by incubation with FITC-conjugated anti-rat IgG (Cappel, Organon Teknika Corpo-ration, Durham, NC, USA). Positive immunoreactivitieswere detected with fluorescence microscopy (BX-9000;KEYENCE, Osaka, Japan).DNA helicase and ATPase activities of the DNAhelicasesA human MCM4/6/7 complex was prepared, and its DNAhelicase activity was measured, as reported previously,except for some minor modifications [9]. The standard reac-tion mixture (20 lL) contained 50 mm Tris/HCl (pH 7.9),20 mm 2-mercaptoethanol, 10 mm ATP, 10 mm magnesiumacetate, 0.5 mgÆmL)1bovine serum albumin, 1–2.5 fmol ofa 17-mer oligonucleotide annealed to M13mp18 DNA andan approximately100 ng sample of human MCM4/6/7 com-plex, a 25 ng sample of SV40 T antigen or a 1.25 ng sampleof Werner protein, in the presence or absence of heliquino-mycin at the indicated concentrations. This mixture wasincubated at 37 °C for 40 min, and the products were anal-ysed using 12% PAGE. The ATPase activity was measuredby incubating either the MCM proteins (120–340 ng) or theSV40 T antigen (200 ng) at 37 °C for 30 min in the pres-ence of 74 kBq [c-32P]ATP in a solution containing 50 mmTris/HCl (pH 7.9), 20 mm 2-mercaptoethanol, 0.5 mgÆmL)1bovine serum albumin, 10 mm magnesium acetate, 10 mmATP and heliquinomycin at the indicated concentrations inthe presence or absence of 5 lg of single-stranded DNA(heat-denatured). Further, 0.5 lL of the reaction mixturewas spotted onto a poly (ethyleneimine)-cellulose thin layerchromatography plate (Cellulose F; Merck, Darmstadt,Germany). Chromatography was performed at 4 °C for aperiod of 2 h using a solution of 0.8 m acetic acid and0.8 m LiCl. The radioactivity on the plate was detectedusing a Bio-Image Analyser (FLA3000; Fuji, Tokyo,Japan).Formation of the MCM4/6/7 complexThe reaction mixture (10 lL) containing 50 mm Tris/HCl(pH 7.5), 20 mm 2-mercaptoethanol, 5 mm MgCl2,5mmATP, 100 lgÆmL)1bovine serum albumin and the MCM4/6/7 complex (170 ng) was incubated at 37 °C for 30 min in thepresence or absence of heliquinomycin. The resulting solu-tion was analysed on a 5% acrylamide gel in 50 mm Tris/HCl (pH 8.0) and 50 mm glycine. Subsequently, the gel wasimmersed in a solution containing 49 mm Tris, 38 mm gly-cine and 0.25% SDS, and was incubated at 80 °C for 1 h inorder to achieve protein denaturation. The proteins in the gelwere then transferred onto a membrane filter (Immobilon;Inhibition of MCM4/6/7 helicase with heliquinomycin Y. Ishimi et al.3388 FEBS Journal 276 (2009) 3382–3391 ª 2009 The Authors Journal compilation ª 2009 FEBSMillipore, Billerica, MA, USA) and anti-MCM4 IgG wereused to detect MCM4 on the filter [9].RNA priming and DNA synthesis with DNApolymerase-a/primaseThe DNA polymerase-a/primase complex was purified fromHeLa cells by immunoadsorption, followed by elution froma column coated with a monoclonal antibody (SJK237), asreported previously [28]. DNA polymerase activity wasmeasured using a reaction mixture (20 lL) containing20 mm Tris/HCl (pH 7.9), 3.3 mm 2-mercaptoethanol,0.2 mgÆmL)1bovine serum albumin, 5 mm MgCl2,0.25 mgÆmL)1activated DNA, 100 lm each of dATP,dGTP and dTTP, 50 lm of dCTP, 111 kBq [a-32P]dCTPand 85 ng DNA polymerase-a/primase complex in the pres-ence of heliquinomycin at the indicated