TY - JOUR
AU1 - Zhang,, Bo
AU2 - Zhang,, Ai-hua
AU3 - Chen,, Lei
AU4 - Xi, Xu, Guang
AB - Abstract RecQ helicases play an essential role in maintaining genetic integrity in all organisms from Escherichia coli to humans. Defects to these enzymes are responsible for three distinct human diseases: Werner syndrome, Bloom syndrome and Rothmund–Thomson syndrome. All three diseases are characterized by a predisposition to cancer due to increased genomic instability. Previous studies on the effects of non-covalent DNA modifications on the catalytic activity of purified Werner and Bloom DNA helicases have shown that both enzymes have similar sensitivity profiles to these DNA-binding agents and are most strongly inhibited by the minor groove binder distamycin A. In this study, we show that the sensitivity profiles of E. coli RecQ to a number of DNA-binding ligands are different to those observed for WRN and Bloom helicases. These observations may give insights into the differences in molecular mechanisms underlying efficient motor function of RecQ helicases. helicase, human diseases, DNA modification, minor groove binder, molecular motor Helicases are ubiquitous enzymes that are involved in most aspects of various nucleic acid metabolic pathways, such as DNA replication, DNA repair, recombination, transcription, RNA processing and protein translation (1). Abnormalities in three members of the RecQ helicase family have been shown to cause three different human diseases that are associated with inherent genomic instability: Bloom syndrome, Werner syndrome and Rothmund–Thomson syndrome (2). All RecQ helicases have seven highly conserved motifs that contain an ATP-binding sequence (Walker A and B box) and the DEXH box, which is characteristic of the RecQ helicase family. Also, Escherichia coli and Bloom RecQ helicases have a zinc finger motif that is highly conserved structurally and functionally. This motif plays an essential role in DNA binding and DNA substrate discrimination (3, 4). In living cells, helicases co-ordinately interact with others proteins to perform their different functions. It has been reported that E. coli RecQ helicase unwinding activity is greatly enhanced by the single-stranded DNA-binding protein (SSB) (5, 6). Recently, E. coli RecQ helicase has been shown to physically and functionally interact with SSB (7). Although the mechanism of helicase-mediated unwinding is still poorly understood, the biochemical properties of numerous helicases from various organisms are well known. Helicases are molecular motors that convert the chemical energy derived from nucleoside triphosphate hydrolysis to the mechanical energy to unwind double-stranded (ds) DNA and to translocate along DNA. Recent studies have revealed that the roles of helicases extend beyond simply separating double stranded DNA (dsDNA). Several helicases have been shown to dislodge proteins bound to DNA and streptavidin bound to biotinylated oligonucleotides (8, 9). The E. coli RecQ helicase is 609-amino acids long, whereas most eukaryotic RecQ helicases are more than 1 000-amino acids long, with long N-terminal and C-terminal regions on either side of the helicase domain. These structural differences may confer additional functions to eukaryotic RecQ helicases. Indeed, the N-terminal sequence of the WRN protein, which has been shown to be similar to the N-terminal sequence of DNase D, possesses an intrinsic exonuclease activity (10, 11). We wondered whether the structural differences between helicases from prokaryotes and eukaryotes might influence their reactivity with chemotherapeutic drugs used as antibiotic and anti-tumour agents. Previous studies have shown that both Werner and Bloom helicases are potently inhibited by drugs that bind the DNA minor groove (12). Here, we report the effect of various DNA-binding chemotherapeutic agents on the DNA binding, ATPase activity and helicase