Inhibition of unwinding and ATPase activities of pea MCM6 DNA helicase by actinomycin and nogalamycin

Abstract

Pea mini-chromosome maintenance 6 (MCM6) single subunit (93 kDa) forms homohexamer (560 kDa) and contains an ATP-dependent and replication fork stimulated 3′ to 5′ DNA unwinding activity along with intrinsic DNA-dependent ATPase and ATP-binding activities¹ (Plant Mol Biol 2010; DOI: 10.1007/s11103-010-9675-7). Here, we have determined the effect of various DNA-binding agents, such as actinomycin, nogalamycin, daunorubicin, doxorubicin, distamycin, camptothecin, cyclophosphamide, ellipticine, VP-16, novobiocin, netropsin, cisplatin, mitoxantrone and genistein on the DNA unwinding and ATPase activities of the pea MCM6 DNA helicase. The results show that actinomycin and nogalamycin inhibited the DNA helicase (apparent Ki values of 10 and 1 µM, respectively) and ATPase (apparent Ki values of 100 and 17 µM, respectively) activities. Although, daunorubicin and doxorubicin also inhibited the DNA helicase activity of pea MCM6, but with less efficiency; however, these could not inhibit the ATPase activity. These results suggest that the intercalation of the inhibitors into duplex DNA generates a complex that impedes translocation of MCM6, resulting in the inhibitions of the activities. This study could be useful in our better understanding of the mechanism of plant nuclear DNA helicase unwinding.

Introduction

Eukaryote genome is organized into multiple chromosomes, and DNA replication initiates at a large number of origins (10³–10⁵) to ensure that the entire genome is fully replicated. But these origins must be strictly regulated to make sure that genomic DNA is replicated completely and accurately once during S phase in a single cell cycle.^2,3 The mini-chromosome maintenance (MCM) protein complex (MCM2-7) plays an important role in initiation of DNA replication by providing DNA helicase function which helps in unwinding the duplex near to origin.^4,5 In general, the DNA helicases catalyse the unwinding of duplex DNA in an ATP-dependent manner and play an essential role in most of the DNA metabolism including DNA replication.^6,7 Studies on MCMs have shown that all six MCM proteins are essential for DNA replication.⁵ Moreover, all the six MCM proteins interact with each other to form several stable subassemblies including heterohexamer. The complexes of MCM4/7,⁸ MCM4/6/7,⁹ or MCM2-7,¹⁰ have been shown to contain helicase and ATPase activities in vitro. However, very recently we have shown that only a single subunit MCM6 from a pea can form the homohexamer and function as DNA helicase.¹ In order to understand the mechanism of action of unwinding by helicases, many DNA-binding agents have been shown to modulate DNA metabolism by interacting to the DNA and disrupting the enzymatic machinery that interacts with it.^11,12 It is therefore of interest to determine the effect of these agents on the DNA helicase, which is likely to be the first component of the “protein machines” during replication and other DNA structural transitions. Here we report studies of the effect of various DNA-binding agents on the helicase and ATPase activities of MCM6 DNA helicase. Various DNA-binding agents used were actinomycin, nogalamycin, daunorubicin, doxorubicin, distamycin, camptothecin, cyclophosphamide, ellipticine, VP-16, novobiocin, netropsin, cisplatin, mitoxantrone and genistein. Out of these actinomycin and nogalamycin were found to be the potent inhibitors for pea MCM6.

Results

Effects of DNA-binding agents on DNA unwinding activity of PsMCM6 and their kinetics of inhibition.

The DNA unwinding activity of MCM6 in presence of 50 µM of different DNA-binding agents is shown in Figure 1A. The results clearly indicate that the DNA-binding agents, actinomycin (lane 1) and nogalamycin (lane 10) inhibited the DNA unwinding activity effectively, while daunorubicin and doxorubicin inhibited less effectively (Fig. 1A and lane 5 and 6, respectively). The other compounds such as distamycin, camptothecin, cyclophosphamide, ellipticine, VP-16, novobiocin, netropsin, cisplatin, mitoxantrone and genistein were unable to inhibit the DNA unwinding activity of MCM6 (Fig. 1A and lanes 2–4, 7–9, 11–14, respectively). The two most effective compounds (actinomycin and nogalamycin), which inhibited the DNA unwinding activity, were investigated for the kinetics of inhibition. Each inhibitor was included in the helicase reaction at final concentrations ranging from 1 to 100 µM. The results of concentration curves are shown in Figure 1B, which indicated the apparent Ki values of 1 and 10 µM for nogalamycin and actinomycin, respectively. The structures of the MCM6 inhibitory compounds actinomycin and nogalamycin are shown in the Figure 1E and F, respectively.

Figure 1.

(A) The effect of inhibitors on MCM6 DNA helicase activity. Lane + is the heat denatured substrate, lane − is reaction without MCM6 enzyme, lane 0 is the reaction with MCM6 enzyme without any compound and lanes 1 to 14 are the enzyme reaction in presence of different compounds mentioned on top. (B) Kinetics of inhibition by nogalamycin and actinomycin. (C) The effect of inhibitors on MCM6 ATPase activity. Lane 1 ATPase reaction without MCM6 enzyme, lane 2 is the reaction with MCM6 enzyme without any compound and lanes 3 to 15 are the enzyme reaction in presence of different compounds mentioned on top. (D) Kinetics of inhibition by nogalamycin and actinomycin. (E) Structure of actinomycin. (F) Structure of nogalamycin.