concentrations. Thereaction was terminated by the addition of 30 lL of sodiumpyrophosphate (0.17 m) and 50 lL of sperm DNA(1 mgÆmL)1). Further, 1 mL of 5% trichloroacetic acid wasadded, and the acid-insoluble radioactive material trappedon a glass fibre filter was measured in a liquid scintillationcocktail. The reaction mixture (10 lL) used for the mea-surement of the RNA priming activity contained 40 mmTris/HCl (pH 7.5), 10 mm magnesium acetate, 1 mm dithio-threitol, 100 lgÆ mL)1bovine serum albumin, 0.1 mm ATP,185 kBq [a-32P]ATP and 10 lm (dT)50in the presence ofheliquinomycin at the indicated concentrations. This mix-ture was incubated at 37 °C for 40 min and then heated at95 °C for 5 min. Thereafter, bacterial alkaline phosphatase(0.6 units) was added, and the mixture was further incu-bated at 65 °C for 30 min. The mixture was heated at98 °C for 5 min in the presence of 3 lL of loading buffer(0.1% bromophenol blue, 0.1% xylene cyanol, 10 mmEDTA and 98% formamide), and the products were analy-sed on a 25% polyacrylamide gel containing 7 m urea. Theoligonucleotides, A10, 17-mer and oligo-dT50, were labelledat their 5¢ ends and used as markers. The gel was dried andthe radioactivity was detected using a Bio-Image Analyser.Preparation of the RPA complexcDNAs for human RPA1, RPA2 and RPA3 were synthe-sized from mRNA extracted from HeLa cells by the reversetranscription-polymerase chain reaction (RT-PCR) method(Invitrogen, Carlsbad, CA, USA), and were cloned into thebaculovirus vectors pVL1393, pAcUW31 and pVL1393,respectively. RPA1 was cloned to be expressed as a (His)6-RPA1 fusion protein, and RPA2 as a flag-RPA2 fusionprotein. High-5 cells were co-infected with the three virusesexpressing the RPA1, RPA2 and RPA3 proteins for 2 days.The recombinant RPA proteins in the lysates of theinfected cells were purified by performing nickel-nitrilotri-acetic acid (Qiagen, Hilden, Germany) affinity columnchromatography as follows. The purification involved thesuspension of the infected cells in lysis buffer consistingof 10 mm Tris/HCl (pH 7.5), 130 mm NaCl, 1% TritonX-100, 10 mm NaF, 10 mm sodium phosphate buffer,10 mm Na4P2O7and protease inhibitors (Pharmingen BD,San Jose, CA, USA). The mixture was incubated for40 min on ice, and insoluble components were separated bycentrifugation at 137 000 g (TLS55; Beckman, Fullerton,CA, USA) for 40 min at 4 °C. To 1 vol of the clarifiedlysate, 1/10 vol of nickel-nitrilotriacetic acid-agarose wasadded, and the mixture was incubated for 1 h at 4 °Conarocking platform. Agarose beads were then collected bycentrifugation and thoroughly washed with buffer A[50 mm sodium phosphate buffer (pH 6.0), 300 mm NaCland 10% glycerol] containing 20 mm imidazole. Next, thebeads were washed once with buffer B [50 mm sodiumphosphate buffer (pH 8.0), 300 mm NaCl and 10% glyc-erol] containing 20 mm imidazole, and the proteins boundto the beads were eluted by adding buffer B containing300 mm imidazole at a volume equivalent to 1 bed. Thiswas followed by incubation for 5 min at 4 °C on a rockingplatform and separation of the beads by centrifugation.The proteins were eluted twice more. The eluates werepooled and diluted to decrease the NaCl concentration to50 mm, and the solution thus obtained was