activity of E. coli RecQ helicase. Our results show that the sensitivity profiles of this enzyme to some DNA-binding ligands are different to those observed for WRN and Bloom helicases. These observations may give insights into the different molecular mechanisms underlying efficient motor function of RecQ helicase family members. MATERIALS AND METHODS Oligonucleotides PAGE-purified, unlabelled and fluoresein-labelled synthetic oligonucleotides were purchased from Proligo (France). As some of the compounds used in this study preferentially bind to DNA duplex tracts containing a 4–5 A-T tract, we used the following DNA sequences: Oligo A:5′-ATGCTGATGCAAATCCAATCGCAAGACA(T)20-3′ and Oligo B: 5′-TGTCTTGCGATTGGATTTGCATCAGCAT-Fluorescein-3′. Double-stranded oligonucleotides were prepared in a 20 mM Tris–HCl buffer, pH 7.2, containing 100 mM NaCl. The mixture was heated to 85°C for 5 min and annealed by slow cooling to room temperature. Preparation of DNA-Binding Compounds All chemicals were obtained from Sigma and were prepared as previously described (12). The concentrations of the DNA-binding compounds were determined spectrophotometrically according to published extinction coefficients (12). Enzyme His-6-tagged E. coli RecQ helicase was expressed from the pET-15b plasmid in E. coli strain BL21 (DE3) and purified as previously described (13). Briefly, the over-expressed protein was purified under native conditions using chromatography on Ni2+-nitrilotriacetic acid columns (Qiagen, Valencia, CA, USA), followed by FPLC size exclusion chromatography (Superdex 200, Pharmacia) and ion-exchange chromatography (DEAE Sephadex A-50). The purity of the RecQ preparation was determined as >95% based on Sypro Orange-stained SDS–PAGE and electrospray mass spectrometry analyses. Helicase Assay We carried out an unwinding assay using a Beacon 2000 polarization instrument, according to Xu et al. (14). An appropriate quantity of fluorescein-labelled duplex oligonucleotide (1 nM) was added to the helicase unwinding buffer containing 25 mM Tris–HCl (pH 8.0), 30 mM NaCl, 3 mM (CH3CO2)2Mg, 0.1 mM DTT (150 µl total) in a temperature-controlled cuvette at 25°C. The anisotropy was continuously measured until it stabilized. Helicases were then added to the cuvette. When the anisotropy again became stable, the unwinding reaction was started by the rapid addition of ATP solution to a final concentration of 1 mM. The anisotropy decrease was recorded every 8 s until it stabilized. We determined the apparent catalytic constants from the equation: At = A exp(–kobst), where At is the anisotropy at time t, and kobs is the observed rate constant. For some experiments, Helicase activity was also confirmed by radiometric assay (4). DNA unwinding activity in the presence of distamycin A was also measured by a radiometric assay. The reaction was carried out in a reaction mixture containing 25 mM HEPES-NaOH, pH 7.5, 25 mM NaOAc, 7.5 mM Mg(OAc)2, 2 mM ATP, 1 mM DTT, 0.1 mg/ml BSA, 32P-labelled partial DNA duplex substrate (10 fmol, 3 000 c.p.m/fmol) and the indicated inhibitor concentrations. The partial DNA duplex substrate is the same as that used for the fluorimetric assay, except that the oligo B was 32P-radiolabelled at the 5′ position, rather than fluorescein labelled at the 3′ position. The reaction was initiated by addition of 80 nM RecQ helicase at 37°C for 20 min. The reaction was terminated by the addition of 5 µl of 5× loading buffer (50 mM EDTA, 0.5% SDS, 0.1% xylene cyanol, 0.1% bromophenol blue and 50% glycerol). The reaction products were resolved on a 12% (w/v) PAGE run in a TBE buffer (90 mM Tris, 90 mM boric acid, pH 8.3 and 1 mM EDTA) at 100 V for 2 h at 4°C. ATPase Assay ATPase activity was measured from the amount of radioactive γ−32P liberated during hydrolysis (15). Briefly, the experiment was carried out at 37°C in a reaction mixture containing 1.5 µM of nucleotide