Effect of heliquinomycin on the activity of human minichromosome maintenance 4/6/7 helicase Yukio Ishimi 1,2 , Takafumi Sugiyama 1 , Ryou Nakaya 1 , Makoto Kanamori 3 , Toshiyuki Kohno 2 , Takemi Enomoto 3 and Makoto Chino 4 1 College of Science*, Ibaraki University, Japan 2 Macromolecular Structure Research Group*, Mitsubishi Kagaku Institute of Life Sciences, Tokyo, Japan 3 Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Miyagi, Japan 4 Pharmaceuticals Group, Nippon Kayaku Co. Ltd., Tokyo, Japan Minichromosome maintenance (MCM) proteins are essential factors for the prevention of the loss of extra- chromosomal DNA in Saccharomyces cerevisiae [1–3]. A heterohexameric MCM2–7 protein complex has been identified as a component of the DNA replication licensing system that ensures a single round of DNA replication per cell cycle [4–7]. This complex functions as a replicative DNA helicase that drives the unwinding of the DNA duplex prior to semiconservative DNA synthesis at the replication forks. This notion is sup- ported by the following findings. First, all of the MCM2–7 proteins possess DNA-dependent ATPase motifs that are common features of DNA helicases [8]. Second, the MCM4/6/7 subcomplex forms the core of the MCM2–7 hexamer and exhibits intrinsic DNA helicase 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], and these proteins migrate on the genome together with the replication forks [14,15]. One of the intricacies related to the function of the MCM2–7 complex is that an iso- lated MCM2–7 complex does not exhibit definite DNA helicase activity in vitro, but the MCM4/6/7 hexamer does. Further, the interaction between the MCM2 protein and the MCM4/6/7 hexamer, or between the MCM3/5 proteins and the MCM4/6/7 hexamer, Keywords anticancer drug; DNA replication; MCM 4/6/7 helicase Correspondence Y. Ishimi, Ibaraki University, 2-1-1 Bunkyo, Mito, Ibaraki 310-8512, Japan Fax: +81 29 228 8439 Tel: +81 29 228 8439 E-mail: ishimi@mx.ibaraki.ac.jp (Received 9 March 2009, revised 13 April 2009, accepted 16 April 2009) doi:10.1111/j.1742-4658.2009.07064.x The antibiotic heliquinomycin, which inhibits cellular DNA replication at a half-maximal inhibitory concentration (IC 50 ) 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 IC 50 value of 2.4 lm. In contrast, 14 lm heliqui- nomycin did not inhibit significantly either the DNA helicase activity of the SV40 T antigen and Werner protein or the oligonucleotide displacement activity of human replication protein A. At IC 50 values 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 the enzymes studied, the MCM4/6/7 complex was the most sensitive to heliqui- nomycin; this suggests that MCM helicase is one of the main targets of heliquinomycin in vivo. It was observed that heliquinomycin did not inhibit the ATPase activity of the MCM4/6/7 complex to a great extent in the absence of single-stranded DNA. In contrast, heliquinomycin at an IC 50 value of 5.2 lm inhibited the ATPase activity of the MCM4/6/7 complex in the pres- ence of single-stranded DNA. This suggests that heliquinomycin interferes with the interaction of the MCM4/6/7 complex with single-stranded DNA. Abbreviations BrdU, bromodeoxyuridine; FITC, fluorescein isothiocyanate; IC 50, half-maximal inhibitory concentration; MCM, minichromosome maintenance; RPA, replication protein A. *[Corrections added on 18 May 2009 after first online publication: in affiliation 1, ‘Macromolecular Structure Research Group’ has been replaced 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 FEBS inhibits helicase activity [16,17]. On the basis of these previous reports, we propose that a structural change in the MCM2–7 complex may generate the MCM4/6/7 hexamer, which, in turn, exhibits helicase activity. Another possibility is that the DNA helicase activity of the MCM2–7 complex may be attributed to the inter- action of this complex with other proteins. It has been reported that the CMG complex, which consists of the Cdc45 protein, MCM2–7 hexamer and the GINS com- plex purified from Drosophila embryo