- Email: [email protected]
ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method
Accepted Manuscript ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method Mu Yang, Ganggang Wang PII:
S0003-2697(16)30163-4
DOI:
10.1016/j.ab.2016.06.028
Reference:
YABIO 12430
To appear in:
Analytical Biochemistry
Received Date: 2 May 2016 Revised Date:
24 June 2016
Accepted Date: 27 June 2016
Please cite this article as: M. Yang, G. Wang, ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method, Analytical Biochemistry (2016), doi: 10.1016/j.ab.2016.06.028. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
ATPase Activity Measurement of DNA Replicative Helicase from Bacillus stearothermophilus by Malachite Green Method Mu Yang a,b,c, Ganggang Wang a,b* a
Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of
Sciences, Chengdu, 610041, China;
c
Key Laboratory of Environmental Microbiology of Sichuan Province, Chengdu, 610041, China;
University of Chinese Academy of Sciences, Beijing, 100049, China.
*Corresponding author’s:
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SEND CORRESPONDENCE TO:
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b
Ganggang Wang
Sciences, Chengdu, 610041, China
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Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of
Tel: 86-28-82890828; E-mail: [email protected]
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Subject Category: Electrophoresis Enzymatic assays and analysis
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Abstract The DnaB helicase from Bacillus stearothermophilus (DnaBBst) was a model protein for studying the bacterial DNA replication. In this work, a non-radioactive method for measuring ATPase activity of DnaBBst helicase was described. The working parameters and conditions were optimized. Furthermore, this method was applied to investigate effects of DnaG primase, ssDNA and
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helicase loader protein (DnaI) on ATPase activity of DnaBBst. Our results showed this method was sensitive and efficient. Moreover, it is suitable for the investigation of functional interaction between DnaB and related factors.
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Key words: ATPase Activity; DnaB; Malachite Green; Optimization
ACCEPTED MANUSCRIPT 1. Introduction DnaB protein is a ring-shaped RecA-type helicase that unwinds duplex DNA at replication fork by utilizing the energy of ATP hydrolysis[1-3]. The measurement on ATPase activity of DnaB helicase is crucial for deeper understanding the function of DnaB helicase in DNA replication as well
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as screening for specific inhibitors of bacterial DnaB[4, 5]. Traditionally,γ-32P radiolabelled ATP was used as the substrate for measuring ATPase activity of DnaB helicase and homologous proteins, after reaction, the released
32
Pi was separated using
thin-layer chromatography, followed by autoradiography and then quantified. This method was [6, 7]
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relatively inefficient and time-consuming, especially for the application to high-throughput screening . Moreover, rigorous safety measures should be applied to prevent experimenters from
radioactive contamination. In this way, the high cost, low efficiency and the harm of the radiation
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limited the wider application of radioactive method.
The malachite green method was a non-radioactive procedure for determination of phosphate released from ATP hydrolysis. This method was widely used to analyze the organic and inorganic phosphate in water as well as the activities of various phosphatases for advantages of being efficient, sensitive, cost effective and nonhazardous[4, 8-10].
This method was also applied to characterize ATPase activities of helicases like WRN-1 RecQ
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helicase[11, 12] and DnaB helicase from Klebsiell apneumoniae[13]. However, the working conditions were variable in previous reports, it was essential to optimize the parameters of ATPase activity assay. The DnaB helicase from Bacillus stearothermophilus (DnaBBst) was a model protein for studying the bacterial DNA replication[1, 14-17]. Up till now, there still lacks a detailed report on the
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ATPase activity of DnaBBst helicase measured by malachite green method. In this study, the malachite green method was applied to determine the ATPase activity of
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DnaBBst. Several parameters were optimized for high-throughput measurement. In addition, under the optimized condition, this method was applied for the first time to study on the functional interaction between DnaB and other related factors (DnaG primase, DnaI helicase loader and ssDNA,). Results showed that the optimized malachite green method was efficient in measuring the ATPase activity of DnaB helicase from B.stearothermophilus. It was also suitable for studying the interplay between DnaB and its partners. Moreover, this method might be used for high-throughput screening of specific DnaB inhibitors.
2.Results and discussions 2.1 Optimization of the parameters of Malachite Green Method In an effort to apply the Malachite green-Molybdate method for efficiently measuring ATPase activity of DnaBBst helicase, the maximum absorption wavelength was firstly determined (Fig.1A).