Effects of DNA-binding agents on ATPase activity of PsMCM6 and their kinetic of inhibition.

The ATPase activity of MCM6 in the presence of 50 µM of different DNA-binding agents is shown in Figure 1C. The results clearly indicate that the DNA-binding agents nogalamycin (Fig. 1C and lane 9) and actinomycin (Fig. 1C and lane 13) inhibited the ATPase activity. The other compounds such as camptothecin, daunorubicin, VP-16, netropsin, novobiocin, doxorubicin, cyclophosphamide, ellipticine, mitoxantrone, distamycin and genistein were unable to inhibit the ATPase activity of PsMCM6 (Fig. 1C and lanes 1–8, 10–12, respectively). The two effective compounds (actinomycin and nogalamycin), which inhibited the ATPase activity, were further investigated for the kinetics of inhibition. Each inhibitor was included in the ATPase reaction at final concentrations ranging from 1 to 250 µM. The results of concentration curves are shown in Figure 1D, which indicated the apparent Ki values of 17 and 100 µM for nogalamycin and actinomycin, respectively.

Discussion

It is well known that DNA binding agents modulate its metabolism by binding to DNA and disrupting its interacting enzymatic machinery. Therefore, in order to understand the mechanism of DNA unwinding we have also tested the effect of various DNA-binding agents on the DNA unwinding and ATPase activities of PsMCM6 protein. The effective agents that inhibited both the activities of PsMCM6 were nogalamycin and actinomycin. Actinomycin is a polypeptide containing antibiotic and inhibitor of nucleic acid synthesis. It binds to the double-helical DNA specifically and is a known inhibitor for almost all the helicases. It has been reported to inhibit helicase activity of pea chloroplast DNA helicase I,¹³ human DNA helicase II,¹⁴ PfDH60,¹⁵ and PfH45,¹⁶ but not the other helicases such as PDH45, PDH65, WRN, BLM, UvrD and PcDDH45.^17,18

Nogalamycin is an anthracycline antibiotic and it is a dumbell-shaped molecule that intercalates into both the major and the minor grooves of DNA. It has also been reported to inhibit the helicase and ATPase activities of the tested helicases, PcDDH45; PfDH60 and PfH45.^13–18 The mechanism by which these ligands inhibit the helicase activity might be through intercalation into the major or minor groove of the duplex DNA strand (or hairpin structure present in ssDNA), probably providing a physical block to continued translocation by the helicase, causing the unwinding reaction to be inhibited.

Materials and Methods

Cloning of pea MCM6 cDNA.

A cDNA library was constructed from 5 µg poly (A) RNA (isolated from top four leaves of 7 days old pea) in a UniZAP XR vector using a UniZAP cDNA synthesis kit (Stratagene) following the manufacturer's protocol. The resulting phage library was screened to isolate the MCM6 cDNA (Accession number: AY169793) as described earlier review in reference ¹.

Expression and purification of MCM6 protein.

The complete ORF of MCM6 cDNA (2.48 kb) was PCR amplified by using gene specific primers, cloned into pET-14b (Clontech) vector followed by transformation into E. coli BL21(DE3) and the expressed MCM6 protein was purified using nickel-NTA (Qiagen GmbH, Cologne, Germany) column chromatography as described earlier review in reference ¹.

Preparation of helicase substrate, DNA helicase and ATPase assays.

The substrate used in the DNA-unwinding assay consisted of a ³²P labeled 47-mer DNA oligo [5′-(T)₁₅GTT TTC CCA GTC ACG AC(T)₁₅-3′] annealed to M13mp19 phage ssDNA. This partial duplex DNA substrate contained hanging tails of 15 nucleotides at both the 5′ and 3′ ends. It was prepared as described earlier in reference 19. The DNA helicase reaction was performed in 10 µl of mixture consisting of 20 mM Tris-HCl (pH 8.0), 2 mM ATP, 0.5 mM MgCl₂, 75 mM KCl or NaCl, 8 mM DTT, 4% (w/v) sucrose, 80 µg/ml BSA, about 1 ng of ³²P-labeled substrate (approximately 2,000 cpm.; 40 pM or 0.40 fmol/10 µl) and the purified MCM6 protein (250 ng). The reaction mixture was incubated for 60 min at 37°C and the reaction was stopped and analyzed as previously described in reference ²⁰. The ATPase reaction conditions were identical, except that the ³²P-labeled helicase substrate and 2 mM ATP were omitted and instead 1,665 Bq [γ-³²P]-ATP was included. This reaction was run for 2 h at 37°C and analyzed as described in reference ¹⁹ and ²⁰. Unless otherwise specified, all the DNA-binding agents were added to the helicase reaction mixture prior to the addition of the enzyme. In this study the Ki values represent the inhibitor concentration necessary to inhibit enzyme activity by 50 ± 5%.