and heat-denatured HindIII-cut pGEM-7Zf linear DNA at the same concentration as ATP. The reactions were started by the addition of RecQ helicase into 100 µl of reaction mixture. Every 30-s aliquot (80 µl) was pipetted from one of the reaction mixtures into a hydrochloric solution of ammonium molybdate. The liberated radioactive 32P was extracted with a solution of 2-butanol-benzene-acetone-ammonium molybdate (150 µM) (750:750:15:1). An aliquot of the organic phase was counted in 6 ml of Aquasol. DNA-Binding Assays DNA-binding assays were carried out using a Beacon 2000 polarization instrument (PanVera Corp.) (16). An appropriate quantity of fluorescein-labelled dsDNA was added to a standard titration buffer (150 μl total volume) in a temperature-controlled cuvette at 25°C. The anisotropy of the fluorescein-labelled DNA was measured continuously in the presence of different concentrations of the compounds until it stabilized. An appropriate quantity of RecQ helicase was then added. After each addition of RecQ helicase the anisotropy was measured continuously until a stable plateau was reached. The relative DNA-binding activity was determined as: (An/A0) × 100%, where An is the fluorescence anisotropy at a given concentration of different compounds and A0 is the fluorescence anisotropy in the absence of the compounds. RESULTS AND DISCUSSION Based on their chemical structure, the DNA-binding agents used in this study can be divided into three kinds: DNA intercalators (ethidium bromide, mitoxantrone, m-AMSA and actinomycin D), minor groove binders (Hoechst 33258, DAPI and distamycin A) and topoisomerase inhibitors (camptothecin, 10-hydrocamptothecin and VP16) (Fig. 1). Here we have used our previously developed rapid, real-time helicase assay (14) to determine the effect of the DNA-binding agents on the helicase activity. This assay is based on the observation that fluorescein-labelled free oligonucleotides tumble more rapidly due to their very rapid rotational diffusion, than helicase–oligonucleotide complexes. Therefore, the DNA unwinding reaction can be followed in real time by measuring the change in fluorescence polarization. This approach has been used successfully in a number of studies and the obtained data were comparable to the data acquired with the conventional radiometric assay (13). Here we have determined the apparent catalytic constant of E. coli RecQ helicase in the presence of increasing concentrations of the different agents by measuring the fluorescence anisotropy of the reaction mixtures. The apparent unwinding constant can be derived from fitting an exponential function to the kinetic curves. An example for mitoxantrone is shown in Fig. 2A. We also systematically determined the DNA binding and ATPase activities under the same experimental conditions in order to better understand the mechanism of action of these agents. Fig. 1. Open in new tabDownload slide Chemical structures of DNA binding compounds. Fig. 1. Open in new tabDownload slide Chemical structures of DNA binding compounds. Fig. 2. Open in new tabDownload slide Effect of the intercalators used to study DNA unwinding, ATPase and DNA-binding activities of E. coli RecQ helicase. (A) The kinetic unwinding curve of E. coli RecQ helicase in the absence (square) and presence of 0.5 µM (diamond), 1 µM (closed circle), 3 µM (triangle) and 5 µM (open circle) mitoxantrone. DNA and the protein concentrations were 1 nM of 3′-fluorecent-labelled partial duplex DNA and 50 nM helicase. The apparent catalytic constants were determined according to the equation: At = A exp(–kobst), where At is the anisotropy amplitude at time t, and kobs is the observed rate constant. (B) RecQ helicase activity was determined as in Fig. 2A in the presence of different compounds at different concentrations. All experiments were carried as per the conditions indicated in Materials and Methods section. DNA and