ACCEPTED MANUSCRIPT Fig. 1A showed the absorption spectra of standard sodium dihydrogen phosphate, in complex with dye reagent mentioned above, from the wavelength of 550 to 750nm. The spectra exhibited a characteristic absorption peak approximately at 650nm, which deviated slightly from previously proposed 620nm[18, 19] and 630nm[20]. Then the standard curve was determined in the 0-300uM range of sodium dihydrogen phosphate (Fig. S1). The standard curve was linear (R2=0.9929) between 0
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and 300uM phosphate concentrations to guarantee accuracy measurement of absorbances between 0.2 and 1. It was highly reproducible with the coefficient of variation less than 3%.
The self-hydrolysis of ATP in the reaction buffer led to remarkable background absorbance[21, 22]
, here the ATP concentration was optimized to minimize the background. As shown in Fig.1B, at
low concentrations of ATP-Na2 between 0 and 500uM, the background signals were negligible. In
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contrast, at concentrations of ATP-Na2 higher than 500uM, the absorbances increased up dramatically. The high background may severely influence the accuracy of measurement. Based on
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these results, 500uM ATP-Na2 was selected for further optimization.
The next step was the application of this method to ATPase activity of DnaB helicase. Firstly, the background signals of various concentrations of DnaB was investigated and confirmed to be minimal (data not shown). Besides, the acidic malachite green-molybdate straining reagent was also proved to be effective to terminate ATPase reaction at 37
(data not shown). Then the optimum
reaction time of ATP hydrolysis by helicase was investigated (Fig.1C). The variation of absorbance
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with time of ATPase reaction was exhibited in Fig.1C. The absorbance at 650nm increased considerably within the first 15 min, followed by gradually increasing until the plateau was reached at 22 min. In pursuit of high efficiency and accuracy for the measurement of initial velocity, a reaction time of 8 min was adopted for further investigations.
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The ultimate colorimetric complex is formed after the formation of phosphomolybdate, indicating enough time is required for full color development[23]. Hence, the staining time was
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optimized (Fig.1D). As shown in Fig.1D, the curve of absorbance increased sharply within 8 min, indicating a rapid chemical reaction between dye reagent and free phosphates at the beginning stage. A considerable level off occurred after 10 min until a maximum value was reached at 15min, suggesting the saturation of staining reaction. For the insurance of full color development, the minimum staining time will be 15min. In summary, the Malachite green method was finally optimized as follows: the concentration of ATP-Na2 was 500uM. After incubation with DnaB helicase (5-30nM) at 37 prepared dye reagent was added for further incubation at 37
for 8 min, freshly
for 15min, the ATPase reaction will
be terminated and the complex will be fully stained. The reaction solutions were finally transferred to 96-well plates and the absorptions were detected at the wavelength of 650nm.
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Fig.1 Optimization of the parameters. (A) The Absorption spectra of sodium dihydrogen phosphate from 550-750nm. (B) The Absorption of self-hydrolyzed ATP at 650nm with the concentration of 125, 250, 375, 500, 625, 875, 1125, 1500, 1875uM. (C) The Absorption at various reaction time (0, 2, 4, 6, 8, 10, 14, 18, 22, 26 and 30min) of ATP hydrolysis. Reactions were carried out in buffer A with 500uM ATP and 20nM DnaB, the staining time was 15min. (D) The Absorption at various staining time (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 18, 22 and 26min) of staining reaction. Reactions were carried out in buffer A with 500uM ATP and 20nM DnaB, the reaction time was 10min.