extracts, exhibits DNA helicase activity in vitro [18]. Furthermore, it has been demonstrated recently that the MCM2–7 complex prepared from S. cerevisiae exhibits DNA helicase activity in the presence of potassium acetate or gluta- mate. These results suggest that the MCM2–7 complex functions as a replicative DNA helicase in vivo [19]. Heliquinomycin, which is an antibiotic [20,21], inhib- its cellular DNA replication and RNA synthesis. To elucidate its cellular targets for the inhibition of DNA synthesis, we examined the effects of heliquinomycin on 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 protein A (RPA), and on the RNA priming and DNA poly- merization activities of the DNA polymerase-a/primase complex. The results indicated that, among all the enzymes examined, the MCM4/6/7 helicase was the most sensitive to heliquinomycin. It was observed that, in the absence of single-stranded DNA, heliquinomycin did not inhibit the ATPase activity of the MCM4/6/7 complex to a great extent; in contrast, in the presence of DNA, this antibiotic inhibited the ATPase activity. This result suggests that heliquinomycin inhibits the helicase activity of MCM4/6/7 by interfering with the interaction of this complex with single-stranded DNA. Results Sensitivity of cellular DNA replication to heliquinomycin Heliquinomycin with a relative molecular mass of 698 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 transformed cells at a half-maximal inhibitory concentration (IC 50 ) of 1.4–4 lm [21]. In these experiments, DNA synthesis was measured by the incorporation of labelled thymi- dine into DNA. To confirm this, human HeLa cells were pulse labelled with bromodeoxyuridine (BrdU) in the presence of increasing concentrations of heliquino- mycin (Fig. 1A). BrdU incorporated into DNA was detected by staining the cells with anti-BrdU Ig and A B Fig. 1. Effect of heliquinomycin (HQ) on the incorporation of BrdU into DNA in HeLa cells. (A) Logarithmically growing HeLa cells were incubated with the indicated concentrations of heliquinomycin for 1 h and then pulse labelled with BrdU for 20 min. The incorporated BrdU in the cells was detected by incubation of the cells with anti- BrdU Ig, followed by FITC-labelled anti-rat Ig. MCM7 was detected by incubation of the cells with anti-MCM7 Ig, followed by Cy3- labelled anti-mouse Ig. (B) One hundred Cy3-stained cells were selected, and the fluorescence intensity of FITC in the cells was quantified. The average level of intensity in the cells cultured in the presence of heliquinomycin was expressed in comparison with that in the cells cultured in the absence of heliquinomycin. Y. Ishimi et al. Inhibition of MCM4/6/7 helicase with heliquinomycin FEBS Journal 276 (2009) 3382–3391 ª 2009 The Authors Journal compilation ª 2009 FEBS 3383 then with fluorescein isothiocyanate (FITC)-labelled second Ig. The cells were also stained with specific Ig to detect MCM7 protein in the nucleus. In the absence of heliquinomycin, approximately 27% of the cells were stained with anti-BrdU Ig. As the concentration of heliquinomycin was increased, the proportion of BrdU-positive cells and the intensity of the signal grad- ually decreased. Almost no BrdU-positive cells were detected in the presence of 14 lm heliquinomycin. In contrast, staining with anti-MCM7 Ig was not changed by the presence of heliquinomycin. The fluorescence derived from the incorporated BrdU was quantified in these experiments, and the IC 50 value was determined to be 2.3 lm (Fig. 1B), which is similar to the value reported previously [21]. Sensitivity of MCM4/6/7 helicase to heliquinomycin Heliquinomycin at an IC 50 value of 7–14 lm inhibits the activity of the cellular DNA helicase called DNA helicase I [22], but scarcely affects the activities of topoisomerases and the replication of the SV40 chromosome in vitro [21]. To understand the cellular targets of this antibiotic during DNA replication, the effects of this antibiotic on the helicase activities of the human MCM4/6/7 complex, SV40 T antigen and Werner protein