concentratedusing Centricon 30 (Millipore). The concentrated proteinswere loaded onto a MonoQ column (GE Healthcare, Pis-cataway, NJ, USA), and the bound proteins were elutedusing a linear NaCl gradient (0.1–0.6 m). The RPA1(70 kDa), RPA2 (34 kDa) and RPA3 (14 kDa) proteinswere co-eluted with approximately 0.3 m NaCl, and wereconcentrated using Microcon 30 after the salt concentrationhad decreased to 0.1 m. The oligonucleotide displacementactivity of RPA was measured using the same reaction mix-ture as that employed to assess the DNA helicase activity,except that the reaction mixture contained 200 ng of RPAcomplex.Purification of Werner helicaseHigh-5 cells infected with recombinant virus encoding(His)6-WRN were cultured for 3 days and then collected bycentrifugation. The cells were lysed with 0.5% Nonidet P-40 in buffer C [50 mm Tris/HCl (pH 7.9), 150 mm NaCl,10% glycerol, 1 mm phenylmethylsulfonyl fluoride and20 lgÆmL)1leupeptin) for 10 min on ice, and NaCl wasadded to the lysate at a final concentration of 0.5 m. Afterincubation for 30 min on ice, the cell lysate was centrifugedat 265 070 g (TLA 100.3; Beckman) for 30 min at 4 °C.The supernatant was passed through a DE52 (Whatman,Maidstone, Kent, UK) column equilibrated with 0.5 mNaCl in buffer C to remove nucleic acids. Flow-throughfractions were loaded on to a nickel-nitrilotriacetic acidaffinity column equilibrated with buffer D [20 mm KPi(pH 7.5), 1 mm phenylmethylsulfonyl fluoride and20 lgÆmL)1leupeptin] containing 0.5 m NaCl. After loadingY. Ishimi et al. Inhibition of MCM4/6/7 helicase with heliquinomycinFEBS Journal 276 (2009) 3382–3391 ª 2009 The Authors Journal compilation ª 2009 FEBS 3389onto the nickel-nitrilotriacetic acid column, the column waswashed with buffer D containing 0.2 m NaCl and 25 mmimidazole, and eluted with buffer D containing 0.2 m NaCland 200 mm imidazole. The fractions containing Wernerprotein were determined by performing SDS–PAGE.Pooled fractions were loaded onto a MonoS column (GEHealthcare). After the column had been washed with bufferH [25 mm Hepes/NaOH (pH 7.8), 1 mm EDTA, 10% glyc-erol, 0.01% Nonidet P-40, 1 mm phenylmethylsulfonyl fluo-ride and 20 lgÆmL)1leupeptin] containing 0.2 m NaCl, thebound proteins were eluted with buffer H containing 0.5 mNaCl. Fractions around the main peak were pooled,concentrated using Vivaspin 20 (Sartorius, Hanover,Germany) and then fractionated on Superdex 200 HR inbuffer H containing 0.1 m NaCl. The purified protein wasconcentrated with Vivaspin and dialysed against buffer Hcontaining 0.1 m NaCl.Other materialsThe SV40 T antigen was prepared as reported previously[28]. Heliquinomycin was purified from Streptomyces sp.MJ929-SF2, as reported previously [29], and 1 mg of thepurified heliquinomycin was dissolved in 0.1 mL ofdimethyl sulfoxide to prepare a 10 mgÆmL)1stock solution.To prepare activated DNA, calf thymus DNA (30 mg) wasdigested for 30 min at 37 °C with DNase I (1 lg) in a mix-ture (10 mL) containing 50 mm Tris/HCl (pH 7.5), 5 mmMgCl2and 0.5 mgÆmL)1bovine serum albumin. The mix-ture was heated for 5 min at 77 °C to stop the reaction andthen dialysed against 50 mm Tris/HCl (pH 8.1) and 5 mmMgCl2.AcknowledgementsThis study was supported in part by a Grant-in-Aidfor Scientific Research