• Bulletin of the Korean Chemical Society
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• 제27권2호
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• pp.224-230
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• 2006

Escherichia coli transcription termination factor Rho catalyzes the unwinding of RNA/DNA duplex in reactions that are coupled to ATP binding and hydrolysis. We report here the kinetic mechanism of presteady state ATP binding and hydrolysis by the Rho-RNA complex. Presteady state chemical quenched-flow technique under multiple turnover condition was used to probe the kinetics of ATP binding and hydrolysis by the Rho-RNA complex. The quenched-flow presteady state kinetics of ATP hydrolysis studies show that three ATPs are bound to the Rho-RNA complex with a rate of $4.4\;{\times}\;10^5M^{-1}s^{-1}$, which are subsequently hydrolyzed at a rate of $88s^{-1}$ and released during the initiation process. Global fit of the presteady state ATP hydrolysis kinetic data suggests that a rapid-equilibrium binding of ATP to Rho-RNA complex occurs prior to the first turnover and the chemistry step is not reversible. The initial burst of three ATPs hydrolysis was proposed to be involved in the initialization step that accompanies proper complex formation of Rho-RNA. Based on these results a kinetic model for initiation process for Rho-RNA complex was proposed relating the mechanism of ATP binding and hydrolysis by Rho to the structural transitions of Rho-RNA complex to reach the steady state phase, which is implicated during translocation along the RNA.

• Bulletin of the Korean Chemical Society
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• 제30권8호
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• pp.1724-1728
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• 2009

Severe acute respiratory syndrome coronavirus (SARS-CoV) helicase separates the double-stranded nucleic acids using the energy from ATP hydrolysis. We have measured ATPase activity of SARS-CoV helicase in the presence of various types of nucleic acids. Steady state ATPase analysis showed that poly(U) has two-times higher turnover number than poly(C) with lower Michaelis constant. When M13 single-stranded DNA is used as substrate, the Michaelis constant was about twenty-times lower than poly(U), whereas turnover numbers were similar. However, stimulation of ATPase activity was not observed in the presence of double-stranded DNA. pH dependent profiles of ATP hydrolysis with the helicase showed that the optimal ATPase activities were in a range of pH 6.2 ~ 6.6. In addition, ATP hydrolysis activity assays performed in the presence of various divalent cations exhibited that $Mg^{2+}$ stimulated the ATPase activity with the highest rate and $Mn^{2+}$ with about 40% rate as compared to the $Mg^{2+}$.

• Journal of Microbiology
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• 제37권2호
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• pp.59-65
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• 1999

RecA protein can promote strand assimilation, homologous pairing, and strand exchange. All these reactions require DNA-dependent ATP hydrolysis by recA protein, and the activities of recA protein are affected by the ionic environment. In this experiment, DNA-dependent ATPase activity showed different sensitivity to anionic species. ATP hydrolysis and strand exchange were relatively sensitive to salt in the reactions with NaCl, strongly inhibited at 100 mM NaCl. However, the inhibition by sodium acetate or sodium glutamate was not observed at 50∼100 mM concentration. Addition of sodium glutamate to the standard reaction condition increased the apparent efficiency of ATP hydrolysis during strand exchange. The condition including 50∼100 mM sodium-glutamate might be similar to the physiological condition.

• 대한약학회:학술대회논문집
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• 대한약학회 2002년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2
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• pp.336.1-336.1
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• 2002

HSP100/Clp family functions as molecular chaperone and ATP dependent protease. The Streptococcus pneumoniae ClpL. a homologue of bacterial ClpB and yeast cytosolic HSP 104. is one of major heat shock proteins but its biochemical properties are unknown. In this study. ClpL in Streptococcus pneumoniaewas characterized using histidine tagged recombinant ClpL. When ATP hydrolysis activity was compared in the presence or absence of a variety of nucleotides or divalent ions. either ATP or Mn$2^＋$ ion was found to increase significantly the rate of ATP hydrolysis. Furthermore. glutaraldehyde cross-linking and subsequent native-PAGE analfysis showed that ClpL forms dimer. but in the presence of 4 mM concentration of $Mn^{2＋}$ion as a cofactor for ATP hydrolysis and oligormerization in vitro.

• 미생물학회지
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• 제46권4호
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• pp.313-318
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• 2010

D. radiodurans RecA 단백질은 DNA에 결합한 DNA-단백질 복합체만이 ATPase 활성을 나타내며. 보통의 낮은 염 농도조건에서는 DNA가 존재하지 않으면 RecA 단백질에 의한 ATP 가수분해는 거의 일어나지 않았으나 이러한 ATP 가수분해현상은 높은 농도의 염을 첨가하게 되면 1,000배 활성화 되었으며 1.6 M KCl이 존재할 때 ATP 혹은 dATP를 가수분해 하였다. DNA가 존재하지 않을 때 염에 의해 촉진되는 활성은 RecA 단백질 농도에 비례하였고, 더 높은 염농도에서 더 높은 ATP 가수분해 활성이 나타났다. 이러한 활성화 현상을 다양한 종류의 이온 형태에서 분석하였을 때 1.6 M Cl 음이온이 존재할 때 양이온의 형태에 따른 활성화 정도는 $K^+{\geq}Na^+$> $NH_4^+$의 경향을 보였으며, 1.6 M의 K 양이온 존재할 때 음이온의 형태에 따른 활성화는 glutamate > $Cl^-$ > acetate > $PO_4^-$의 순서로 높게 나타났다. 고농도의 염이 존재하는 조건에서 DNA 비의존성 ATPase의 활성은 비교적 넓은 범위 최적 조건인 pH7과 pH 8 사이에서 최대 활성을 보였고, 기질에 대한 친화도면에서도 외가닥 DNA 의존성 활성보다는 이중가닥 DNA 의존성 활성형태를 보였다. 고농도의 염이 첨가되고 DNA가 존재하지 않을 때 RecA 단백질에 의한 ATP 가수분해를 위한 RecA 단백질의 활성 종 형태는 최소 3개의 RecA 단백질이 결합되어 있는 과량체로 작용하는 것으로 나타났다.