were examined. In addition to these three proteins, the human DNA polymerase-a/primase complex and the human RPA complex were purified to near homogeneity (Fig. S2). Some unidentified pro- teins were found in the purified DNA polymerase-a/ primase complex and the human RPA complex. It has been reported that MCM3 interacts with DNA polymerase-a/primase [23]. We examined the presence of MCM4, 5 and 6 proteins in the purified DNA + MCM4/6/7 A B + Tag 0 (μ M) 0.43 1.4 4.3 14 43 HQ 0 0.43 1.4 4.3 14 43 % 17-mer 17-mer/M13 0 (μ M) HQ 0.43 1.4 4.3 14 43 Tag MCM 0 (μ M) HQ 0.43 120 100 80 60 40 20 0 1.4 4.3 14 43 % + Werner 0 0.43 1.4 4.3 14 43 (μM) HQ 17-mer 17-mer/M13 Fig. 2. Effect of heliquinomycin on DNA helicase activity. (A) Top: effects of increasing concentrations of heliquinomycin (HQ) on the DNA helicase activities of the MCM4/6/7 complex and the SV40 T antigen. Dimethyl sulfoxide solution (0.4 lL) containing or lacking heliquinomycin was added to the reaction mixture. The final con- centrations of heliquinomycin added to the reaction mixture are indicated at the top. The DNA helicase activity was measured as the activity that displaces 17-mer oligonucleotides annealed to M13mp18 single-stranded DNA. Bottom: the proportion of dis- placed 17-mer oligonucleotides in total DNA was considered to be 100% in the control reaction mixture lacking heliquinomycin, and the 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 were performed 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 an average of the values was plotted. (B) Top: effects of increasing concentrations of heliquinomycin on the DNA helicase activity of the Werner protein. Bottom: proportion of displaced 17-mer oligo- nucleotides. Two independent experiments were performed, and an 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 FEBS polymerase-a/primase complex (Fig. S3). Only small amounts of MCM4 (0.1% of total protein) and MCM5 (0.9%) proteins were detected, and MCM6 was not found. DNA helicases were added to the DNA helicase reaction mixtures at the minimum amounts required to displace almost all of the 17-mer oligonucleotides. Heliquinomycin at a concentration of 14 lm did not inhibit the helicase activity of the SV40 T antigen to a great extent, but inhibited that of the MCM4/6/7 com- plex at an IC 50 value of 2.4 lm (Fig. 2A). Heliquino- mycin (14 lm) did not inhibit the helicase activity of the Werner protein to a great extent (Fig. 2B). It should be noted that the mobility of displaced frag- ments in the absence or presence of heliquinomycin was different. The RPA complex displaces oligonucleo- tides annealed to M13 single-stranded DNA without triggering ATP hydrolysis [24]. We found that heliqui- nomycin scarcely affected the oligonucleotide displace- ment activity of RPA (Fig. S4). We also examined the effect of heliquinomycin on the reactions of RNA priming (Fig. 3A) and DNA polymerization activity (Fig. 3B) of the DNA polymerase-a/primase complex. When dT 50 was used as a template, an RNA primer of approximately 10 nucleotides was synthesized only in the presence of the above complex. The synthesis of the RNA primer was inhibited in the presence of heliquinomycin at an IC 50 value of 25 lm. The DNA polymerization activity of the DNA polymerase-a/ primase complex was measured using activated DNA as a template and a primer. The observed reduction in the level of the incorporated nucleotides indicates that heliquinomycin at an IC 50 value of 6.5 lm inhibits the DNA polymerization activity of the complex. These results indicate that, among the enzymes studied, MCM4/6/7 is the most sensitive to heliquinomycin and the DNA polymerase-a/primase complex is also rela- tively sensitive to this antibiotic (Table 1). Sensitivity of MCM4/6/7 helicase to heliquinomycin To understand the mechanism by which heliquino- mycin inhibits the activity of MCM4/6/7 helicase, we 0 0.43 1.4 4.3 14 43 HQ (μ M) % 0 (μ M) 0.43 1.4 4.3 14 43 HQ 17 10 50 nt + Polα-primase A B 0 (μ M) HQ 0.43 1.4 4.3 14 43 % 120 100 80 60 40 20 0 100 80 60 40 