At the optimized condition, the ATPase activity of DnaBBst helicase was measured. Fig.2A showed the ATPase activity increased with the elevation of the concentration of DnaB helicases (5-30nM) . A linear fashion (R2=0.9830) was shown in Fig.2A, the curve was highly reproducible and stable in the DnaB concentration range of 5-30nM. Additionally, this method exhibited high sensitivity since the ATPase activity could be detected at the DnaB concentration as low as 5nM, compared with previously reported 24nM DnaB using enzyme coupled essays[1]
2.2 Investigations on the effect of DnaGBst , DnaIBsu and ssDNA on the ATPase activity of DnaB helicase
ACCEPTED MANUSCRIPT The malachite green ATPase assay was applied, for the first time, to investigate functional interaction between DnaB and related factors. The effect of DnaIBsu, DnaGBst and ssDNA on ATPase activity of DnaBBst helicase was studied. Firstly, the background signals of diverse concentrations of DnaG, DnaI and ssDNA were verified to be undetectable (data not shown). In the absence of additional components, DnaB exhibited the ATPase activity lower than 2 (s-1)
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(Fig.2B). Upon adding DnaGBst primase at concentrations range from 0 to 150nM (equivalent to the molar ratio of DnaG monomer and DnaB hexamer from 0 to 7.5:1), the ATPase activity increased rapidly at the DnaG concentrations range from 0 to 60nM and then increased slightly at higher concentrations, indicating a saturation of stimulatory effect of DnaG on ATPase activity at high concentration of DnaG (Fig.2B). This was consistent with biochemical and structural analysis that stimulation of DnaB’s ATPase activity[7, 16, 24, 25].
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three molecules of DnaG interacted with one molecule of DnaB hexamer, inducing a significantly
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A similar result was observed in the addition of ssDNA (0-25uM, equivalent to a 0 to 750-fold excess of ssDNA), the ATPase activity rose sharply until a maximum value of 4 s-1 was reached at 2.5uM concentration of ssDNA, then the ATPase activity didn’t increase anymore (Fig.2C). The stimulatory effect of ssDNA on ATPase activity of DnaB was in consistent with that in enzyme-coupled assays[14, 26], however, the assay here was much easier. Interestingly, high concentration of DnaIBsu (DnaI and DnaB with the molar ratio higher than
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3:1, DnaB referred to was monomer here) showed inhibition on the ATPase activity of DnaB, the increasing of the DnaI concentration resulted in further inhibition of ATPase activity (Fig.2D). However, at the molar ratio of 1:1, which was supposed to be functional in vivo by structural information[27-29], DnaI showed no inhibition effect on ATPase activity of DnaB(data not shown).
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This may be explained by an assumption of competition between DnaI and ATP, since ATP hydrolysis was supposed to promote release of DnaC (homologous to DnaIBst) from DnaB in E.coli , conversely, the binding of ATP to DnaB may be inhibited by high concentration of DnaI.
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[30, 31]
However, this assumption requires more work to be done.
3.Conclusions
In conclusion, the malachite green based colorimetric technique was well adopted to measuring ATPase activity of DnaBBst helicase with high efficiency. Additionally, this assay has potentials for investigating the interplay between DnaB and its partners and for high-throughput screening of helicase inhibitors with anti-bacterial activity as well.
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Fig.2 The application of the optimized malachite green method.
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(A) The ATPase activity of DnaBBst helicase. Reactions were carried out at the optimized condition with various concentrations of DnaB helicase (0, 5, 10, 15, 20, 25 and 30nM). (B) Effect of DnaGBst primase on ATPase activity of DnaB helicase, reactions were carried out in optimized condition in buffer A containing 20nM DnaB and various concentrations of DnaG (0, 10, 20, 30, 40, 50, 60, 100 and 150nM). (C) Effect of 14nt OligodT ssDNA on ATPase activity of DnaB helicase, reactions were carried out in optimized condition in buffer A containing 20nM DnaB and various concentrations of ssDNA (0, 0.5, 1, 1.5, 2.5, 12.5 and 25uM). (D) Effect of DnaIBsu on ATPase activity of DnaB helicase, reactions were carried out in optimized condition in buffer A containing 20nM DnaB and various concentrations of DnaIBsu (0, 180, 360, 540, 720 and 900nM).
Acknowledgments
This work was supported by the National Natural Science Foundation of China (31270783) and the initial Grants from The 100 Talents Program of the Chinese Academy of Sciences.
