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

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.Key words: ATPase, actinomycin, DNA-binding agents, DNA helicase, nogalamycin, pea MCM6     

The Gly-Arg-rich C-terminal domain of pea nucleolin is a DNA helicase that catalytically translocates in the 5'- to 3'-direction

Nucleolin is a major nucleolar phosphoprotein of exponentially growing eukaryotic cells. Here we report the cloning, purification, and characterization of the C-terminal glycine/arginine-rich (GAR) domain of pea nucleolin. The purified recombinant protein (17 kDa) shows ATP-/Mg(2+)-dependent DNA helicase and ssDNA-/Mg(2+)-dependent ATPase activities. The enzyme unwinds DNA in the 5'- to 3'-direction, which is the first report in plant for this directional activity. It unwinds forked/non-forked DNA with equal efficiency. The anti-nucleolin antibodies immunodepleted the activities of the enzyme. The DNA interacting ligands nogalamycin, daunorubicin, actinomycin C1, and ethidium bromide were inhibitory to DNA unwinding (with K(i) values of 0.40, 2.21, 8.0, and 9.0 microM, respectively) and ATPase (with K(i) values of 0.43, 1.65, 4.6, and 7.0 microM, respectively) activities of the enzyme. This study confirms that the unwinding and ATPase activities of pea nucleolin resided in the GAR domain. This study should make important contribution to our better understanding of DNA transaction in plants, mechanism of DNA unwinding, and the mechanism by which these ligands can disturb genome integrity.     

Replication fork-stimulated eIF-4A from Plasmodium cynomolgi unwinds DNA in the 3' to 5' direction and is inhibited by DNA-interacting compounds

Plasmodium cynomolgi DEAD-box DNA helicase 45 (PcDDH45) is an ATP-dependent DNA-unwinding enzyme with intrinsic DNA-dependent ATPase activity and is highly homologous to eIF-4A. In this study, we have further characterized and tested the effect of various DNA-interacting compounds on the DNA-unwinding activity of PcDDH45. The results show that PcDDH45 translocates in the 3' to 5' direction along the bound strand, a replication fork-like structure of the substrate stimulates its DNA-unwinding activity, and it failed to unwind blunt-ended duplex DNA. Of various compounds tested, only cisplatin, 4',6'-diamidino-2-phenylindole, daunorubicin, and nogalamycin were inhibitory to the unwinding activity of PcDDH45 with apparent IC(50) values of 1.0, 4.0, 7.5, and 1.7 microM, respectively. These results suggest that the interaction of these compounds with duplex DNA generate a complex that probably impedes the translocation of PcDDH45, resulting in inhibition of unwinding activity. This study is one of the first to demonstrate the effect of various DNA-binding compounds on a malaria parasite DNA helicase and should make an important contribution to our better understanding of the nucleic acid transactions in the parasite.     

A novel nuclear DNA helicase with high specific activity from Pisum sativum catalytically translocates in the 3'-->5' direction.

A novel ATP-dependent nuclear DNA unwinding enzyme from pea has been purified to apparent homogeneity and characterized. This enzyme is present at extremely low abundance and has the highest specific activity among plant helicases. It is a heterodimer of 54 and 66 kDa polypeptides as determined by SDS/PAGE. On gel filtration chromatography and glycerol gradient centrifugation it gives a native molecular mass of 120 kDa and is named as pea DNA helicase 120 (PDH120). The enzyme can unwind 17-bp partial duplex substrates with equal efficiency whether or not they contain a fork. It translocates unidirectionally along the bound strand in the 3'-->5' direction. The enzyme also exhibits intrinsic single-stranded DNA- and Mg2+-dependent ATPase activity. ATP is the most favoured cofactor but other NTPs and dNTPs can also support the helicase activity with lower efficiency (ATP > GTP = dCTP > UTP > dTTP > CTP > dATP > dGTP) for which divalent cation (Mg2+ > Mn2+) is required. The DNA intercalating agents actinomycin C1, ethidium bromide, daunorubicin and nogalamycin inhibit the DNA unwinding activity of PDH120 with Ki values of 5.6, 5.2, 4.0 and 0.71 micro Ms, respectively. This inhibition might be due to the intercalation of the inhibitors into duplex DNA, which results in the formation of DNA-inhibitor complexes that impede the translocation of PDH120. Isolation of this new DNA helicase should make an important contribution to our better understanding of DNA transaction in plants.     