20 0 Fig. 3. Effect of heliquinomycin on the RNA priming activity of the DNA polymerase-a/primase complex. (A) Top: effect of increasing concentrations of heliquinomycin (HQ) on the RNA priming action of the DNA polymerase-a/primase. RNA priming activity was mea- sured by the analysis of oligoA synthesis when dT 50 was used as the template. The products were electrophoresed under denaturing conditions. The three oligonucleotides of A 10 , 17-mer and dT 50 were labelled at their 5¢ ends and electrophoresed to determine the size of the synthesized oligoA fragment. The arrow indicates the position of the RNA primer synthesized by DNA polymerase-a/prim- ase. Bottom: radioactivity of the synthesized RNA primer. The radioactivity recorded for RNA in the control reaction mixture lack- ing heliquinomycin was considered to be 100%, and that recorded for the reaction mixtures containing heliquinomycin was presented in relation to this control value. Two independent experiments were performed, and an average of the values was plotted together with error bars. (B) Effect of increasing concentrations of heliquinomycin on the DNA polymerization activity of the DNA polymerase-a/prim- ase. The reaction was performed using activated DNA as a primer and template. The acid-insoluble radioactive material trapped on the glass fibre filter was measured. The radioactivity recorded in the case of the control reaction mixture lacking heliquinomycin was considered to be 100%, and that recorded for the reaction mixture containing heliquinomycin was presented in relation to this control value. 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 heliquinomycin FEBS Journal 276 (2009) 3382–3391 ª 2009 The Authors Journal compilation ª 2009 FEBS 3385 examined the effect of heliquinomycin on the formation of the MCM4/6/7 complex (Fig. 4). In the absence of heliquinomycin, the MCM4/6/7 complex, as detected using anti-MCM4 IgG, exhibits a trimeric or hexameric structure, depending on its mobility in the gel. We observed that the hexamer–trimer proportion increased slightly with an increase in the heliquinomycin concen- tration. The hexameric form of the MCM4/6/7 complex was 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 that higher concentrations of heliquinomycin affect signifi- cantly the formation of the MCM4/6/7 complex. We also examined the sensitivity of the ATPase activities of the MCM4/6/7 complex and the SV40 T antigen to heliquinomycin in the absence of single-stranded DNA (Fig. 5). Heliquinomycin inhibited the ATPase 0 (μ M) HQ 0.43 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 heliquinomycin and subsequently electrophoresed on a native polyacrylamide gel. The proteins in the gel were transferred onto a filter and the MCM4 protein was detected by incubating the filter with rabbit anti-MCM4 IgG, followed by horseradish peroxidase-conjugated anti-rabbit IgG. Finally, the bound antibodies were examined for chemiluminescence using West Pico chemiluminescent substrate (Thermo Scientific, Rockford, IL, USA). The positions to which thyroglobulin (669 kDa) and ferritin (440 kDa) migrated in the gel are indicated. Tag MCM 0 (μ M) HQ 0.43 1.4 4.3 14 43 0 (μ M) 0.43 1.4 4.3 14 43 HQ 0 0.43 1.4 4.3 14 43 + MCM4/6/7 + Tag P i ATP 120 100 80 60 % 40 20 0 Fig. 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-stranded DNA. After incubation under these conditions, an aliquot of the mix- ture was subjected to thin layer chromatography. The radioactivity at the sites to which P i and ATP migrated was measured, and the ratio of the released P i to ATP was calculated. The ratio obtained in the case of the reaction performed without the enzymes was subtracted from that obtained in the reactions performed with the enzymes. Bottom: the ratio obtained in the case of the control reaction mixture which lacked heliquinomycin was considered to be 100%, and that recorded for the reaction mixture that contained heliquinomycin was presented in relation to this control value. Two independent experi- ments were performed for the