• Journal of Photoscience
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• 제2권1호
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• pp.27-29
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• 1995

Site directed mutagenesis has been introduced to determine active site(s) and molecular structure of E. coli RecA protein. Recombinant DNAs were constructed by point mutation of Tyr-264 to Phe which assumed active site for binding and hydrolysis of ATP. RecA proteins were purified from recombinants containing wild type and mutant genes and analyzed for ATPase activity assay. Result suggests that Tyr-264 is involved in ATP binding rather than ATP hydrolysis.

• Journal of Microbiology
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• 제44권5호
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• pp.508-514
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• 2006

In this study, the double-stranded DNA-dependent activities of Deinococcus radiodurans RecA protein (Dr RecA) were characterized. The interactions of the Dr RecA protein with double-stranded DNA were determined, especially dsDNA-dependent ATP hydrolysis by the Dr RecA protein and the DNA strand exchange reaction, in which multiple branch points exist on a single RecA protein-DNA complex. A nucleotide cofactor (ATP or dATP ) was required for the Dr RecA protein binding to duplex DNA. In the presence of dATP, the nucleation step in the binding process occurred more rapidly than in the presence of ATP. Salts inhibited the binding of the Dr RecA protein to double-stranded DNA. Double-stranded DNA-dependent ATPase activities showed a different sensitivity to anion species. Glutamate had only a minimal effect on the double-stranded DNA-dependent ATPase activities, up to a concentration of 0.7 M. In the competition experiment for Dr RecA protein binding, the Dr RecA protein manifested a higher affinity to double-stranded DNA than was observed for single-stranded DNA.

• Journal of Microbiology and Biotechnology
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• 제25권12호
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• pp.2007-2010
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• 2015

A new chemical inhibitor against severe acute respiratory syndrome (SARS) coronavirus helicase, 7-ethyl-8-mercapto-3-methyl-3,7-dihydro-1H-purine-2,6-dione, was identified. We investigated the inhibitory effect of the compound by conducting colorimetry-based ATP hydrolysis assay and fluorescence resonance energy transfer-based double-stranded DNA unwinding assay. The compound suppressed both ATP hydrolysis and double-stranded DNA unwinding activities of helicase with IC₅₀ values of 8.66 ± 0.26 μM and 41.6 ± 2.3 μM, respectively. Moreover, we observed that the compound did not show cytotoxicity up to 80 μM concentration. Our results suggest that the compound might serve as a SARS coronavirus inhibitor.

• Journal of Microbiology and Biotechnology
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• 제27권11호
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• pp.2070-2073
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• 2017

We have discovered a novel chemical compound, (E)-3-(furan-2-yl)-N-(4-sulfamoylphenyl) acrylamide, that suppresses the enzymatic activities of SARS coronavirus helicase. To determine the inhibitory effect, ATP hydrolysis and double-stranded DNA unwinding assays were performed in the presence of various concentrations of the compound. Through these assays, we obtained $IC_{50}$ values of $2.09{\pm}0.30{\mu}M$ (ATP hydrolysis) and $13.2{\pm}0.9{\mu}M$ (DNA unwinding), respectively. Moreover, we found that the compound did not have any significant cytotoxicity when $40{\mu}M$ of it was used. Our results showed that the compound might be useful to be developed as an inhibitor against SARS coronavirus.

• Molecules and Cells
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• 제44권2호
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• pp.79-87
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• 2021

Chromatin dynamics is essential for maintaining genomic integrity and regulating gene expression. Conserved bromodomain-containing AAA+ ATPases play important roles in nucleosome organization as histone chaperones. Recently, the high-resolution cryo-electron microscopy structures of Schizosaccharomyces pombe Abo1 revealed that it forms a hexameric ring and undergoes a conformational change upon ATP hydrolysis. In addition, single-molecule imaging demonstrated that Abo1 loads H3-H4 histones onto DNA in an ATP hydrolysis-dependent manner. However, the molecular mechanism by which Abo1 loads histones remains unknown. Here, we investigated the details concerning Abo1-mediated histone loading onto DNA and the Abo1-DNA interaction using single-molecule imaging techniques and biochemical assays. We show that Abo1 does not load H2A-H2B histones. Interestingly, Abo1 deposits multiple copies of H3-H4 histones as the DNA length increases and requires at least 80 bp DNA. Unexpectedly, Abo1 weakly binds DNA regardless of ATP, and neither histone nor DNA stimulates the ATP hydrolysis activity of Abo1. Based on our results, we propose an allosteric communication model in which the ATP hydrolysis of Abo1 changes the configuration of histones to facilitate their deposition onto DNA.