Effects of functional group changes in the EcoRI recognition site on the cleavage reaction catalyzed by the endonuclease

McLaughlin, Larry W.; Benseler, Fritz; Graeser, Erika; Piel, Norbert; Scholtissek, Stephan

doi:10.1021/bi00397a007

Cited by 164 publications

(126 citation statements)

References 63 publications

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“…From a linear regression of initial slopes at different substrate concentrations, it was confirmed that EcoRI catalysis obeys the Michaelis-Menten equation (Fig. 5), as already shown by others (30)(31)(32)(33). From an Eadie-Hofstee plot (Fig.…”

Section: Resultssupporting

confidence: 82%

“…5 Inset) a K M of 14 Ϯ 1 nM was derived. As pointed out by McLaughlin et al (31) the K M value of EcoRI depends sensitively on the size of the substrate. Although most studies cannot be compared due to varying detection methods and reaction conditions (temperature, ionic strength, etc.…”

Section: Resultsmentioning

confidence: 85%

“…Although most studies cannot be compared due to varying detection methods and reaction conditions (temperature, ionic strength, etc. ), plasmid substrates usually revealed K M values between 1 and 10 nM (25,(34)(35)(36), whereas the K m was remarkably higher (80-230 nM, in one case up to 7 M) when short synthetic DNA oligomers were used (30,31,33,37,38). Compared with these values, the K M obtained here is rather low, and this is most probably caused by the relatively large size of the substrate.…”

Section: Resultsmentioning

confidence: 99%

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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: Resultssupporting

confidence: 82%

Section: Resultsmentioning

confidence: 85%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

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

View full text Add to dashboard Cite

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“…2-AP can substitute for adenine and pair with thymine without changing much of the physicochemical properties of double-stranded polynucleotide (22)(23)(24)(25)(26). 2-AP acts as an adenine and pairs with thymine when present in the template strand during polynucleotide synthesis by DNA polymerase (see the experiments mentioned in the Fig.…”

Section: Resultsmentioning

confidence: 99%

A “master” in base unpairing during isomerization of a promoter upon RNA polymerase binding

Lim

Lee

Roy

et al. 2001

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

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Isomerization of a closed to open complex of a promoter upon RNA polymerase binding involves base unpairing at the ؊10 region. After potassium permanganate sensitivity of unpaired thymine residues, we studied base unpairing at the ؊10 region during isomerization upon RNA polymerase binding at the P1 and P3 promoters of the gal operon. Substitution of adenine by 2-amino purine (2-AP) at the invariable A⅐T base pair at the ؊11 position of P1 and P3 prevented unpairing not only at that position but also at the other downstream positions, suggesting a ''master'' role of the adenine base at ؊11 of the template strand in overall base unpairing. 2-AP at ؊11 did not inhibit the formation of RNA polymerase⅐promoter complex and subsequent isomerization of the polymerase. Substitution of adenine by 2-AP at several other positions did not affect thymine unpairing. Changing the position of the amino group from C6 in adenine to C2 in 2-AP is mutational only at the master switch position, ؊11. U nder ordinary physiological conditions, transcription inEscherichia coli is executed by the RNA polymerase holoenzyme comprising ␣ 2 ␤␤Ј 70 subunits. Transcription initiation follows specific binding of RNA polymerase to the promotor. The initial binary complex (closed complex) undergoes structural changes (isomerization) in both proteins and DNA to make an open complex that is competent for subsequent templatedependent polymerization of ribonucleotides (1, 2). Whether isomerization of RNA polymerase and DNA occurs simultaneously or one after the other is unknown. The 70 subunit plays a key role in recognizing and making specific contacts with base pairs at the Ϫ35 and Ϫ10 regions of the promoter (3). In the open complex, DNA at the Ϫ10 region presumably becomes singlestranded for RNA polymerase to start reading the exposed template strand and polymerizing ribonucleotides (4-10). The bases in the nontemplate strand make specific interactions with amino acid residues of 70 (11). The consequence of these interactions is believed to stabilize the unstable nature of the unpaired DNA region (11-13). The consensus DNA sequence of the Ϫ10 region is 5Ј-TATAAT-3Ј in the nontemplate strand, with the first T being at the Ϫ12 position (1). To elucidate the molecular procedures of base unpairing during open complex formation, altered forms of DNA (depurinated, nicked, forked, or with single-stranded gap) in the Ϫ10 region were used as templates (10,(14)(15)(16)(17)(18)(19). These studies suggested that base unpairing involves an initial distortion or localized melting event around Ϫ11 (16,20). In this paper, we provide results by using intact duplex DNA to demonstrate that the adenine residue itself at the position Ϫ11 of the nontemplate strand plays a master role in the initiation of base unpairing. Materials and MethodsMaterials. E. coli RNA polymerase holoenzyme was purchased from Epicentre Technologies (Madison, WI). cAMP receptor protein (CRP), purified to 98% homogeneity by FPLC (Amersham Pharmacia) from an E. coli strain carrying the crp gen...