Appendix A. Supplementary data
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References
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
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Bird, L.E., et al., Mapping protein-protein interactions within a stable complex of DNA primase and DnaB helicase from Bacillus stearothermophilus. Biochemistry, 2000. 39(1): p. 171-82. Soni, R.K., et al., Helicobacter pylori DnaB helicase can bypass Escherichia coli DnaC function in vivo. Biochemical Journal, 2005. 389: p. 541-548. Thirlway, J. and P. Soultanas, In the Bacillus stearothermophilus DnaB-DnaG complex, the activities of the two proteins are modulated by distinct but overlapping networks of residues. J Bacteriol, 2006. 188(4): p. 1534-9. Chang, L., et al., High-throughput screen for small molecules that modulate the ATPase activity of the molecular chaperone DnaK. Anal Biochem, 2008. 372(2): p. 167-76. Lebowitz, J.H. and R. Mcmacken, The Escherichia-Coli Dnab Replication Protein Is a DNA Helicase. Journal of Biological Chemistry, 1986. 261(10): p. 4738-4748. Velten, M., et al., A two-protein strategy for the functional loading of a cellular replicative DNA helicase. Mol Cell, 2003. 11(4): p. 1009-20. Wang, G., et al., The structure of a DnaB-family replicative helicase and its interactions with primase. Nature Structural & Molecular Biology, 2008. 15(1): p. 94-100. D'Angelo, E., J. Crutchfield, and M. Vandiviere, Rapid, sensitive, microscale determination of phosphate in water and soil. J Environ Qual, 2001. 30(6): p. 2206-9. Havriluk, T., et al., Colorimetric determination of pure Mg(2+)-dependent phosphatidate phosphatase activity. Anal Biochem, 2008. 373(2): p. 392-4. Sherwood, A.R., et al., A malachite green-based assay to assess glucan phosphatase activity. Anal Biochem, 2013. 435(1): p. 54-6. Hyun, M., V.A. Bohr, and B. Ahn, Biochemical characterization of the WRN-1 RecQ helicase of Caenorhabditis elegans. Biochemistry, 2008. 47(28): p. 7583-93. Janscak, P., et al., Characterization and mutational analysis of the RecQ core of the bloom syndrome protein. J Mol Biol, 2003. 330(1): p. 29-42. Lin, H.H. and C.Y. Huang, Characterization of flavonol inhibition of DnaB helicase: real-time monitoring, structural modeling, and proposed mechanism. J Biomed Biotechnol, 2012. 2012: p. 735368. Thirlway, J., et al., DnaG interacts with a linker region that joins the N- and C-domains of DnaB and induces the formation of 3-fold symmetric rings. Nucleic Acids Res, 2004. 32(10): p. 2977-86. Bird, L.E. and D.B. Wigley, The Bacillus stearothermophilus replicative helicase: cloning, overexpression and activity. Biochim Biophys Acta, 1999. 1444(3): p. 424-8. Bailey, S., W.K. Eliason, and T.A. Steitz, Structure of hexameric DnaB helicase and its complex with a domain of DnaG primase. Science, 2007. 318(5849): p. 459-463. Itsathitphaisarn, O., et al., The hexameric helicase DnaB adopts a nonplanar conformation during translocation. Cell, 2012. 151(2): p. 267-77. Biswas, T., et al., A novel non-radioactive primase-pyrophosphatase activity assay and its application to the discovery of inhibitors of Mycobacterium tuberculosis primase DnaG. Nucleic Acids Res, 2013. 41(4): p. e56. Biswas, T., et al., DNA-dependent ATPase activity of bacterial XPB helicases. Biochemistry, 2009. 48(12): p. 2839-48. Bernal, C., et al., A colorimetric assay for the determination of 4-diphosphocytidyl-2-C-methyl-D-erythritol 4-phosphate synthase activity. Analytical Biochemistry, 2005. 337(1): p. 55-61. Repen, B., E. Schneider, and U. Alexiev, Optimization of a malachite green assay for detection of ATP hydrolysis by solubilized membrane proteins. Anal Biochem, 2012. 426(2): p. 103-5. Wu, Z.L., Phosphatase-coupled universal kinase assay and kinetics for first-order-rate coupling reaction. PLoS One, 2011. 6(8): p. e23172. Monroy, C.A., D.I. Mackie, and D.L. Roman, A high throughput screen for RGS proteins using steady state monitoring of free phosphate formation. PLoS One, 2013. 8(4): p. e62247. Marians, K.J., Understanding how the replisome works. Nat Struct Mol Biol, 2008. 15(2): p. 125-7. Soultanas, P., The bacterial helicase-primase interaction: a common structural/functional module. Structure, 2005. 13(6): p. 839-44. Chintakayala, K., et al., Conserved residues of the C-terminal p16 domain of primase are involved in modulating the activity of the bacterial primosome. Mol Microbiol, 2008. 68(2): p. 360-71. Liu, B., W.K. Eliason, and T.A. Steitz, Structure of a helicase-helicase loader complex reveals insights into the mechanism of bacterial primosome assembly. Nat Commun, 2013. 4: p. 2495. Soultanas, P., Loading mechanisms of ring helicases at replication origins. Mol Microbiol, 2012. 84(1): p. 6-16. Arias-Palomo, E., et al., The bacterial DnaC helicase loader is a DnaB ring breaker. Cell, 2013. 153(2): p. 438-48.