Inhibition of DNA helicase II unwinding and ATPase activities by DNA-interacting ligands. Kinetics and specificity.

Although DNA helicases play important roles in the processing of DNA, little is known about the effects of DNA-interacting ligands on these helicases. Therefore, the effects of a wide variety of DNA-binding ligands on the unwinding and ATPase reactions catalyzed by Escherichia coli DNA helicase II were examined. DNA minor groove binders and simple DNA intercalators did not inhibit helicase II. However, DNA intercalators, such as mitoxantrone and nogalamycin, which position functionalities in the major groove upon binding duplex DNA, were potent inhibitors of helicase II. To determine the mechanism by which mitoxantrone inhibited helicase II, the unwinding and DNA-dependent ATPase activities of helicase II were measured using a spectrum of double- and single-stranded DNA substrates. Using either a 71-base pair (bp) M13mp7 partially duplexed DNA substrate or a 245-bp bluntended, fully duplexed DNA substrate, the apparent Ki value for inhibition by mitoxantrone of both the unwinding and ATPase reactions was approximately 1 microM for both substrates, suggesting that the mechanism of inhibition of helicase II by mitoxantrone is the same for both substrates and requires the presence of double-stranded structure. To strengthen this conclusion, the ability of mitoxantrone to inhibit the DNA-dependent ATPase activity of helicase II was determined using two single-stranded substrates, poly(dT) and the 245-bp substrate after heat denaturation. Using either substrate, mitoxantrone inhibited the ATPase activity of helicase II far less effectively. Thus, these results indicate that the intercalation of mitoxantrone into double-stranded DNA, with accompanying placement of functionalities in the major groove, generates a complex that impedes helicase II, resulting in both inhibition of ATP hydrolysis and unwinding activity. Furthermore, we report here that DNA-binding ligands inhibit the unwinding activity of helicases I and IV and Rep protein from E. coli, demonstrating that the inhibition observed for helicase II is not unique to this enzyme.     

Characterization of replication fork and phosphorylation stimulated Plasmodium falciparum helicase 45

Helicases are essential enzymes, which play important role in the metabolism of nucleic acids. In the present study we report further characterization of PfH45 (Plasmodium falciparum helicase 45), which is an essential enzyme for parasite survival. The results show that the helicase activity of PfH45 is significantly stimulated by replication fork like structure. The studies using truncated derivatives of PfH45 show that its nucleic acid dependent ATPase activity resides in the N-terminal one third of the protein and its RNA and DNA-binding activity predominantly resides in the C-terminal two third of the protein. The phosphorylation of PfH45 by protein kinase C at Ser and Thr residues stimulated its DNA and RNA helicase and ssDNA and RNA-dependent ATPase activities. DNA-interacting compounds actinomycin, DAPI, daunorubicin, ethidium bromide, netropsin and nogalamycin were able to inhibit the helicase and ssDNA-dependent ATPase activity with apparent IC50 values ranging from 0.5 to 5.0 microM respectively. These compounds distinctively inhibit the helicase activity probably by forming complex with DNA and obstructing enzyme movement.     

甲磺酸培氟沙星抑制大肠杆菌RecQ解旋酶的体外DNA结合、解链和ATPase活性

RecQ家族解旋酶是DNA解旋酶中高度保守的一个重要家族,参与DNA复制、修复、重组、转录及维持端粒稳定等细胞代谢过程,在维持染色体稳定性与完整性中起着重要作用．甲磺酸培氟沙星(pefloxacin mesylate,PFM)是一种新型氟喹诺酮类抗菌药物,对一些革兰氏阴性菌具有明显的杀菌效果,临床上已广泛使用．本研究利用荧光偏振、自由磷检测技术研究PFM对大肠杆菌RecQ解旋酶的DNA结合活性、解链活性、ATPase活性的影响．结果表明,低浓度PFM可促进大肠杆菌RecQ解旋酶与ssDNA、dsDNA结合,达到一定量后PFM则抑制酶与DNA底物的结合,这种影响与DNA底物有关;PFM对RecQ解旋酶的DNA解链活性和ATP酶活性都具有抑制作用,但其抑制的效果有极显著差异(P<0.01)：比较PFM对两种活性抑制的Ci值(对解链活性抑制的Ci值为(1.5±0.2) μmol/L,对ATP酶活性抑制的Ci值为(0.010±0.005) μmol/L)可知,PFM对大肠杆菌RecQ解旋酶ATPase活性的抑制强于其解链活性. 这些结果可为研究以DNA解旋酶为药物靶标的分子机理奠定相关理论基础．     

