Kinetic Analysis of <i>Escherichia Coli</i> Ribonuclease HI Using Oligomeric DNA/RNA Substrates Suggests an Alternative Mechanism for the Interaction between the Enzyme and the Substrate

Kanaya, Eiko; Kanaya, Shigenori

doi:10.1111/j.1432-1033.1995.0557d.x

Cited by 48 publications

(25 citation statements)

References 30 publications

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“…Thus, the observed values will always be lower than the maximum values. Therefore, our current results are consistent with a 9 -10-bp binding size (22,38).…”

Section: Discussionsupporting

confidence: 82%

“…Kinetic analyses, under conditions in which the RNA-DNA hybrid was cleaved at a unique site (22), or using RNA-DNA hybrids with 2Ј-O-methylnucleosides, which limits the cleavage at a single site (38), suggest that RNase HI interacts with 9 -10 bp of RNA-DNA hybrid. Using these values, one can expect the maximum number of RNase HI molecules able to bind to the 18-, 24-, 30-, and 36-bp hybrid to be 1-2, 2, 3, and 3-4, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, kinetic analyses using RNA-DNA hybrids, under conditions in which the hybrid was cleaved at a unique site (22), suggest that DNA residues complementary to the RNA residues located six or seven residues upstream of the cleavage site interact with the basic protrusion region of the enzyme. Such an interaction seems to require a conformational change in the enzyme or substrate, or in both.…”

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confidence: 99%

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Kinetic and Stoichiometric Analysis for the Binding of Escherichia coli Ribonuclease HI to RNA-DNA Hybrids Using Surface Plasmon Resonance

Haruki¹,

Noguchi²,

Kanaya³

et al. 1997

Journal of Biological Chemistry

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To understand how ribonucleases H recognize RNA-DNA hybrid substrates, we analyzed kinetic parameters of binding of Escherichia coli RNase HI to RNA-DNA hybrids ranging in length from 18 to 36 base pairs (bp) using surface plasmon resonance (BIAcore). The k on and k off values for the binding of the enzyme to the 36-bp substrate were 1. 5 (5) in the catalytic function was established by site-directed mutagenesis for the catalytic function of the enzyme. Two alternative mechanisms have been proposed: one is a two-metal ion mechanism (6) and the other is a carboxyl-hydroxyl relay mechanism (4, 7-9).X-ray crystallographic analyses of E. coli RNase HI (6, 10, 11) and the RNase H domain of HIV-1 1 reverse transcriptase (12) showed that these RNases H have a similar structural topology, with the exception of the presence of a handle (6) or basic protrusion region (11) in E. coli RNase HI. The importance of this region for substrate binding has been demonstrated in several studies. The RNase HI domain isolated from HIV-1 reverse transcriptase is enzymatically inactive (13,14), whereas that from murine leukemia virus reverse transcriptase, which has a part of this region, is active (15, 16). Site-directed mutagenesis indicated that the positively charged residues in this region are important for substrate binding (17). Incorporation of the basic protrusion of E. coli RNase HI at the equivalent position of the RNase H domain of HIV-1 reverse transcriptase resulted in the production of the active HIV-1 RNase H domain (18,19 However, it is not fully understood how RNase H binds to its substrate. A kinetic study using synthetic nucleosides with modifications of their 2Ј-hydroxyl groups revealed the importance of the 2Ј-hydroxyl group of the nucleoside on the 3Ј-side of the cleaved phosphodiester and that of the second nucleoside 5Ј to the cleaved phosphodiester for hydrogen bonding (20). Models for the binding of the enzyme to an RNA-DNA hybrid have been proposed based upon computer docking of the structure of E. coli RNase HI (free from its substrate) with an RNA-DNA hybrid whose structure was assumed to be an A form (6, 11), was found by NMR to be an A form (7), or that was neither A nor B (21). In these models, the RNA strand upstream of the cleavage site interacts with the enzyme. None of these models assumes that either the enzyme or the substrate alters its conformation upon binding.Recently, kinetic analyses using RNA-DNA hybrids, under conditions in which the hybrid was cleaved at a unique site (22), suggest that DNA residues complementary to the RNA residues located six or seven residues upstream of the cleavage site interact with the basic protrusion region of the enzyme. Such an interaction seems to require a conformational change in the enzyme or substrate, or in both. Determination of kinetic parameters and stoichiometry of RNase HI molecules bound to substrates of various lengths would provide additional information about the binding of enzyme to substrate.Magnesium ions may also modulate protein-nucleic acid ...