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30. Weller, S.K. and R.D. Kuchta, The DNA helicase-primase complex as a target for herpes viral infection. Expert Opin Ther Targets, 2013. 17(10): p. 1119-32. 31. Wahle, E., R.S. Lasken, and A. Kornberg, The dnaB-dnaC replication protein complex of Escherichia coli. I. Formation and properties. J Biol Chem, 1989. 264(5): p. 2463-8.
S0003-2697(16)30163-4
DOI:
10.1016/j.ab.2016.06.028
Reference:
YABIO 12430
To appear in:
Analytical Biochemistry
Received Date: 2 May 2016 Revised Date:
24 June 2016
Accepted Date: 27 June 2016
Please cite this article as: M. Yang, G. Wang, ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method, Analytical Biochemistry (2016), doi: 10.1016/j.ab.2016.06.028. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
ATPase Activity Measurement of DNA Replicative Helicase from Bacillus stearothermophilus by Malachite Green Method Mu Yang a,b,c, Ganggang Wang a,b* a
Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of
Sciences, Chengdu, 610041, China;
c
Key Laboratory of Environmental Microbiology of Sichuan Province, Chengdu, 610041, China;
University of Chinese Academy of Sciences, Beijing, 100049, China.
*Corresponding author’s:
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SEND CORRESPONDENCE TO:
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b
Ganggang Wang
Sciences, Chengdu, 610041, China
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Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of
Tel: 86-28-82890828; E-mail: [email protected]
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Subject Category: Electrophoresis Enzymatic assays and analysis
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Abstract The DnaB helicase from Bacillus stearothermophilus (DnaBBst) was a model protein for studying the bacterial DNA replication. In this work, a non-radioactive method for measuring ATPase activity of DnaBBst helicase was described. The working parameters and conditions were optimized. Furthermore, this method was applied to investigate effects of DnaG primase, ssDNA and
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helicase loader protein (DnaI) on ATPase activity of DnaBBst. Our results showed this method was sensitive and efficient. Moreover, it is suitable for the investigation of functional interaction between DnaB and related factors.
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Key words: ATPase Activity; DnaB; Malachite Green; Optimization
ACCEPTED MANUSCRIPT 1. Introduction DnaB protein is a ring-shaped RecA-type helicase that unwinds duplex DNA at replication fork by utilizing the energy of ATP hydrolysis[1-3]. The measurement on ATPase activity of DnaB helicase is crucial for deeper understanding the function of DnaB helicase in DNA replication as well
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as screening for specific inhibitors of bacterial DnaB[4, 5]. Traditionally,γ-32P radiolabelled ATP was used as the substrate for measuring ATPase activity of DnaB helicase and homologous proteins, after reaction, the released
32
Pi was separated using
thin-layer chromatography, followed by autoradiography and then quantified. This method was [6, 7]
SC
relatively inefficient and time-consuming, especially for the application to high-throughput screening . Moreover, rigorous safety measures should be applied to prevent experimenters from
radioactive contamination. In this way, the high cost, low efficiency and the harm of the radiation
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limited the wider application of radioactive method.
The malachite green method was a non-radioactive procedure for determination of phosphate released from ATP hydrolysis. This method was widely used to analyze the organic and inorganic phosphate in water as well as the activities of various phosphatases for advantages of being efficient, sensitive, cost effective and nonhazardous[4, 8-10].
This method was also applied to characterize ATPase activities of helicases like WRN-1 RecQ
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helicase[11, 12] and DnaB helicase from Klebsiell apneumoniae[13]. However, the working conditions were variable in previous reports, it was essential to optimize the parameters of ATPase activity assay. The DnaB helicase from Bacillus stearothermophilus (DnaBBst) was a model protein for studying the bacterial DNA replication[1, 14-17]. Up till now, there still lacks a detailed report on the
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ATPase activity of DnaBBst helicase measured by malachite green method. In this study, the malachite green method was applied to determine the ATPase activity of
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DnaBBst. Several parameters were optimized for high-throughput measurement. In addition, under the optimized condition, this method was applied for the first time to study on the functional interaction between DnaB and other related factors (DnaG primase, DnaI helicase loader and ssDNA,). Results showed that the optimized malachite green method was efficient in measuring the ATPase activity of DnaB helicase from B.stearothermophilus. It was also suitable for studying the interplay between DnaB and its partners. Moreover, this method might be used for high-throughput screening of specific DnaB inhibitors.