the protein concentrations were 1 nM 3′-fluorescent-labelled duplex DNA and 50 nM helicase. (C), Experiments were carried out as indicated in Materials and Methods section. Helicase concentrations used were 50 nM. (D) DNA and the protein concentrations were 1 nM 3′-fluorescent-labelled duplex DNA and 50 nM RecQ helicase. The experimental conditions were as described in Materials and Methods section. Fig. 2. Open in new tabDownload slide Effect of the intercalators used to study DNA unwinding, ATPase and DNA-binding activities of E. coli RecQ helicase. (A) The kinetic unwinding curve of E. coli RecQ helicase in the absence (square) and presence of 0.5 µM (diamond), 1 µM (closed circle), 3 µM (triangle) and 5 µM (open circle) mitoxantrone. DNA and the protein concentrations were 1 nM of 3′-fluorecent-labelled partial duplex DNA and 50 nM helicase. The apparent catalytic constants were determined according to the equation: At = A exp(–kobst), where At is the anisotropy amplitude at time t, and kobs is the observed rate constant. (B) RecQ helicase activity was determined as in Fig. 2A in the presence of different compounds at different concentrations. All experiments were carried as per the conditions indicated in Materials and Methods section. DNA and the protein concentrations were 1 nM 3′-fluorescent-labelled duplex DNA and 50 nM helicase. (C), Experiments were carried out as indicated in Materials and Methods section. Helicase concentrations used were 50 nM. (D) DNA and the protein concentrations were 1 nM 3′-fluorescent-labelled duplex DNA and 50 nM RecQ helicase. The experimental conditions were as described in Materials and Methods section. The RecQ Helicase Sensitivity Profiles to DNA-Intercalators The curves for the relative DNA unwinding rate constants of E. coli RecQ helicase in the presence of four different DNA-intercalators showed that both ethidium bromide and mitoxantrone inhibited unwinding more strongly than actinomycin D and m-AMSA (Fig. 2B), as a 50% unwinding was achieved with 2–5 µM ethidium bromide or mitoxantrone versus 40 µM m-AMSA or 50 µM actinomycin D. We then examined the ATPase activity as a function of increasing concentrations of the DNA-binding agents to study their mechanism of enzyme inhibition. Consistent with the previous results, ethidium bromide and mitoxantrone inhibited ATPase activity by 50% at a concentration of <30 µM, whereas at least 200 μM were required to inhibit the ATPase activity by 10% for m-AMSA or 60% for actinomycin D (Fig. 2C). As RecQ helicase is a DNA-stimulated ATPase and an ATP-dependent helicase, DNA binding is essential for regulating ATPase and helicase activities. Therefore, we determined the DNA-binding activities of RecQ helicase in the presence of the DNA-binding agents, at concentrations identical to those used for the ATPase and helicase assays. The DNA-binding activity of the enzyme was completely inhibited by 50 µM ethidium bromide or mitoxantrone (Fig. 2D), with apparent Ki values of 10 µM for ethidium bromide and 12 µM for mitoxantrone. In contrast, AMSA had no detectable inhibitory effect up to a concentration of 150 µM, and at the same concentration actinomycin D only inhibited by about 20% the DNA-binding activity. These results suggested that ethidium bromide and mitoxantrone inhibit DNA binding, which in turn affects the ATPase and helicase activities. These observations are consistent with the crystal structure of the ethidium bromide–DNA complex (17, 18). Ethidium bromide is a potent inhibitor of DNA synthesis and may bend and distort the DNA structure through electrostatic binding to the phosphodiester backbone of DNA. It is possible that E. coli RecQ helicase is extremely sensitive to this modification of the DNA structure and therefore fails to bind to DNA. Also, the sensitivity profiles of the enzyme to ethidium bromide and mitoxantrone are almost identical, a result