MCM4/6/7 complex, and an average of the values was plotted together with error bars. Table 1. Sensitivity of the enzymes to heliquinomycin. The IC50 values indicated are those calculated in the present study as well as in a previous investigation [21]. Those determined in the pre- vious investigation are marked by an asterisk. IC 50 (lM) Human cellular DNA synthesis* 1.4–4 HeLa DNA synthesis 2.5 SV40 chromosome replication in vitro*>72 T antigen helicase 43 HeLa helicase I* 7–14 hMCM4/6/7 helicase 2.4 Werner helicase > 43 Human RPA > 43 DNA polymerase a 6.5 DNA primase 25 Topoisomerase I* 145 Topoisomerase II* 43 Inhibition 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 FEBS activities of these two helicases only slightly. We also examined its effect on these activities in the presence of heat-denatured, single-stranded DNA (Fig. 6); under these conditions, the ATPase activity of the MCM4/6/7 complex increased manifold [9]. Heliquinomycin scar- cely inhibited the ATPase activity of the T antigen, but inhibited that of the MCM4/6/7 helicase at an IC 50 value of 5.2 lm. The ATPase activity of MCM4/6/7 was also stimulated in the presence of M13mp18 single- stranded DNA in place of heat-denatured DNA, and the stimulated activity was also inhibited in the pres- ence of heliquinomycin (Fig. S5). These results suggest that heliquinomycin interferes with the interaction of the MCM4/6/7 complex with single-stranded DNA, which is required to increase the ATPase activity of this complex. Discussion Heliquinomycin was first characterized as a compound that inhibits bacterial cell growth, and was found to inhibit DNA synthesis in several cancer cells at an IC 50 value of 1.4–4 lm [21]. In the present study, we found that heliquinomycin inhibits BrdU incorporation into DNA at an IC 50 value of 2.3 lm; this result is consistent with the previous findings. The reported study also indicated that the cell cycle progression of HeLa cells was retarded during the S phase and the cells were arrested in the G2 phase in the presence of heliquinomycin [21]. Heliquinomycin inhibits the cellular DNA helicase, helicase I, at an IC 50 value of 7–14 lm, but does not inhibit the activity of topoi- somerases. Our study indicates that heliquinomycin inhibits the activity of human MCM4/6/7 helicase at an IC 50 value of 2.4 lm, but scarcely inhibits the DNA helicase activity of the SV40 T antigen and the Werner protein, or the oligonucleotide displacement activity of human RPA. Further, it inhibits the RNA priming and DNA polymerization activities of the human DNA polymerase-a/primase at IC 50 values 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 at an IC 50 value of 14 lm (data not presented). Thus, among the enzymes studied, MCM4/6/7 helicase was found to be the most sensitive to heliquinomycin. These results suggest that MCM helicase and DNA polymerases may be the critical targets of heliquinomy- cin during cellular DNA replication. Further, we observed that the checkpoint system that is induced by the inhibition of DNA polymerases during DNA repli- cation is not induced in HeLa cells treated with 4.3 lm heliquinomycin (data not presented). This suggests that the 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 the single-stranded DNA-dependent ATPase activity of the complex. Heliquinomycin suppressed the ATPase activity of the complex in the absence of single- stranded DNA, but the enzymatic activity was significantly less sensitive to heliquinomycin. Thus, heliquinomycin may inhibit the ATPase activity and DNA helicase activity of the MCM4/6/7 complex by affecting the ability of this complex to interact with single-stranded DNA. The finding that the activities 0 (μ M)0.43 1.4 4.3 14 43 HQ 0 0.43 1.4 4.3 14 43 + MCM4/6/7 A B + Tag P i ATP Tag MCM 0 (μ M) HQ 0.43 1.4 4.3 14 43 120 100 80 60 % 40 20 0 Fig. 