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“…The self-complementary oligomer d(CTGAnPTTCAG)2 was prepared as described (McLaughlin et al, 1988) using the segmental solid-phase phosphotriester method. Stock solutions were made up in distilled water.…”

Section: Materlals and Methodsmentioning

confidence: 99%

Thermodynamics of interaction of a fluorescent DNA oligomer with the anti‐tumour drug netropsin

Patel

Berglund

Nilsson

et al. 1992

European Journal of Biochemistry

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Fluorescence spectroscopy was used to study the interaction between the minor-groove-binding drug netropsin and the self-complementary oligonucleotide d(CTGAnPTTCAG)2 containing the fluorescent base analogue 2-aminopurine (nP). The binding of netropsin to this oligonucleotide causes strong quenching of the 2-aminopurine fluorescence, observed by steady-state as well as time-resolved spectroscopy. From fluorescence titrations, binding isotherms were recorded and evaluated. The parameters showed one netropsin binding site/oligonucleotide duplex and an association constant of about 10' M -' at 25"C, 3-4 orders of magnitude weaker than for an exclusive adenine/thymine host sequence. From the temperature dependence of the association constant the thermodynamic parameters were obtained as AG = -29 kJ/mol, A H = -12 kJ/mol and A S = + 55 J . mol-' . K-' at 25°C. These parameters resemble those of the interaction of poly [(dG-dC) . (dG-dC)] with netropsin, indicating a mainly entropy-driven reaction. The amino group of 2-aminopurine, like that of guanine, resides in the minor groove of DNA. Therefore the relatively weak binding of netropsin to d(CTGAnPTTCAG), is probably related to partial blockage of the tight fit of netropsin into the preferred minor groove of an exclusive adenine/thymine host sequence.Base-sequence-dependent selectivity and specificity in the interaction of DNA with low-molecular-mass ligands, such as anti-tumour drugs, or with macromolecules such as proteins, is an important biological recognition problem. It is important to understand the detailed molecular background of this recognition and to develop new strategies for experimental studies of specific interactions between DNA and binding ligands. This study concerns the interaction between netropsin and a fluorescent DNA oligomer.Netropsin is an established anti-viral anti-tumour antibiotic (Zimmer and Wiihnert, 1986) which, however, is too toxic for clinical use. Its biological activity is considered to be a consequence of its tight binding to double-helical B-DNA.The interaction of netropsin with DNA has involved a variety of experiments. Foot-printing techniques (Taylor et al., 1984; Dervan, 1986) have established that netropsin binds A + T-rich sites of 5 5 1 base pairs in naturally occurring DNA polymers. A preference for the B conformation of the DNA has been established (Zimmer, 1975). Viscosimetric studies indicate that netropsin binding increases the contour length of A + T-rich DNA helices and makes them more rigid (Reinert et al., 1980). The thermal stability of the DNA is enhanced on netropsin binding and combined spectroscopic and calorimetric techniques have shown that the thermodynamics for netropsin binding to A + T-rich sequences are very favourable, involving an entirely enthalpy-driven interaction Correspondence to A. Grislund,

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Effects of functional group changes in the EcoRI recognition site on the cleavage reaction catalyzed by the endonuclease

Cited by 164 publications

References 63 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

A “master” in base unpairing during isomerization of a promoter upon RNA polymerase binding

Thermodynamics of interaction of a fluorescent DNA oligomer with the anti‐tumour drug netropsin

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