also seen for the WRN and BLM proteins (12). The Sensitivity Profiles to Minor Groove Binders When the minor groove binders were studied, (Fig. 3), we found that the most effective unwinding inhibitors were DAPI and H33258, with apparent Ki of 15 and 20 µM, respectively (these and the other results are given in Table 1). Although, the apparent Ki value for distamycin A was estimated to be about 200 µM, the maximum inhibition for the enzyme unwinding activity reached only 70% of the control and the inhibition curve never reached an asymptotic minimum even at concentrations of 500 µM. These observations were further confirmed by radiometric assay (Fig. 3A, insert). Thus, the sensitivity of E. coli RecQ helicase to distamycin A is similar to that reported for UvrD helicase (19), but is completely different from that measured for the WRN and BLM helicases (12). In this latter study, distamycin A potently inhibited both WRN and BLM helicase activity at a concentration ranging between 0.5 and 1 µM. These three minor groove binders, which inhibit helicase activity by different extents, all inhibit the ATPase activity by similar amounts, about 70% (Fig. 3B), suggesting that these compounds do not markedly inhibit DNA binding. Indeed, the binding studies showed that distamycin A had no detectable inhibition effect on DNA binding (Fig. 3C), whereas both H33258 and DAPI had similar inhibition profiles to that for ATPase activity. These results suggested that distamycin A does not prevent the helicase from binding to the DNA substrate, consistent with a previous study (12), showing that distamycin A efficiently inhibits the helicase activity of WRN and BLM while having no detectable effect on ATPase activity. It is possible that the WRN and BLM helicases are more sensitive to distamycin A than the E. coli RecQ helicase despite the fact that these enzymes belong to the same RecQ helicase family and have high sequence similarities. Escherichia coli RecQ helicase and human RecQ helicase are however considerably different with respect to their structure and enzymatic activity. The N-terminal and C-terminal extension sequences of the WRN and BLM helicases certainly confer different enzymatic activities to these helicases compared to E. coli helicase. More importantly, the ATPase and helicase activities of E. coli RecQ helicase are higher than those of WRN and BLM (20–23). As shown in Table 2, E. coli RecQ helicase efficiently catalyses the hydrolysis of ATP than its homologues eukaryotic RecQ family helicases. The binding of distamycin A to DNA may physically block the continued translocation of WRN and BLM helicases. Escherichia coli RecQ helicase may overcome this inhibitory effect through its highly efficient ATP hydrolysis and high helicase activity, whereas the low ATPase activities of the human RecQ helicases (WRN and BLM) do not provide enough energy to overcome this obstacle. Table 1. Comparison of DNA-binding compounds on the inhibition of DNA unwinding by RecQ helicase family from human and E. coli. Drug . Apparent Ki (μM) . . WRNa . BLMa . E. coli RecQ . Intercalator     Ethidium bromide 10 10 2     Mitoxantrone 10 10 5     m-AMSA 100 100 40     Actinomycin 100 100 50 Minor groove     DAPI 10 10–10 15     Hoescht 33258 10 10–10 20     Distamycin A 0.5–2.5 0.5–2.5 ≥200 Topoisomerase inhibitors     Camptothecin 100 100 ND     10-Hydrocamptothecin ND     VP16 (Etoposide) 100 100 ND Drug . Apparent Ki (μM) . . WRNa . BLMa . E. coli RecQ . Intercalator     Ethidium bromide 10 10 2     Mitoxantrone 10 10 5     m-AMSA 100 100 40     Actinomycin 100 100 50 Minor groove     DAPI 10 10–10 15     Hoescht 33258 10 10–10 20     Distamycin A 0.5–2.5 0.5–2.5 ≥200 Topoisomerase inhibitors     Camptothecin 100 100 ND     10-Hydrocamptothecin ND     VP16 (Etoposide) 100 100 ND ND, non detectable. aWerner and Bloom helicases (12). Open in new tab Table 1. Comparison of