FRET-based kinetic analysis of highly reactive heterochiral DNA toward EcoRI endonuclease

Urata, Hidehito; Tamaki, Chihiro; Matsuno, Miki; Wada, Shun‐ichi; Akagi, Masao

doi:10.1016/j.bbrc.2009.08.164

Cited by 12 publications

(9 citation statements)

References 21 publications

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“…Furthermore, by plotting the reaction velocities with respect to the substrate concentrations and fitting these data to the hyperbolic curves, kinetic parameters K m and V max were estimated to be 97.6 nM and 0.103 nM/s, respectively. These values were compatible with the data reported previously with small margins [19], [22]. It has been known that the lengths of the duplex substrates as well as the nucleotides flanking the recognition sequence at 5′- or 3′-end could exert direct or indirect effects on the kinetic values, e.g.…”

Section: Resultssupporting

confidence: 92%

“…FRET approach has been established previously for monitoring the restriction endonucease cleavage activity [18], [19], [20], [21]. To validate this method and to determine the enzyme concentration adequate for experiments in the current study, we tested the effects of the enzyme concentration on the cleavage efficiency for the native nucleotide sequence.…”

Section: Resultsmentioning

confidence: 99%

“…2′-fluoro-2′-deoxyguanosine (2′-F-dG) and 2′-chloro-2′-deoxyguanosine (2′-Cl-dG) have been the only two 2′-modified nucleotide derivatives so far, and only the G1 residue of the Eco RI sequence has been modified. Modification of the residue flanking the recognition sequence at the 3′-end by L-nucleotide (ribose in L-homochirality) has shown different enzymatic behavior from the normal one [19]. In structure biology, six torsion angles along the phosphate backbone -P-O5′-C5′-C4′-C3′-O3′-P- of DNA strand, namely α, β, γ, δ, ε and ζ, are used to characterize the backbone conformations of DNA and RNA, and each one has been restricted in a well defined range for different conformations of DNA duplexes.…”

Section: Discussionmentioning

confidence: 99%

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Effects of 2′-O-Methyl Nucleotide Substitution on EcoRI Endonuclease Cleavage Activities

Zhao

Tong

et al. 2013

PLoS ONE

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To investigate the effect of sugar pucker conformation on DNA-protein interactions, we used 2′-O-methyl nucleotide (2′-OMeN) to modify the EcoRI recognition sequence -TGAATTCT-, and monitored the enzymatic cleavage process using FRET method. The 2′-O-methyl nucleotide has a C3′-endo sugar pucker conformation different from the C2′-endo sugar pucker conformation of native DNA nucleotides. The initial reaction velocities were measured and the kinetic parameters, Km and Vmax were derived using Michaelis-Menten equation. Experimental results showed that 2′-OMeN substitutions for the EcoRI recognition sequence decreased the cleavage efficiency for A2, A3 and T4 substitutions significantly, and 2′-OMeN substitution for T5 residue inhibited the enzymatic activity completely. In contrast, substitutions for G1 and C6 could maintain the original activity. 2′-fluoro nucleic acid (2′-FNA) and locked nucleic acid (LNA) having similar C3′-endo sugar pucker conformation also demonstrated similar enzymatic results. This position-dependent enzymatic cleavage property might be attributed to the phosphate backbone distortion caused by the switch from C2′-endo to C3′-endo sugar pucker conformation, and was interpreted on the basis of the DNA-EcoRI structure. These 2′-modified nucleotides could behave as a regulatory element to modulate the enzymatic activity in vitro, and this property will have potential applications in genetic engineering and biomedicine.

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Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Effects of 2′-O-Methyl Nucleotide Substitution on EcoRI Endonuclease Cleavage Activities

Zhao

Tong

et al. 2013

PLoS ONE

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“…The endonucleases, because of their capability to bind and cleave specific DNA sequences, also play a key role in DNA recombinant and cloning methods (1,2). The EcoR1-DNA equilibria and reaction dynamics have been probed extensively by a number of established methods, such as, but not limited to: FRET (3)(4)(5), gel electrophoresis (6)(7)(8)(9)(10)(11)(12)(13)(14)(15), surface plasmon resonance (16), microfluidic trapping (17), two-color cross-correlation spectroscopy (18), and isothermal calorimetry (19).…”

mentioning

confidence: 99%

Binding and cleavage of DNA with the restriction enzyme EcoR1 using time-resolved second harmonic generation

