How the EcoRI endonuclease recognizes and cleaves DNA

Heitman, Joseph

doi:10.1002/bies.950140704

Cited by 52 publications

(44 citation statements)

References 74 publications

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“…Different mechanisms have been proposed for the action of class II restriction endonucleases: either the reactions can occur simultaneously on each strand, or the two reactions occur sequentially, with or without the release of an intermediate. Several mechanistic studies have revealed that EcoRI usually cuts both strands sequentially without releasing an intermediate, and that the dissociation of the final product is the rate-limiting step (23,24), although altered reaction kinetics have been observed that depend on the substrate characteristics as well as on the assay conditions (24-29). The method described in this study detects cleavage reactions by observing the separation of two different fluorescent labels, resulting from scissions in both strands of a single molecule; consequently, the kinetics that is observed refers to an overall reaction rate.…”

Section: Resultsmentioning

confidence: 99%

Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy

Kettling

Koltermann

Schwille

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

291

224

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A method for sensitively monitoring enzyme kinetics and activities by using dual-color f luorescence crosscorrelation spectroscopy is described. This universal method enables the development of highly sensitive and precise assays for real-time kinetic analyses of any catalyzed cleavage or addition reaction, where a chemical linkage is formed or cleaved through an enzyme's action between two f luorophores that can be discriminated spectrally. In this work, a homogeneous assay with restriction endonuclease EcoRI and a 66-bp double-stranded DNA containing the GAATTC recognition site and f luorophores at each 5 end is described. The enzyme activity can be quantified down to the low picomolar range (>1.6 pM) where the rate constants are linearly dependent on the enzyme concentrations over two orders of magnitude. Furthermore, the reactions were monitored online at various initial substrate concentrations in the nanomolar range, and the reaction rates were clearly represented by the Michaelis-Menten equation with a K M of 14 ؎ 1 nM and a k cat of 4.6 ؎ 0.2 min ؊1 . In addition to kinetic studies and activity determinations, it is proposed that enzyme assays based on the dual-color f luorescence cross-correlation spectroscopy will be very useful for high-throughput screening and evolutionary biotechnology.Kinetic studies on enzymes are among the most important tools for understanding biological interactions at the molecular level. In combination with new approaches in genetic engineering and structure determination, there have been major efforts in recent years to develop more sensitive and precise techniques for characterizing the kinetics of enzyme reactions. These techniques have made it possible to develop efficient assays for analyzing catalytic parameters such as turnover rates, substrate specificity, as well as regio-and stereospecificity; they constitute a major part of the biochemical and pharmaceutical research. Moreover, the alteration of catalytic properties, either by evolutionary biotechnology (1-4) or by rational approaches, is of special interest for medical and industrial applications. The desired features are as follows: accelerated reaction rates; higher, relaxed, or even new substrate specificities; tolerance to nonnatural environments; and novel types of reactions. Modern rational design is the interplay between computer modeling and experimental testing of designed molecules by sensitive and quantitative assays. On the other hand, evolutionary approaches make use of rapid and highly sensitive screening assays to detect minute quantities of better adapted individuals among a large excess of alternatives. For all these purposes, fluorescence correlation spectroscopy (FCS) (5-8), with its ability to quantify molecular interactions at nanomolar and lower concentrations, has been proposed as an ideal tool (2). Most contemporary applications of FCS that are performed in confocal microscope (9) are based on the analysis of the molecular dynamics and the reaction kinetics of fluorescently labeled ...

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Section: Resultsmentioning

confidence: 99%

Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy

Kettling

Koltermann

Schwille

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

291

224

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“…The question of whether protein conformational changes occur during EcoRI catalysis has been unresolved for several years (10,57,58). The magnitude of the activation volumes for catalysis at both GAATTC and TAATTC sites clearly indicates that significant conformational changes do occur during the catalytic steps of the reaction (48,59).…”

Section: Biochemistry: Robinson and Sligarmentioning

confidence: 99%

Changes in solvation during DNA binding and cleavage are critical to altered specificity of the Eco RI endonuclease

Robinson

Sligar

1998

Proc. Natl. Acad. Sci. U.S.A.