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-stranded DNA. The radioactivity at the sites to which P i and ATP migrated was measured, and the ratio of the released P i to ATP was calcu- lated. The ratio in the case of the reaction performed without the enzyme was subtracted from that in the case of the reactions per- formed with these enzymes. (B) The ratio obtained in the case of the control reaction mixture which lacked heliquinomycin was con- sidered to be 100%, and that recorded for the reaction mixture that contained heliquinomycin was presented in relation to this control value. Two independent experiments were performed for the MCM4/6/7 complex, and an average of the values was plotted together with error bars. Y. Ishimi et al. Inhibition of MCM4/6/7 helicase with heliquinomycin FEBS Journal 276 (2009) 3382–3391 ª 2009 The Authors Journal compilation ª 2009 FEBS 3387 of DNA polymerase-a/primase are also inhibited at higher concentrations of heliquinomycin may suggest that heliquinomycin interacts with single-stranded DNA to interfere with the activities. However, there is no evidence for the interaction of heliquinomycin with single-stranded DNA. In contrast, the formation of the MCM4/6/7 complex was inhibited by heliquinomycin at higher concentrations. Although these concentra- tions are higher than those that inhibit MCM4/6/7 helicase activity, it is possible that heliquinomycin interacts directly with the MCM4/6/7 complex to inhi- bit the interaction of this complex with single-stranded DNA, even at low concentrations. MCM proteins are considered to be one of the most sensitive diagnostic markers for the detection of cancer cells in human tissues [25]. The expression of MCM proteins appears to be critical for the development of cancer cells, as this expression shows a strong correlation with the malignant transforma- tion of cells. The finding that MCM2–7 proteins are overexpressed in transformed cancer cells [26] suggests that the upregulation of MCM protein expression may play a role in the development of cancer cells. Consistent with this notion, it has recently been reported that deregulated expression of the MCM7 protein accelerates the transformation of cells [27]. Thus, MCM proteins are among the most critical targets for achieving the inhibition of cancer cell growth. Furthermore, heliquinomycin may have useful applications in the development of MCM-specific anticancer drugs. Materials and methods BrdU labelling of HeLa cells HeLa cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 7% fetal calf serum. Cells cul- tured on coverslips were incubated with dimethyl sulfoxide or increasing concentrations of heliquinomycin for 1 h and then pulse labelled with 20 lm BrdU for 20 min. After being washed with NaCl/P i , the cells were fixed by incubation with 4% paraformaldehyde in NaCl/P i for 5 min at room tem- perature. The cells were washed with NaCl/P i , and then permeabilized and blocked by incubation with 0.1% Triton X-100, 0.02% SDS and 2% nonfat dried milk in NaCl/P i for 1 h at 37 °C. The cells were incubated overnight with anti-MCM7 mouse Ig (sc-9966; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C in the above-mentioned blocking solution. The cells were washed with the same solution and then incubated with cyanine-3 (Cy3)-conju- gated anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) for 1.5 h at 37 °C in the blocking solu- tion. They were then re-fixed, treated with 4 m HCl for 30 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 immunoreactivities were detected with fluorescence microscopy (BX-9000; KEYENCE, Osaka, Japan). DNA helicase and ATPase activities of the DNA helicases A human MCM4/6/7 complex was prepared, and its DNA helicase 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 magnesium acetate, 0.5 mgÆmL )1 bovine serum albumin, 1–2.5 fmol of a 17-mer oligonucleotide annealed to M13mp18 DNA and an approximately100 ng sample of human MCM4/6/7 com- plex, a 25 ng sample of SV40 T antigen or a 1.25 ng sample of Werner protein, in the presence or absence of heliquino- mycin at the indicated concentrations. This mixture was incubated at 37 °C for 40 min, and the products were anal- ysed using 12% PAGE. The ATPase activity was measured by