Doughty

Kazer

Eisenthal

2011

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

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The binding of EcoR1 to a 90-bp DNA duplex attached to colloidal microparticles and the subsequent cleavage by the enzyme was observed in real time and label-free with time-resolved second harmonic (SH) spectroscopy. This method provides a unique way to investigate biomolecular interactions based on its sensitivity to changes in structure and electrical charge on formation of a complex and subsequent dynamics. The binding of EcoR1 to the recognition sequence in DNA appears as a rapid increase in the SH signal, which is attributed to the enzyme-induced change in the DNA conformation, going from a rod-like to a bent shape. In the presence of the cofactor Mg 2 , the subsequent decay in the SH signal was monitored in real time as the following processes occurred: cleavage of DNA, dissociation of the enzyme from the DNA, and diffusion of the 74-bp fragment into the bulk solution leaving the 16-bp fragment attached to the microparticle. The observed decay was dependent on the concentration of Mg 2 , which functions as a cofactor and as an electrolyte. With SH spectroscopy the rehybridization dynamics between the rehybridized microparticle bound and free cleaved DNA fragments was observed in real time and label-free following the cleavage of DNA. Collectively, the experiments reported here establish SH spectroscopy as a powerful method to investigate equilibrium and time-dependent biological processes in a noninvasive and label-free way.cleavage kinetics | DNA-endonuclease | rehybridization kinetics | nonspecific binding T he highly selective binding of proteins to specific nucleotide sequences in DNA is an essential step in many biological processes, including gene regulation, gene expression, and DNA hybridization (1). The family of type II restriction enzymes, which includes EcoR1, have been, and continue to be, of major importance in serving as model systems of protein-DNA interactions. The endonucleases, because of their capability to bind and cleave specific DNA sequences, also play a key role in DNA recombinant and cloning methods (1, 2). The EcoR1-DNA equilibria and reaction dynamics have been probed extensively by a number of established methods, such as, but not limited to: FRET (3-5), gel electrophoresis (6-15), surface plasmon resonance (16), microfluidic trapping (17), two-color cross-correlation spectroscopy (18), and isothermal calorimetry (19).The essential characteristic of the method used here is the application of second harmonic (SH) spectroscopy to selectively probe the interface of a polystyrene carboxylate (PSC) microparticle, to which target DNA molecules have been attached. Significant effort and progress has been made in hybridizing artificial colloidal nano-and micromaterials with naturally occurring biological reagents to build self-assembled superstructured materials (20-26), and to develop biologically coded probes for protein detection and disease treatment (27,28). The present work builds on these pioneering studies, and on studies of DNA supported on flat surfaces using SH and sum-frequ...

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“…Radio-labeled substrates and electrophoresis are traditional methods, but the processes are tedious. The fluorescencebased analytical approaches have advantages of real-time analysis, convenience, and precise quantization, and found more and more applications in various enzyme assays, such as polymerase assays and endonuclease assays [8][9][10][11][18][19][20]. In comparison, only a few fluorescence-based exonuclease activity assays have been developed, and are mainly for kinetic analysis; however, they require special instruments [21][22][23][24].…”

Section: Effects Of Monovalent Metal Ions and Dntpmentioning

confidence: 99%

Effects of cations on small fragment of DNA polymerase I using a novel FRET assay

Zhao¹,

Tang²,

Li³

et al. 2014

ABBS

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DNA polymerase I (PolI) digested by protease produces a small fragment (SF) containing 5 0 !3 0 exonuclease activity. The 5 0 !3 0 exonuclease activity of polI cleaves the downstream RNA primer strands during DNA replication in vivo. Previous in vitro studies suggested its capability of cleaving duplex from 5 0 terminal and a flap-structure-specific endonuclease activity. From the crystal structures of other nucleases and biochemical data, a two-metal-ion mechanism has been proposed but has not been determined. In this study, we cloned, expressed, and purified the SF protein, and established a novel fluorescence resonance energy transfer (FRET)

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FRET-based kinetic analysis of highly reactive heterochiral DNA toward EcoRI endonuclease

Cited by 12 publications

References 21 publications

Effects of 2′-O-Methyl Nucleotide Substitution on EcoRI Endonuclease Cleavage Activities

Effects of 2′-O-Methyl Nucleotide Substitution on EcoRI Endonuclease Cleavage Activities

Binding and cleavage of DNA with the restriction enzyme EcoR1 using time-resolved second harmonic generation

Effects of cations on small fragment of DNA polymerase I using a novel FRET assay

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