from the Ministry of Education,Science, Sports and Culture of Japan.References1 Tye BK (1999) MCM proteins in DNA replication.Annu Rev Biochem 68, 649–686.2 Bell SP & Dutta A (2002) DNA replication in eukary-otic cells. Annu Rev Biochem 71, 333–374.3 Forsburg SL (2004) Eukaryotic MCM proteins: beyondreplication initiation. Microbiol Mol Biol Rev 68, 109–131.4 Kubota Y, Mimura S, Nishimoto S, Takisawa H. &Nojima H (1995) Identification of the yeastMCM3-related protein as a component of XenopusDNA replication licensing factor. Cell 81, 601–609.5 Chong JPJ, Mahbubani HM, Khoo C-Y & Blow JJ(1995) Purification of an MCM-containing complex as acomponent of the DNA replication licensing system.Nature 375, 418–421.6 Madine MA, Khoo C-Y, Mills AD & Laskey RA(1995) MCM3 complex required for cell cycle regulationof DNA replication in vertebrate cells. Nature 375,421–424.7 Prokhorova TA & Blow JJ (2000) Sequential MCM/P1subcomplex assembly is required to form a heterohex-amer with replication licensing activity. J Biol Chem275, 2491–2498.8 Koonin EV (1993) A common set of conserved motifsin a vast variety of putative nucleic acid-dependentATPases including MCM proteins involved in the initia-tion of eukaryotic DNA replication. Nucleic Acids Res21, 2541–2547.9 Ishimi Y (1997) A DNA helicase activity is associatedwith an MCM4, -6, and -7 protein complex. J BiolChem 272, 24508–24513.10 Lee J-K & Hurwitz J (2000) Isolation and characteriza-tion of various complexes of the minichromosomemaintenance protein of Schizosaccharomyces pombe.J Biol Chem 275, 18871–18878.11 Kaplan DL, Davey MJ & O’Donnell M (2003)Mcm4,6,7 uses a ‘pump in ring’ mechanism to unwindDNA by steric exclusion and actively translocate alonga duplex. J Biol Chem 278, 49171–49182.12 Bochman M & Schwacha A (2007) Differences in thesingle-stranded DNA binding activities of MCM2-7 andMCM467: MCM2 and 5 define a slow ATP-dependentstep. J Biol Chem 282, 33759–33804.13 Labib K, Tercero JA & Diffley JF (2000) UninterruptedMCM2–7 function required for DNA replication forkprogression. Science 288, 1643–1647.14 Aparicio OM, Weinstein DM & Bell SP (1997) Compo-nents and dynamics of DNA replication complexes inS. cerevisiae: redistribution of MCM proteins andCdc45p during S phase. Cell 91, 59–69.15 Katou Y, Kanoh Y, Bando M, Noguchi H, Tanaka H,Ashikari T, Sugimoto K & Shirahige K (2003) S-phasecheckpoint proteins Tof1 and Mrc1 form astable replication-pausing complex. Nature 424,1078–1083.16 Ishimi Y, Komamura Y, You Z & Kimura H (1998)Biochemical function of mouse minichromosome main-tenance 2 protein. J Biol Chem 273, 8369–8375.17 Sato M, Gotow T, You Z, Komamura-Kohno Y,Uchiyama Y, Yabuta N, Nojima H & Ishimi Y (2000)Electron microscopic observation and single-strandedDNA binding activity of Mcm4,6,7 complex. J Mol Biol300, 421–431.18 Moyer SE, Lewis PW & Botchan MR (2006) Isolationof the Cdc45–MCM2–7–GINS (CMG) complex, a can-didate for the eukaryotic DNA replication fork helicase.Proc Natl Acad Sci USA 103, 10236–10241.Inhibition of MCM4/6/7 helicase with heliquinomycin Y. Ishimi et al.3390 FEBS Journal 276 (2009) 3382–3391 ª 2009 The Authors Journal compilation ª 2009 FEBS19 Bochman ML & Schwacha A (2008) The Mcm2–7complex has in vitro helicase activity. Mol