Specific binding by EcoRV endonuclease to its DNA recognition site GATATC

Engler, Lisa E.; Welch, Karlla K; Jen‐Jacobson, Linda

doi:10.1006/jmbi.1997.1027

Cited by 99 publications

(166 citation statements)

References 67 publications

(128 reference statements)

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“…Using a lower EcoRV concentration could diminish this problem, but as mentioned before we detected fluctuations in the actual amount of enzymes present in the flow chamber if we decreased the EcoRV concentration Ͻ1 nM. In reaction buffers containing 150 and 200 mM NaCl, target site location rates were dramatically lowered for all DNA configurations, probably attributable to the reduced affinity of EcoRV for both specific and nonspecific DNA in these buffers (35) (Figs. 4 Inset and 5).…”

Section: Resultssupporting

confidence: 51%

How DNA coiling enhances target localization by proteins

Broek

Lomholt²,

Kalisch

et al. 2008

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

188

197

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Many genetic processes depend on proteins interacting with specific sequences on DNA. Despite the large excess of nonspecific DNA in the cell, proteins can locate their targets rapidly. After initial nonspecific binding, they are believed to find the target site by 1D diffusion (''sliding'') interspersed by 3D dissociation/reassociation, a process usually referred to as facilitated diffusion. The 3D events combine short intrasegmental ''hops'' along the DNA contour, intersegmental ''jumps'' between nearby DNA segments, and longer volume ''excursions.'' The impact of DNA conformation on the search pathway is, however, still unknown. Here, we show direct evidence that DNA coiling influences the specific association rate of EcoRV restriction enzymes. Using optical tweezers together with a fast buffer exchange system, we obtained association times of EcoRV on single DNA molecules as a function of DNA extension, separating intersegmental jumping from other search pathways. Depending on salt concentration, targeting rates almost double when the DNA conformation is changed from fully extended to a coiled configuration. Quantitative analysis by an extended facilitated diffusion model reveals that only a fraction of enzymes are ready to bind to DNA. Generalizing our results to the crowded environment of the cell we predict a major impact of intersegmental jumps on target localization speed on DNA.A n essential feature in biological processes on DNA is the ability of proteins to quickly locate specific DNA sequences in a vast surplus of nonspecific DNA (1, 2). A protein's search for the target site is thought to be accelerated by facilitated diffusion along nonspecific DNA (3-6). Recent work has yielded considerable insight in the possible search strategies of sitespecific proteins (7-15). Assisted by DNA looping some proteins can, for instance, intermittently bind to two DNA segments simultaneously. This way they can directly move from one to another chemically remote segment (16). This intersegmental transfer accelerates target finding on DNA because it assumes a constantly changing random configuration (11). Here, we demonstrate and quantify a similar mechanism, intersegmental jumping, for proteins with only one DNA-binding site.Little experimental work on facilitated diffusion is available. To date, most studies have been investigating DNA cleavage by restriction enzymes in bulk assays, measuring association times as a function of DNA length, or monitoring processivity on DNA constructs with two sites (7,(17)(18)(19)(20)(21). Although in these biochemical assays valuable information can be obtained, association rates of proteins to specific sites are difficult to measure, and the underlying kinetics of the target search mechanism are often obscured. Furthermore, it remains experimentally challenging to distinguish 1D and 3D search pathways. In previous singlemolecule assays only pure 1D protein search has been addressed (21-24). Here, we present single-molecule measurements of DNA cleavage by EcoRV on individual plasm...

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Section: Resultssupporting

confidence: 51%

How DNA coiling enhances target localization by proteins

Broek

Lomholt²,

Kalisch

et al. 2008

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

188

197

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“…Under certain reaction conditions, many restriction endonucleases cleave DNA at sites that differ from the canonical site by one base pair (2). The star activity of EcoRV and EcoRI restriction endonucleases had been investigated thoroughly (16)(17)(18)(19)(20)(21)(22). The efficiency of cleavage at different star sites varies depending on the nature of the noncanonical base and its location within the sequence (16)(17)(18).…”

mentioning

confidence: 99%

Self-generated DNA termini relax the specificity of Sgr AI restriction endonuclease

Bitinaite

Schildkraut²

2002

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

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The primary target of SgrAI restriction endonuclease is a multiple sequence of the form 5 -CPu2CCGGPyG. Previous work had indicated that SgrAI must bind two recognition sites simultaneously for catalysis [Bilcock, D. T., Daniels, L. E., Bath, A. J. & Halford, S. E. (1999) J. Biol. Chem. 274, 36379 -36386]. In the present study, SgrAI is shown to cleave not only its canonical sequences, but also the sequences 5 -CPuCCGGPy(A,T,C) and 5 -CPuCCGGGG, both referred to as secondary sequences. On plasmid pSK7, SgrAI cleaves secondary sites 26-fold slower than the canonical site. However, the same plasmid, but without the canonical site, is cleaved 200-fold slower. We show that DNA termini generated by cleaving the canonical site for SgrAI assist in the cleavage of secondary sites. The SgrAI-termini in cis with respect to secondary site are markedly preferred over those in trans. The SgrAI-termini provided in a form of oligonucleotide duplex are also shown to stimulate canonical site cleavage. At a 40-fold molar excess of the SgrAI-termini over substrate, the SgrAI specificity is shown to improve by two orders of magnitude, because of concurrent 10-fold increase in the cleavage of canonical site and 50-fold decrease in the cleavage of secondary sites. The unconventional reaction pathway by which SgrAI utilizes the self-generated DNA termini to cleave its DNA targets has not been observed hitherto among type II restriction endonucleases. Based on our work and previous reports, a pathway of DNA binding and cleavage by the SgrAI restriction endonuclease is proposed.

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“…A single base substitution in the recognition sequence reduces the overall cutting rate by at least a factor 10 6 . 26 The X-ray crystallography structure of EcorV bound to its cognate DNA (Fig. 1) shows the DNA kinked by approximately 50 0 away from the straight conformation, 21 the enzyme mostly enveloping the outer side of the kink, like a hand pressing and bending the DNA between thumb and fingers.…”

Section: Introductionmentioning

confidence: 99%

EcoRV catalysis with a pre-bent substrate

Sanchez

Zocchi

2015

AIP Advances

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Enzymes are deformable molecules which often adapt their conformation to the substrate’s geometry. In the case of restriction enzymes acting on DNA, the substrate (DNA) is deformable also. While it is well established that external mechanical stress exerted on an enzyme modulates the enzymatic activity, the effect of mechanically stressing the substrate is less explored. Here we present the restriction enzyme EcoRV with a series of pre-bent DNA substrates and observe modulation of the overall speed of the enzymatic reaction by up to a factor 50. While in all cases the reaction is slowed down compared to the unstressed substrate, we observe peaks in the reaction rate as we vary the state of stress of the DNA substrate. Both bending moments and torsional moments on the DNA substrate affect the reaction rate.

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Specific binding by EcoRV endonuclease to its DNA recognition site GATATC

Cited by 99 publications

References 67 publications

How DNA coiling enhances target localization by proteins

How DNA coiling enhances target localization by proteins

Self-generated DNA termini relax the specificity of Sgr AI restriction endonuclease

EcoRV catalysis with a pre-bent substrate

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