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“…Thus, the observed values will always be lower than the maximum values. Therefore, our current results are consistent with a 9 -10-bp binding size (22,38).…”

Section: Discussionsupporting

confidence: 82%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Kinetic and Stoichiometric Analysis for the Binding of Escherichia coli Ribonuclease HI to RNA-DNA Hybrids Using Surface Plasmon Resonance

Haruki¹,

Noguchi²,

Kanaya³

et al. 1997

Journal of Biological Chemistry

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“…(The reason for including at least part of this cost is that with Asp132 unprotonated, process I did not complete in our calculations.) For a kinetic prefactor of ~1/ps, the experimental rates of ~100 min −1 and ~1 min −1 for E. coli 37,38 and human 37,39 RNase H1 at 37°C correspond to barrier heights of ~16.7 and ~19.5 kcal mol −1 , respectively, assuming further that product release is fast. We expect the rate and barrier of the Bh enzyme to be closer to that of the faster E. coli RNase H1.…”

Section: Resultsmentioning

confidence: 99%

Catalytic Mechanism of RNA Backbone Cleavage by Ribonuclease H from Quantum Mechanics/Molecular Mechanics Simulations

Rosta¹,

Nowotny

Yang³

et al. 2011

J. Am. Chem. Soc.

180

306

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We use quantum mechanics/molecular mechanics (QM/MM) simulations to study the cleavage of the ribonucleic acid (RNA) backbone catalyzed by ribonuclease H. This protein is a prototypical member of a large family of enzymes that use two-metal catalysis to process nucleic acids. By combining Hamiltonian replica exchange with a finite-temperature string method, we calculate the free energy surface underlying the RNA cleavage reaction and characterize its mechanism. We find that the reaction proceeds in two steps. In a first step, catalyzed primarily by magnesium ion A and its ligands, a water molecule attacks the scissile phosphate. Consistent with thiol-substitution experiments, a water proton is transferred to the downstream phosphate group. The transient phosphorane formed as a result of this nucleophilic attack decays by breaking the bond between the phosphate and the ribose oxygen. In the resulting intermediate, the dissociated but unprotonated leaving group forms an alkoxide coordinated to magnesium ion B. In a second step, the reaction is completed by protonation of the leaving group, with a neutral Asp132 as a likely proton donor. The overall reaction barrier of ~15 kcal mol−1, encountered in the first step, together with the cost of protonating Asp132, is consistent with the slow measured rate of ~1–100/min. The two-step mechanism is also consistent with the bell-shaped pH dependence of the reaction rate. The non-monotonic relative motion of the magnesium ions along the reaction pathway agrees with X-ray crystal structures. Proton transfer reactions and changes in the metal ion coordination emerge as central factors in the RNA cleavage reaction.

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“…Traditional techniques, including gel electrophoresis, high-performance liquid chromatography (HPLC), electrochemical studies, and the acid-soluble release of RNA fragments have been established in assays of enzyme activities and evaluations of the kinetic parameters. [11][12][13][14] These protocols share the drawbacks of being time-intensive, discontinuous, and laborious, and usually require isotope labeling. Many of these limitations are now being addressed by the development of fluorescence assays based on resonance energy transfer (FRET) or an excimer mechanism.…”

Section: Introductionmentioning

confidence: 99%

A Quadruplex‐Based, Label‐Free, and Real‐Time Fluorescence Assay for RNase H Activity and Inhibition

Huang

et al. 2010

Chemistry A European J

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We demonstrate a unique quadruplex-based fluorescence assay for sensitive, facile, real-time, and label-free detection of RNase H activity and inhibition by using a G-quadruplex formation strategy. In our approach, a RNA-DNA substrate was prepared, with the DNA strand designed as a quadruplex-forming oligomer. Upon cleavage of the RNA strand by RNase H, the released G-rich DNA strand folds into a quadruplex in the presence of monovalent ions and interacts with a specific G-quadruplex binder, N-methyl mesoporphyrin IX (NMM); this gives a dramatic increase in fluorescence and serves as a reporter of the reaction. This novel assay is simple in design, fast in operation, and is more convenient and promising than other methods. It takes less than 30 min to finish and the detection limit is much better or at least comparable to previous reports. No sophisticated experimental techniques or chemical modification for either RNA or DNA are required. The assay can be accomplished by using a common spectrophotometer and obviates possible interference with the kinetic behavior of the catalysts. Our approach offers an ideal system for high-throughput screening of enzyme inhibitors and demonstrates that the structure of the G-quadruplex can be used as a functional tool in specific fields in the future.

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Kinetic Analysis of Escherichia Coli Ribonuclease HI Using Oligomeric DNA/RNA Substrates Suggests an Alternative Mechanism for the Interaction between the Enzyme and the Substrate

Cited by 48 publications

References 30 publications

Kinetic and Stoichiometric Analysis for the Binding of Escherichia coli Ribonuclease HI to RNA-DNA Hybrids Using Surface Plasmon Resonance

Kinetic and Stoichiometric Analysis for the Binding of Escherichia coli Ribonuclease HI to RNA-DNA Hybrids Using Surface Plasmon Resonance

Catalytic Mechanism of RNA Backbone Cleavage by Ribonuclease H from Quantum Mechanics/Molecular Mechanics Simulations

A Quadruplex‐Based, Label‐Free, and Real‐Time Fluorescence Assay for RNase H Activity and Inhibition

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