incubating either the MCM proteins (120–340 ng) or the SV40 T antigen (200 ng) at 37 °C for 30 min in the pres- ence of 74 kBq [c- 32 P]ATP in a solution containing 50 mm Tris/HCl (pH 7.9), 20 mm 2-mercaptoethanol, 0.5 mgÆmL )1 bovine serum albumin, 10 mm magnesium acetate, 10 mm ATP and heliquinomycin at the indicated concentrations in the presence or absence of 5 lg of single-stranded DNA (heat-denatured). Further, 0.5 lL of the reaction mixture was spotted onto a poly (ethyleneimine)-cellulose thin layer chromatography plate (Cellulose F; Merck, Darmstadt, Germany). Chromatography was performed at 4 °C for a period of 2 h using a solution of 0.8 m acetic acid and 0.8 m LiCl. The radioactivity on the plate was detected using a Bio-Image Analyser (FLA3000; Fuji, Tokyo, Japan). Formation of the MCM4/6/7 complex The reaction mixture (10 lL) containing 50 mm Tris/HCl (pH 7.5), 20 mm 2-mercaptoethanol, 5 mm MgCl 2 ,5mm ATP, 100 lgÆmL )1 bovine serum albumin and the MCM4/6/ 7 complex (170 ng) was incubated at 37 °C for 30 min in the presence 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 was immersed in a solution containing 49 mm Tris, 38 mm gly- cine and 0.25% SDS, and was incubated at 80 °C for 1 h in order to achieve protein denaturation. The proteins in the gel were 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 FEBS Millipore, Billerica, MA, USA) and anti-MCM4 IgG were used to detect MCM4 on the filter [9]. RNA priming and DNA synthesis with DNA polymerase-a/primase The DNA polymerase-a/primase complex was purified from HeLa cells by immunoadsorption, followed by elution from a column coated with a monoclonal antibody (SJK237), as reported previously [28]. DNA polymerase activity was measured using a reaction mixture (20 lL) containing 20 mm Tris/HCl (pH 7.9), 3.3 mm 2-mercaptoethanol, 0.2 mgÆmL )1 bovine serum albumin, 5 mm MgCl 2 , 0.25 mgÆmL )1 activated DNA, 100 lm each of dATP, dGTP and dTTP, 50 lm of dCTP, 111 kBq [a- 32 P]dCTP and 85 ng DNA polymerase-a/primase complex in the pres- ence of heliquinomycin at the indicated concentrations. The reaction was terminated by the addition of 30 lL of sodium pyrophosphate (0.17 m) and 50 lL of sperm DNA (1 mgÆmL )1 ). Further, 1 mL of 5% trichloroacetic acid was added, and the acid-insoluble radioactive material trapped on a glass fibre filter was measured in a liquid scintillation cocktail. The reaction mixture (10 lL) used for the mea- surement of the RNA priming activity contained 40 mm Tris/HCl (pH 7.5), 10 mm magnesium acetate, 1 mm dithio- threitol, 100 lgÆ mL )1 bovine serum albumin, 0.1 mm ATP, 185 kBq [a- 32 P]ATP and 10 lm (dT) 50 in the presence of heliquinomycin at the indicated concentrations. This mix- ture was incubated at 37 °C for 40 min and then heated at 95 °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 at 98 °C for 5 min in the presence of 3 lL of loading buffer (0.1% bromophenol blue, 0.1% xylene cyanol, 10 mm EDTA and 98% formamide), and the products were analy- sed on a 25% polyacrylamide gel containing 7 m urea. The oligonucleotides, A 10 , 17-mer and oligo-dT 50 , were labelled at their 5¢ ends and used as markers. The gel was dried and the radioactivity was detected using a Bio-Image Analyser. Preparation of the RPA complex cDNAs for human RPA1, RPA2 and RPA3 were synthe- sized from mRNA extracted from HeLa cells by the reverse transcription-polymerase chain reaction (RT-PCR) method (Invitrogen, Carlsbad, CA, USA), and were cloned into the baculovirus 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 fusion protein. High-5 cells were co-infected with the three viruses expressing the RPA1, RPA2 and RPA3 proteins for 2 days. The recombinant RPA proteins in the lysates of the infected cells were purified by performing nickel-nitrilotri- acetic acid (Qiagen, Hilden, Germany) affinity column chromatography as follows. The purification involved the suspension of the infected cells in lysis buffer consisting of 10 mm Tris/HCl (pH 7.5), 130 mm NaCl, 1% Triton X-100, 10 mm NaF, 10 mm sodium phosphate buffer, 