Characterization of the adenosinetriphosphatase activity of the Escherichia coli RecBCD enzyme: relationship of ATP hydrolysis to the unwinding of duplex DNA

We find that the rate of dsDNA-dependent ATPase activity is biphasic, with a fast component which represents the unwinding of the dsDNA and a slow component which results from the ssDNA-dependent ATPase activity of recBCD enzyme. Comparison of the ATPase and helicase activities permits evaluation of the efficiency of ATP hydrolysis during unwinding. This efficiency can be calculated from the maximum rates of ATPase and helicase activities and is found to range between 2.0 and 3.0 ATP molecules hydrolyzed per base pair of DNA unwound. The number of ATP molecules hydrolyzed per base pair unwound is not altered by temperature but does increase at low concentrations of DNA and high concentrations of sodium chloride and magnesium acetate. The apparent Km values for the DNA and ATP substrates of recBCD enzyme dsDNA-dependent ATPase activity at 25 degrees C were determined to be 0.13 nM DNA molecules and 85 microM ATP, respectively. The observed kcat value is approximately 45 microM ATP s-1 (microM recBCD enzyme)-1. If this rate is corrected for the measured stoichiometry of recBCD enzyme binding to dsDNA, the kcat for ATPase activity corresponds to an ATP hydrolysis rate of approximately 740 ATP molecules s-1 (functional recBCD complex)-1 at 25 degrees C.     

Cysteine 111 affects coupling of single-stranded DNA binding to ATP hydrolysis in the herpes simplex virus type-1 origin-binding protein

Herpes simplex virus type-1 origin-binding protein (UL9 protein) initiates viral replication by unwinding the origins. It possesses sequence-specific DNA-binding activity, single-stranded DNA-binding activity, DNA helicase activity, and ATPase activity that is strongly stimulated by single-stranded DNA. We have examined the role of cysteines in its action as a DNA helicase. The DNA helicase and DNA-dependent ATPase activities of UL9 protein were stimulated by reducing agent and specifically inactivated by the sulfhydryl-specific reagent N-ethylmaleimide. To identify the cysteine responsible for this phenomenon, a conserved cysteine in the vicinity of the ATP-binding site (cysteine 111) was mutagenized to alanine. UL9C111A protein exhibits defects in its DNA helicase and DNA-dependent ATPase activities and was unable to support origin-specific DNA replication in vivo. A kinetic analysis indicates that these defects are due to the inability of single-stranded DNA to induce high affinity ATP binding in UL9C111A protein. The DNA-dependent ATPase activity of UL9C111A protein is resistant to N-ethylmaleimide, while its DNA helicase activity remains sensitive. Accordingly, sensitivity of UL9 protein to N-ethylmaleimide is due to at least two cysteines. Cysteine 111 is involved in coupling single-stranded DNA binding to ATP-binding and subsequent hydrolysis, while a second cysteine is involved in coupling ATP hydrolysis to DNA unwinding.     

Biochemical characterization of the DNA helicase activity of the escherichia coli RecQ helicase

We demonstrate that RecQ helicase from Escherichia coli is a catalytic helicase whose activity depends on the concentration of ATP, free magnesium ion, and single-stranded DNA-binding (SSB) protein. Helicase activity is cooperative in ATP concentration, with an apparent S(0.5) value for ATP of 200 microm and a Hill coefficient of 3.3 +/- 0.3. Therefore, RecQ helicase utilizes multiple, interacting ATP-binding sites to mediate double-stranded DNA (dsDNA) unwinding, implicating a multimer of at least three subunits as the active unwinding species. Unwinding activity is independent of dsDNA ends, indicating that RecQ helicase can unwind from both internal regions and ends of dsDNA. The K(M) for dsDNA is 0.5-0.9 microm base pairs; the k(cat) for DNA unwinding is 2.3-2.7 base pairs/s/monomer of RecQ helicase; and unexpectedly, helicase activity is optimal at a free magnesium ion concentration of 0.05 mm. Omitting Escherichia coli SSB protein lowers the rate and extent of dsDNA unwinding, suggesting that RecQ helicase associates with the single-stranded DNA (ssDNA) product. In agreement, the ssDNA-dependent ATPase activity is reduced in proportion to the SSB protein concentration; in its absence, ATPase activity saturates at six nucleotides/RecQ helicase monomer and yields a k(cat) of 24 s(-1). Thus, we conclude that SSB protein stimulates RecQ helicase-mediated unwinding by both trapping the separated ssDNA strands after unwinding and preventing the formation of non-productive enzyme-ssDNA complexes.     