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Restriction endonucleases such as EcoRI bind and cleave DNA with great specificity and represent a paradigm for protein-DNA interactions and molecular recognition. Using osmotic pressure to induce water release, we demonstrate the participation of bound waters in the sequence discrimination of substrate DNA by EcoRI. Changes in solvation can play a critical role in directing sequence-specific DNA binding by EcoRI and are also crucial in assisting site discrimination during catalysis. By measuring the volume change for complex formation, we show that at the cognate sequence (GAATTC) EcoRI binding releases about 70 fewer water molecules than binding at an alternate DNA sequence (TAATTC), which differs by a single base pair. EcoRI complexation with nonspecific DNA releases substantially less water than either of these specific complexes. In cognate substrates (GAATTC) k cat decreases as osmotic pressure is increased, indicating the binding of about 30 water molecules accompanies the cleavage reaction. For the alternate substrate (TAATTC), release of about 40 water molecules accompanies the reaction, indicated by a dramatic acceleration of the rate when osmotic pressure is raised. These large differences in solvation effects demonstrate that water molecules can be key players in the molecular recognition process during both association and catalytic phases of the EcoRI reaction, acting to change the specificity of the enzyme. For both the protein-DNA complex and the transition state, there may be substantial conformational differences between cognate and alternate sites, accompanied by significant alterations in hydration and solvent accessibility.The EcoRI endonuclease cleaves double-stranded GAATTC sequences on both strands between G and A, at a rate at least 10 5 faster than the next best nucleic acid sequence as quantitated under standard conditions (1). EcoRI utilizes two ␣-helices to contact this cognate recognition sequence DNA in the major groove via an intricate network of hydrogen bonding interactions, which is thought to give rise to its high specificity (2, 3). The cleavage mechanism of this endonuclease is believed to involve activation of a water molecule by a basic group from the enzyme, followed by nucleophilic attack at the phosphodiester bond. The reaction is thought to involve only one step and is assisted by Mg 2ϩ as cofactor. The rate-limiting step may be release of doubly cleaved product, release of nicked product followed by rebinding and second strand cleavage, or conformational changes in the enzyme-substrate complex (4, 5). Binding constants and catalytic rate constants have been established for all single base substitutions of the canonical sequence and are in good agreement with the structural model for recognition and specificity

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“…Restriction endonucleases, for example, are DNA-binding proteins whose catalytic functions are inactive on nonspecific DNA and active at specific sites. Allosteric effects of DNA sequence on endonuclease activity have been proposed for EcoRI (Heitman 1992) and demonstrated for NaeI, NarI, BspMI, HpaII, and SacII (Oller et al 1991).Might the activities of transcriptional regulatory proteins similarly be governed or influenced by the DNA sequences to which they bind? The magnitude of activation or repression by a given factor commonly varies ~These authors contributed equally to this work.…”

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confidence: 99%

mentioning

confidence: 99%

Influence of a steroid receptor DNA-binding domain on transcriptional regulatory functions.

Lefstin

Thomas²,

Yamamoto³

1994

Genes Dev.

116

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We have isolated two independent mutations in the DNA-binding domain of the rat glucocorticoid receptor, P493R and $459A, that implicate DNA binding in the control of attached transcriptional activation domains, either that of the receptor itself or of VP16. The mutants are capable of activating transcription normally, but unlike wild-type receptors, they interfere with particular transcriptional activators in yeast and mammalian cells, and inhibit growth when overexpressed in yeast. The mutant residues reside at positions within the three-dimensional structure of the receptor that could, in principle, transduce structural changes from the DNA-binding surface of the receptor to other functional domains. These findings, together with the salt dependence of specific and nonspecific DNA binding by these receptors, suggest that specific DNA acts as an aUosteric effector that directs the functional interaction of the receptor with targets of transcriptional activation and that the P493R and $459A mutants mimic the allosteric effect of specific DNA, allowing the receptor to interact with regulatory targets even in the absence of specific DNA binding. The interaction of DNA and site-specific DNA-binding proteins was classically considered as a "rigid body" association determined by the interaction of two preexisting and stable interfaces. However, it is now apparent that many DNA-protein interactions are accompanied by structural changes. DNA structure may be altered by interaction with proteins, and site-specific DNA-binding proteins may undergo conformational changes upon binding to DNA (Spolar and Record 1994). The dynamic nature of protein-DNA interactions raises the possibility that the final structure of the protein might depend upon the particular sequence to which it binds, that is, specific DNA sequences might act as allosteric effectors to determine the catalytic or regulatory activities of the protein. Restriction endonucleases, for example, are DNA-binding proteins whose catalytic functions are inactive on nonspecific DNA and active at specific sites. Allosteric effects of DNA sequence on endonuclease activity have been proposed for EcoRI (Heitman 1992) and demonstrated for NaeI, NarI, BspMI, HpaII, and SacII (Oller et al. 1991).Might the activities of transcriptional regulatory proteins similarly be governed or influenced by the DNA sequences to which they bind? The magnitude of activation or repression by a given factor commonly varies ~These authors contributed equally to this work.with "DNA context." In general, it has not been determined whether such context effects reflect the contributions of other transcriptional regulators bound nearby, or instead indicate a direct effect of DNA sequence on the disposition of a regulator. However, the correlation between site-specific conformations and site-specific activities of the transcription factors PRTF/MCM1 (Tan and Richmond 1990) and the p50 subunit of NF-KB (Fujita et al. 1992;Hay and Nicholson 1993) supports the idea that DNA-induced conformational chan...

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How the EcoRI endonuclease recognizes and cleaves DNA

Cited by 52 publications

References 74 publications

Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy

Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy

Changes in solvation during DNA binding and cleavage are critical to altered specificity of the Eco RI endonuclease

Influence of a steroid receptor DNA-binding domain on transcriptional regulatory functions.

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