2.Results and discussions 2.1 Optimization of the parameters of Malachite Green Method In an effort to apply the Malachite green-Molybdate method for efficiently measuring ATPase activity of DnaBBst helicase, the maximum absorption wavelength was firstly determined (Fig.1A).
ACCEPTED MANUSCRIPT Fig. 1A showed the absorption spectra of standard sodium dihydrogen phosphate, in complex with dye reagent mentioned above, from the wavelength of 550 to 750nm. The spectra exhibited a characteristic absorption peak approximately at 650nm, which deviated slightly from previously proposed 620nm[18, 19] and 630nm[20]. Then the standard curve was determined in the 0-300uM range of sodium dihydrogen phosphate (Fig. S1). The standard curve was linear (R2=0.9929) between 0
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and 300uM phosphate concentrations to guarantee accuracy measurement of absorbances between 0.2 and 1. It was highly reproducible with the coefficient of variation less than 3%.
The self-hydrolysis of ATP in the reaction buffer led to remarkable background absorbance[21, 22]
, here the ATP concentration was optimized to minimize the background. As shown in Fig.1B, at
low concentrations of ATP-Na2 between 0 and 500uM, the background signals were negligible. In
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contrast, at concentrations of ATP-Na2 higher than 500uM, the absorbances increased up dramatically. The high background may severely influence the accuracy of measurement. Based on
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these results, 500uM ATP-Na2 was selected for further optimization.
The next step was the application of this method to ATPase activity of DnaB helicase. Firstly, the background signals of various concentrations of DnaB was investigated and confirmed to be minimal (data not shown). Besides, the acidic malachite green-molybdate straining reagent was also proved to be effective to terminate ATPase reaction at 37
(data not shown). Then the optimum
reaction time of ATP hydrolysis by helicase was investigated (Fig.1C). The variation of absorbance
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with time of ATPase reaction was exhibited in Fig.1C. The absorbance at 650nm increased considerably within the first 15 min, followed by gradually increasing until the plateau was reached at 22 min. In pursuit of high efficiency and accuracy for the measurement of initial velocity, a reaction time of 8 min was adopted for further investigations.
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The ultimate colorimetric complex is formed after the formation of phosphomolybdate, indicating enough time is required for full color development[23]. Hence, the staining time was
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optimized (Fig.1D). As shown in Fig.1D, the curve of absorbance increased sharply within 8 min, indicating a rapid chemical reaction between dye reagent and free phosphates at the beginning stage. A considerable level off occurred after 10 min until a maximum value was reached at 15min, suggesting the saturation of staining reaction. For the insurance of full color development, the minimum staining time will be 15min. In summary, the Malachite green method was finally optimized as follows: the concentration of ATP-Na2 was 500uM. After incubation with DnaB helicase (5-30nM) at 37 prepared dye reagent was added for further incubation at 37
for 8 min, freshly
for 15min, the ATPase reaction will
be terminated and the complex will be fully stained. The reaction solutions were finally transferred to 96-well plates and the absorptions were detected at the wavelength of 650nm.
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ACCEPTED MANUSCRIPT
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Fig.1 Optimization of the parameters. (A) The Absorption spectra of sodium dihydrogen phosphate from 550-750nm. (B) The Absorption of self-hydrolyzed ATP at 650nm with the concentration of 125, 250, 375, 500, 625, 875, 1125, 1500, 1875uM. (C) The Absorption at various reaction time (0, 2, 4, 6, 8, 10, 14, 18, 22, 26 and 30min) of ATP hydrolysis. Reactions were carried out in buffer A with 500uM ATP and 20nM DnaB, the staining time was 15min. (D) The Absorption at various staining time (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 18, 22 and 26min) of staining reaction. Reactions were carried out in buffer A with 500uM ATP and 20nM DnaB, the reaction time was 10min.