Simultaneously monitoring DNA binding and helicase-catalyzed DNA unwinding by fluorescence polarization

A new method for helicase-catalyzed DNA unwinding is described. This assay takes advantage of the substantial change in fluorescence polarization (FP) upon helicase binding and DNA unwinding. The low anisotropy value, due to the fast tumbling of the free oligonucleotide in solution, increases abruptly upon binding of helicase to the fluorescein-labeled oligonucleotide. The high anisotropy of the helicase– DNA complex decreases as the fluorescein-labeled oligonucleotide is released from the complex through helicase-catalyzed DNA unwinding. This FP signal can be measured in real time by fluorescent spectroscopy. This assay can simultaneously monitor DNA binding and helicase-catalyzed DNA unwinding. It can also be used to determine the polarity in DNA unwinding mediated by helicase. This FP assay should facilitate the study of the mechanism by which helicase unwinds duplex DNA, and also aid in screening for helicase inhibitors, which are of growing interest as potential anticancer agents.     

A requirement for MCM7 and Cdc45 in chromosome unwinding during eukaryotic DNA replication

In vertebrates, MCM2-7 and Cdc45 are required for DNA replication initiation, but it is unknown whether they are also required for elongation, as in yeast. Moreover, although MCM2-7 is a prime candidate for the eukaryotic replicative DNA helicase, a demonstration that MCM2-7 unwinds DNA during replication is lacking. Here, we use Xenopus egg extracts to investigate the roles of MCM7 and Cdc45 in DNA replication. A fragment of the retinoblastoma protein, Rb(1-400), was used to neutralize MCM7, and antibodies were used to neutralize Cdc45. When added immediately after origin unwinding, or after significant DNA synthesis, both inhibitors blocked further DNA replication, indicating that MCM7 and Cdc45 are required throughout replication elongation in vertebrates. We next exploited the fact that inhibition of DNA polymerase by aphidicolin causes extensive chromosome unwinding, likely due to uncoupling of the replicative DNA helicase. Strikingly, Rb(1-400) and Cdc45 antibodies both abolished unwinding by the uncoupled helicase. These results provide new support for the model that MCM2-7 is the replicative DNA helicase, and they indicate that Cdc45 functions as a helicase co-factor.     

Modulation of the herpes simplex virus type-1 UL9 DNA helicase by its cognate single-strand DNA-binding protein, ICP8

The mechanism of stimulation of a DNA helicase by its cognate single-strand DNA-binding protein was examined using herpes simplex virus type-1 UL9 DNA helicase and ICP8. UL9 and ICP8 are two essential components of the viral replisome that associate into a complex to unwind the origins of replication. The helicase and DNA-stimulated ATPase activities of UL9 are greatly elevated as a consequence of this association. Given that ICP8 acts as a single-strand DNA-binding protein, the simplest model that can account for its stimulatory effect predicts that it tethers UL9 to the DNA template, thereby increasing its processivity. In contrast to the prediction, data presented here show that the stimulatory activity of ICP8 does not depend on its single-strand DNA binding activity. Our data support an alternative hypothesis in which ICP8 modulates the activity of UL9. Accordingly, the data show that the ICP8-binding site of UL9 constitutes an inhibitory region that maintains the helicase in an inefficient ground state. ICP8 acts as a positive regulator by neutralizing this region. ICP8 does not affect substrate binding, ATP hydrolysis, or the efficiency of translocation/DNA unwinding. Rather, we propose that ICP8 increases the efficiency with which substrate binding and ATP hydrolysis are coupled to translocation/DNA unwinding.     