DNA-binding compounds on the inhibition of DNA unwinding by RecQ helicase family from human and E. coli. Drug . Apparent Ki (μM) . . WRNa . BLMa . E. coli RecQ . Intercalator     Ethidium bromide 10 10 2     Mitoxantrone 10 10 5     m-AMSA 100 100 40     Actinomycin 100 100 50 Minor groove     DAPI 10 10–10 15     Hoescht 33258 10 10–10 20     Distamycin A 0.5–2.5 0.5–2.5 ≥200 Topoisomerase inhibitors     Camptothecin 100 100 ND     10-Hydrocamptothecin ND     VP16 (Etoposide) 100 100 ND Drug . Apparent Ki (μM) . . WRNa . BLMa . E. coli RecQ . Intercalator     Ethidium bromide 10 10 2     Mitoxantrone 10 10 5     m-AMSA 100 100 40     Actinomycin 100 100 50 Minor groove     DAPI 10 10–10 15     Hoescht 33258 10 10–10 20     Distamycin A 0.5–2.5 0.5–2.5 ≥200 Topoisomerase inhibitors     Camptothecin 100 100 ND     10-Hydrocamptothecin ND     VP16 (Etoposide) 100 100 ND ND, non detectable. aWerner and Bloom helicases (12). Open in new tab Table 2. Comparison of the kcat (min−1) for ATP hydrolysis of RecQ family helicases. Helicases . kcat (min−1) . References . hsRECQL 126.3 ± 3.4 (20) hsBLM 1163 ± 358 (12) hsWRN 58.4 ± 17 (22) dmRECQ5 900 ± 3.4 (23) E. coli RECQ 1452-2000 (6) Helicases . kcat (min−1) . References . hsRECQL 126.3 ± 3.4 (20) hsBLM 1163 ± 358 (12) hsWRN 58.4 ± 17 (22) dmRECQ5 900 ± 3.4 (23) E. coli RECQ 1452-2000 (6) Open in new tab Table 2. Comparison of the kcat (min−1) for ATP hydrolysis of RecQ family helicases. Helicases . kcat (min−1) . References . hsRECQL 126.3 ± 3.4 (20) hsBLM 1163 ± 358 (12) hsWRN 58.4 ± 17 (22) dmRECQ5 900 ± 3.4 (23) E. coli RECQ 1452-2000 (6) Helicases . kcat (min−1) . References . hsRECQL 126.3 ± 3.4 (20) hsBLM 1163 ± 358 (12) hsWRN 58.4 ± 17 (22) dmRECQ5 900 ± 3.4 (23) E. coli RECQ 1452-2000 (6) Open in new tab Fig. 3. Open in new tabDownload slide Effects of minor groove compounds on the helicase (A), ATPase (B) and DNA-binding activities (C) of E. coli helicase. The reaction conditions and the enzyme quantities are identical to those in Fig. 2. Insert in Fig. 3A is the radiometric assay of RecQ helicase unwinding activity and the function of increasing distamycin A concentration. The detailed experimental conditions for radiometric assay were indicated in the Materials and Methods section. Fig. 3. Open in new tabDownload slide Effects of minor groove compounds on the helicase (A), ATPase (B) and DNA-binding activities (C) of E. coli helicase. The reaction conditions and the enzyme quantities are identical to those in Fig. 2. Insert in Fig. 3A is the radiometric assay of RecQ helicase unwinding activity and the function of increasing distamycin A concentration. The detailed experimental conditions for radiometric assay were indicated in the Materials and Methods section. Previous studies have shown that SSB stimulates RecQ DNA unwinding (5, 6). In order to determine whether RecQ helicase could overcome the partial inhibitory effect of distamycin A in the presence of the SSB protein, we have measured the helicase activity of E. coli RecQ in the presence of both distamycin and SSB. As shown in Fig. 4, the residual inhibitory effect of distamycin A is completely overcome by RecQ helicase in the presence of SSB protein. In the study by Brosh et al. (12) the overall pattern of distamycin inhibition for WRN or BLM helicases was the same in the presence or absence of human replication protein A (hRPA), suggesting that hRPA does not alleviate the potent inhibition of WRN or BLM unwinding activity by the minor groove binder distamycin A. The different inhibitory effects of distamycin A to the same RecQ family helicases indicate that E. coli RecQ helicase is a powerful molecular motor when works co-ordinately with SSB protein. Fig. 4. Open in new tabDownload slide Escherichia coli RecQ helicase–mediated DNA unwinding activity in the presence of distamycin A and SSB protein. RecQ protein (50 nM) was incubated with 10 