10 mm Na 4 P 2 O 7 and protease inhibitors (Pharmingen BD, San Jose, CA, USA). The mixture was incubated for 40 min on ice, and insoluble components were separated by centrifugation at 137 000 g (TLS55; Beckman, Fullerton, CA, USA) for 40 min at 4 °C. To 1 vol of the clarified lysate, 1/10 vol of nickel-nitrilotriacetic acid-agarose was added, and the mixture was incubated for 1 h at 4 °Cona rocking platform. Agarose beads were then collected by centrifugation and thoroughly washed with buffer A [50 mm sodium phosphate buffer (pH 6.0), 300 mm NaCl and 10% glycerol] containing 20 mm imidazole. Next, the beads were washed once with buffer B [50 mm sodium phosphate buffer (pH 8.0), 300 mm NaCl and 10% glyc- erol] containing 20 mm imidazole, and the proteins bound to the beads were eluted by adding buffer B containing 300 mm imidazole at a volume equivalent to 1 bed. This was followed by incubation for 5 min at 4 °C on a rocking platform and separation of the beads by centrifugation. The proteins were eluted twice more. The eluates were pooled and diluted to decrease the NaCl concentration to 50 mm, and the solution thus obtained was concentrated using Centricon 30 (Millipore). The concentrated proteins were loaded onto a MonoQ column (GE Healthcare, Pis- cataway, NJ, USA), and the bound proteins were eluted using a linear NaCl gradient (0.1–0.6 m). The RPA1 (70 kDa), RPA2 (34 kDa) and RPA3 (14 kDa) proteins were co-eluted with approximately 0.3 m NaCl, and were concentrated using Microcon 30 after the salt concentration had decreased to 0.1 m. The oligonucleotide displacement activity 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 RPA complex. Purification of Werner helicase High-5 cells infected with recombinant virus encoding (His) 6 -WRN were cultured for 3 days and then collected by centrifugation. 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 and 20 lgÆmL )1 leupeptin) for 10 min on ice, and NaCl was added to the lysate at a final concentration of 0.5 m. After incubation for 30 min on ice, the cell lysate was centrifuged at 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 m NaCl in buffer C to remove nucleic acids. Flow-through fractions were loaded on to a nickel-nitrilotriacetic acid affinity column equilibrated with buffer D [20 mm KP i (pH 7.5), 1 mm phenylmethylsulfonyl fluoride and 20 lgÆmL )1 leupeptin] containing 0.5 m NaCl. After loading Y. Ishimi et al. Inhibition of MCM4/6/7 helicase with heliquinomycin FEBS Journal 276 (2009) 3382–3391 ª 2009 The Authors Journal compilation ª 2009 FEBS 3389 onto the nickel-nitrilotriacetic acid column, the column was washed with buffer D containing 0.2 m NaCl and 25 mm imidazole, and eluted with buffer D containing 0.2 m NaCl and 200 mm imidazole. The fractions containing Werner protein were determined by performing SDS–PAGE. Pooled fractions were loaded onto a MonoS column (GE Healthcare). After the column had been washed with buffer H [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 )1 leupeptin] containing 0.2 m NaCl, the bound proteins were eluted with buffer H containing 0.5 m NaCl. Fractions around the main peak were pooled, concentrated using Vivaspin 20 (Sartorius, Hanover, Germany) and then fractionated on Superdex 200 HR in buffer H containing 0.1 m NaCl. The purified protein was concentrated with Vivaspin and dialysed against buffer H containing 0.1 m NaCl. Other materials The 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 the purified heliquinomycin was dissolved in 0.1 mL of dimethyl sulfoxide to prepare a 10 mgÆmL )1 stock solution. 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This supplementary material can be found in the online version of this article. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corre- sponding author for the article. Y. Ishimi et al. Inhibition of MCM4/6/7 helicase with heliquinomycin FEBS Journal 276 (2009) 3382–3391 ª 2009 The Authors Journal compilation ª 2009 FEBS 3391 . compilation ª 2009 FEBS 3385 examined 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