EcoRV

Endonuclease EcoRV
	according to PDB 1AZ0
Mass / length primary structure	29,500 da
Secondary to quaternary structure	Homodimer
Identifier
Gene name (s)	EcoRV (Rebase)
Enzyme classification
EC, category	3.1.21.4 , restriction enzyme
Response type	hydrolysis
Substrate	DNA
Products	Double stranded DNA fragments with terminal 5 'phosphate groups
Occurrence
Parent taxon	Escherichia coli

EcoRV is a restriction enzyme from the bacterium Escherichia coli .

properties

Mechanism of DNA hydrolysis by EcoRV

EcoRV is an endonuclease (type II, subtype P) that cuts DNA at a palindromic DNA recognition sequence . In suboptimal buffer conditions , the affinity of EcoRV for the recognition sequence decreases and the DNA is cut unspecifically ( star activity ). The cut of the DNA by EcoRV results in a blunt end (end without overhang) and a phosphate group at both 5 'ends of the double-stranded DNA products. EcoRV is not inhibited by DAM methylation , DCM or CpG methylation. After restriction of DNA in vitro , EcoRV can be denatured and thus inactivated by heating at 80 ° C for 20 minutes . Eco32I is an isoschizomer from EcoRV and can be used alternatively. Most of the time, EcoRV is produced as a recombinant protein in E.coli .


Recognition sequence	Restriction cut
5'-GATATC-3' 3'-CTATAG-5'	5'-GAT ATC-3' 3'-CTA TAG-5'

structure

EcoRV has at the N -terminus , a dimerization - protein domain , consisting of a short α-helix , two-stranded anti-parallel a β-sheet and a long α-helix. Like all typical endonucleases of type II, it binds to DNA as a homodimer . When cutting, the DNA is bent at an angle of 50 ° at the interface between the bases thymine and adenine , which is unique among the endonucleases of type II. After the EcoRV glides along the DNA, the outer nucleic bases are recognized and the presence of the central and comparatively small bases TA, which are necessary for the DNA to bend, is then checked.

Applications

EcoRV is used for restriction digestions in the context of cloning or restriction analyzes.

Individual evidence

^ ^A ^b Alfred Pingoud , A. Jeltsch: Structure and function of type II restriction endonucleases. In: Nucleic acids research. Volume 29, Number 18, September 2001, pp. 3705-3727, PMID 11557805 , PMC 55916 (free full text).
↑ J. Bitinaite, DA Wah, AK Aggarwal, I. Schildkraut: FokI dimerization is required for DNA cleavage. In: Proceedings of the National Academy of Sciences . Volume 95, Number 18, September 1998, pp. 10570-10575, PMID 9724744 , PMC 27935 (free full text).
↑ M. Zahran, I. Daidone, JC Smith, P. Imhof: Mechanism of DNA recognition by the restriction enzyme EcoRV. In: Journal of molecular biology. Volume 401, number 3, August 2010, pp. 415-432, doi : 10.1016 / j.jmb.2010.06.026 , PMID 20600128 .
↑ Keith Wilson: Principles and Techniques of Biochemistry and Molecular Biology. Cambridge University Press, 2010, ISBN 978-0-521-51635-8 , p. 218.
^ Henri Grosjean: DNA and RNA Modification Enzymes. Taylor & Francis, 2009, ISBN 978-1-587-06329-9 , pp. 82-83.

[Pingoud-1] A ^b Alfred Pingoud , A. Jeltsch: Structure and function of type II restriction endonucleases. In: Nucleic acids research. Volume 29, Number 18, September 2001, pp. 3705-3727, PMID 11557805 , PMC 55916 (free full text).

[2] J. Bitinaite, DA Wah, AK Aggarwal, I. Schildkraut: FokI dimerization is required for DNA cleavage. In: Proceedings of the National Academy of Sciences . Volume 95, Number 18, September 1998, pp. 10570-10575, PMID 9724744 , PMC 27935 (free full text).

[3] M. Zahran, I. Daidone, JC Smith, P. Imhof: Mechanism of DNA recognition by the restriction enzyme EcoRV. In: Journal of molecular biology. Volume 401, number 3, August 2010, pp. 415-432, doi : 10.1016 / j.jmb.2010.06.026 , PMID 20600128 .

[Wilson-4] Keith Wilson: Principles and Techniques of Biochemistry and Molecular Biology. Cambridge University Press, 2010, ISBN 978-0-521-51635-8 , p. 218.

[Grosjean-5] Henri Grosjean: DNA and RNA Modification Enzymes. Taylor & Francis, 2009, ISBN 978-1-587-06329-9 , pp. 82-83.