fmol (3 000 c.p.m./fmol) 32P-labelled partial duplex DNA substrate and 150 µM distamycin A in the presence of increasing concentration of SSB protein, as indicated in the figure. Helicase reaction was conducted as described in Materials and Methods section. Column c represents the boiled DNA substrate. Fig. 4. Open in new tabDownload slide Escherichia coli RecQ helicase–mediated DNA unwinding activity in the presence of distamycin A and SSB protein. RecQ protein (50 nM) was incubated with 10 fmol (3 000 c.p.m./fmol) 32P-labelled partial duplex DNA substrate and 150 µM distamycin A in the presence of increasing concentration of SSB protein, as indicated in the figure. Helicase reaction was conducted as described in Materials and Methods section. Column c represents the boiled DNA substrate. The Sensitivity Profiles to Topoisomerase Inhibitors VP16, camptothecin and hydro-camptothecin did not change the DNA helicase, ATPase and DNA-binding activities (Table 1). These observations are consistent with previous results obtained with WRN, BLM and UvrD helicases (12, 19), suggesting that these topoisomerase inhibitors cannot bind and modulate E. coli RecQ helicase. CONCLUSION RecQ helicases play an essential role in genome integrity. Unlike the RecQ helicases of prokaryotes, the RecQ helicases of eukaryotes usually have extended C-terminals or N-terminals. We compared the behaviour of these enzymes after exposure to chemotherapeutic agents, comparing the inhibition profiles of E. coli RecQ helicase to the human RecQ helicases, WRN and BLM. The results of DNA-binding inhibition for E. coli and human RecQ helicases are summarized in Table 1. Both E. coli and the human RecQ helicases exhibit similar inhibition profiles for the DNA intercalator compounds and are consistent with previous studies on WRN and BLM helicases. However, E. coli RecQ helicase is less sensitive to the minor groove binders, despite the fact that distamycin A strongly inhibited both WRN and BLM unwinding activity. Even at very high concentrations of distamycin A, the inhibition of E. coli RecQ helicase reached only 70% inhibition. The different effects of distamycin A on E. coli and human DNA helicases may reveal mechano-chemical properties of these helicases to DNA-binding agents. Although helicases are mainly known for their ability to unzip DNA or RNA, it has become increasingly clear that helicases can use the energy derived from ATP hydrolysis to disrupt any obstacle when helicases encountered on DNA lattice. The collision between a helicase and an obstacle can lead to stalling of the motor or displacement of the DNA-binding agents, depending on the force imparted by the molecular motor. Considering the fact that E. coli RecQ helicase possesses higher ATPase activity than BLM and WRN helicases it is evident that E. coli RecQ helicase is a stronger molecular motor than the human helicases BLM and WRN, and is able to overcome the physical obstacle of distamycin A to unwind DNA. More importantly, in the presence of SSB protein, E. coli RecQ helicase can completely overcome distamycin inhibitory effect. Taken together, these findings may give insights into the difference in molecular mechanisms underlying efficient motor function of RecQ helicase family. We thank Dr. Xu HQ and Liu J for their contributions at early stage of this work. This work was partially supported by CNRS and INSERM. We are grateful to Dr. Nelly KIEFFER for carefully reading this manuscript. 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TI - Inhibition of DNA Helicase, ATPase and DNA-Binding Activities of E. coli RecQ Helicase by Chemotherapeutic Agents
JF - The Journal of Biochemistry
DO - 10.1093/jb/mvn026
DA - 2008-06-01
UR - https://www.deepdyve.com/lp/oxford-university-press/inhibition-of-dna-helicase-atpase-and-dna-binding-activities-of-e-coli-2rcndwhRgx
SP - 773
EP - 779
VL - 143
IS - 6
DP - DeepDyve
ER -