Cell 31,287–293.20 Chino M, Nishikawa K, Tsuchida T, Sawa R,Nakamura H, Nakamura KT, Muraoka Y, Ikeda D,Naganawa H, Sawa T et al. (1997) Heliquinomycin, anew inhibitor of DNA helicase, produced byStreptomyces sp. MJ929-SF2 II. Structure determinationof heliquinomycin. J Antibiot 50, 143–146.21 Chino M, Nishikawa K, Yamada A, Ohsono M, SawaT, Hanaoka F, Ishizuka M & Takeuchi T (1998) Effectof a novel antibiotic, heliquinomycin, on DNA helicaseand cell growth. J Antibiot 51, 480–486.22 Tuteja N, Tuteja R, Rahman K, Kang L-Y & FalaschiA (1990) A DNA helicase from human cells. NucleicAcids Res 18, 6785–6792.23 Tho¨mmes P, Fett R, Schray B, Burkhart R, Barnes M,Kennedy C, Brown NC & Knippers R (1992) Propertiesof the nuclear P1 protein, a mammalian homologue ofthe yeast Mcm3 protein. Nucleic Acids Res 20, 1069–1074.24 Georgaki A, Strack B, Podust V & Hu¨bscher U (1992)DNA unwinding activity of replication protein A. FEBSLett 308, 240–244.25 Laskey R (2005) The Croonian lecture 2001 hunting theantisocial cancer cell: MCM proteins and their exploita-tion. Philos Trans R Soc Lond B Biol Sci 360, 1119–1132.26 Ishimi Y, Okayasu I, Kato C, Kwon H-J, Kimura H,Yamada K & Song S-Y (2003) Enhanced expressionof MCM proteins in cancer cells derived from uterinecervix. Eur J Biochem 270, 1089–1101.27 Honeycutt KA, Chen Z, Koster MI, Miers M,Nuchtern J, Hicks J, Roop DR & Shohet JM (2006)Deregulated minichromosomal maintenance proteinMCM7 contributes to oncogene driven tumorigenesis.Oncogene 25, 4027–4032.28 Ishimi Y, Claude A, Bullock P & Hurwitz J (1988) Com-plete enzymatic synthesis of DNA containing the SV40origin of replication. J Biol Chem 263, 19723–19733.29 Chino M, Nishikawa K, Umekita M, Hayashi C,Yamazaki T, Tsuchida T, Sawa T, Hamada M &Takeuchi T (1996) Heliquinomycin, a new inhibitor ofDNA helicase, produced by Streptomyces sp. MJ929-SF2 I. Taxonomy, production, isolation, physico-chemi-cal properties and biological activities. J Antibiot 49,752–757.Supporting informationThe following supplementary material is available:Fig. S1. Structure of heliquinomycin.Fig. S2. SDS–PAGE of purified proteins.Fig. S3. Detection of MCM proteins in purified DNApolymerase-a/primase complex.Fig. S4. Effect of heliquinomycin on the oligonucleo-tide displacement activity of RPA.Fig. S5. Effect of heliquinomycin on the ATPaseactivities of MCM4/6/7 in the presence of M13mp18single-stranded DNA.This supplementary material can be found in theonline version of this article.Please note: Wiley-Blackwell is not responsible forthe content or functionality of any supplementarymaterials supplied by the authors. Any queries (otherthan missing material) should be directed to the corre-sponding author for the article.Y. Ishimi et al. Inhibition of MCM4/6/7 helicase with heliquinomycinFEBS Journal 276 (2009) 3382–3391 ª 2009 The Authors Journal compilation ª 2009 FEBS 3391 . compilation ª 2009 FEBS 3385examined the effect of heliquinomycin on the formation of the MCM4/6/7 complex (Fig. 4). In the absence of heliquinomycin, the MCM4/6/7. 440 kDa (4/6/7) 2 (4/6/7) Fig. 4. Effect of increasing concentrations of heliquinomycin (HQ) on the formation of the MCM4/6/7 complex. The MCM4/6/7 com-plex
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