At the optimized condition, the ATPase activity of DnaBBst helicase was measured. Fig.2A showed the ATPase activity increased with the elevation of the concentration of DnaB helicases (5-30nM) . A linear fashion (R2=0.9830) was shown in Fig.2A, the curve was highly reproducible and stable in the DnaB concentration range of 5-30nM. Additionally, this method exhibited high sensitivity since the ATPase activity could be detected at the DnaB concentration as low as 5nM, compared with previously reported 24nM DnaB using enzyme coupled essays[1]
2.2 Investigations on the effect of DnaGBst , DnaIBsu and ssDNA on the ATPase activity of DnaB helicase
ACCEPTED MANUSCRIPT The malachite green ATPase assay was applied, for the first time, to investigate functional interaction between DnaB and related factors. The effect of DnaIBsu, DnaGBst and ssDNA on ATPase activity of DnaBBst helicase was studied. Firstly, the background signals of diverse concentrations of DnaG, DnaI and ssDNA were verified to be undetectable (data not shown). In the absence of additional components, DnaB exhibited the ATPase activity lower than 2 (s-1)
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(Fig.2B). Upon adding DnaGBst primase at concentrations range from 0 to 150nM (equivalent to the molar ratio of DnaG monomer and DnaB hexamer from 0 to 7.5:1), the ATPase activity increased rapidly at the DnaG concentrations range from 0 to 60nM and then increased slightly at higher concentrations, indicating a saturation of stimulatory effect of DnaG on ATPase activity at high concentration of DnaG (Fig.2B). This was consistent with biochemical and structural analysis that stimulation of DnaB’s ATPase activity[7, 16, 24, 25].
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three molecules of DnaG interacted with one molecule of DnaB hexamer, inducing a significantly
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A similar result was observed in the addition of ssDNA (0-25uM, equivalent to a 0 to 750-fold excess of ssDNA), the ATPase activity rose sharply until a maximum value of 4 s-1 was reached at 2.5uM concentration of ssDNA, then the ATPase activity didn’t increase anymore (Fig.2C). The stimulatory effect of ssDNA on ATPase activity of DnaB was in consistent with that in enzyme-coupled assays[14, 26], however, the assay here was much easier. Interestingly, high concentration of DnaIBsu (DnaI and DnaB with the molar ratio higher than
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3:1, DnaB referred to was monomer here) showed inhibition on the ATPase activity of DnaB, the increasing of the DnaI concentration resulted in further inhibition of ATPase activity (Fig.2D). However, at the molar ratio of 1:1, which was supposed to be functional in vivo by structural information[27-29], DnaI showed no inhibition effect on ATPase activity of DnaB(data not shown).
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This may be explained by an assumption of competition between DnaI and ATP, since ATP hydrolysis was supposed to promote release of DnaC (homologous to DnaIBst) from DnaB in E.coli , conversely, the binding of ATP to DnaB may be inhibited by high concentration of DnaI.
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[30, 31]
However, this assumption requires more work to be done.
3.Conclusions
In conclusion, the malachite green based colorimetric technique was well adopted to measuring ATPase activity of DnaBBst helicase with high efficiency. Additionally, this assay has potentials for investigating the interplay between DnaB and its partners and for high-throughput screening of helicase inhibitors with anti-bacterial activity as well.
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Fig.2 The application of the optimized malachite green method.
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(A) The ATPase activity of DnaBBst helicase. Reactions were carried out at the optimized condition with various concentrations of DnaB helicase (0, 5, 10, 15, 20, 25 and 30nM). (B) Effect of DnaGBst primase on ATPase activity of DnaB helicase, reactions were carried out in optimized condition in buffer A containing 20nM DnaB and various concentrations of DnaG (0, 10, 20, 30, 40, 50, 60, 100 and 150nM). (C) Effect of 14nt OligodT ssDNA on ATPase activity of DnaB helicase, reactions were carried out in optimized condition in buffer A containing 20nM DnaB and various concentrations of ssDNA (0, 0.5, 1, 1.5, 2.5, 12.5 and 25uM). (D) Effect of DnaIBsu on ATPase activity of DnaB helicase, reactions were carried out in optimized condition in buffer A containing 20nM DnaB and various concentrations of DnaIBsu (0, 180, 360, 540, 720 and 900nM).
Acknowledgments
This work was supported by the National Natural Science Foundation of China (31270783) and the initial Grants from The 100 Talents Program of the Chinese Academy of Sciences.
Appendix A. Supplementary data
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References
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