Coupling DNA-binding and ATP hydrolysis in Escherichia coli RecQ: role of a highly conserved aromatic-rich sequence

RecQ enzymes are broadly conserved Superfamily-2 (SF-2) DNA helicases that play critical roles in DNA metabolism. RecQ proteins use the energy of ATP hydrolysis to drive DNA unwinding; however, the mechanisms by which RecQ links ATPase activity to DNA-binding/unwinding are unknown. In many Superfamily-1 (SF-1) DNA helicases, helicase sequence motif III links these activities by binding both single-stranded (ss) DNA and ATP. However, the ssDNA-binding aromatic-rich element in motif III present in these enzymes is missing from SF-2 helicases, raising the question of how these enzymes link ATP hydrolysis to DNA-binding/unwinding. We show that Escherichia coli RecQ contains a conserved aromatic-rich loop in its helicase domain between motifs II and III. Although placement of the RecQ aromatic-rich loop is topologically distinct relative to the SF-1 enzymes, both loops map to similar tertiary structural positions. We examined the functions of the E.coli RecQ aromatic-rich loop using RecQ variants with single amino acid substitutions within the segment. Our results indicate that the aromatic-rich loop in RecQ is critical for coupling ATPase and DNA-binding/unwinding activities. Our studies also suggest that RecQ's aromatic-rich loop might couple ATP hydrolysis to DNA-binding in a mechanistically distinct manner from SF-1 helicases.     

Mutations altering the interplay between GkDnaC helicase and DNA reveal an insight into helicase unwinding

Replicative helicases are essential molecular machines that utilize energy derived from NTP hydrolysis to move along nucleic acids and to unwind double-stranded DNA (dsDNA). Our earlier crystal structure of the hexameric helicase from Geobacillus kaustophilus HTA426 (GkDnaC) in complex with single-stranded DNA (ssDNA) suggested several key residues responsible for DNA binding that likely play a role in DNA translocation during the unwinding process. Here, we demonstrated that the unwinding activities of mutants with substitutions at these key residues in GkDnaC are 2-4-fold higher than that of wild-type protein. We also observed the faster unwinding velocities in these mutants using single-molecule experiments. A partial loss in the interaction of helicase with ssDNA leads to an enhancement in helicase efficiency, while their ATPase activities remain unchanged. In strong contrast, adding accessory proteins (DnaG or DnaI) to GkDnaC helicase alters the ATPase, unwinding efficiency and the unwinding velocity of the helicase. It suggests that the unwinding velocity of helicase could be modulated by two different pathways, the efficiency of ATP hydrolysis or protein-DNA interaction.     

Overexpression of a pea DNA helicase 45 in bacteria confers salinity stress tolerance

Salinity stress is one of the major factors negatively affecting growth and productivity in living organisms including plants and bacteria resulting in significant losses worldwide. Therefore, it would be fruitful to develop salinity stress tolerant useful species and also to understand the mechanism of stress tolerance. The pea DNA helicase 45 (PDH45) is a DNA and RNA helicase, homologous to eukaryotic translation initiation factor 4A (eIF-4A) and is involved in various processes including protein synthesis, maintaining the basic activities of the cell, upregulation of topoisomerase I activity and salinity stress tolerance in plant, but its role in salinity stress tolerance in bacteria has not heretofore been studied. This study provides an evidence for a novel function of the PDH45 gene in high salinity (NaCl) stress tolerance in bacteria (Eschericia coli, BL21 cells) also. Furthermore, it has been shown that the functionally active PDH45 gene is required to show the stress tolerance in bacteria because the single mutants (E183G or R363Q) and the double mutant (E183G + R363Q) of the gene could not confer the same function. The response was specific to Na+ ions as the bacteria could not grow in presence of LiCl. This study suggests that the cellular response to high salinity stress across prokaryotes and plant kingdom is conserved and also helps in our better understanding of mechanism of stress tolerance in bacteria and plants. It could also be very useful in developing high salinity stress tolerant useful bacteria of agronomic importance. Overall, this study provides an evidence for a novel function of the PDH45 gene in high salinity stress tolerance in bacteria.Key words: bacteria, cellular stress response, PDH45, pea, plant DNA helicase, salinity stress