Studies on EcoRV methylase
Studies on EcoRV methylase
EcoRV is a type II restriction/modification system consisting of a restriction endonuclease and a modification methylase. Both these enzymes specifically recognise the double stranded DNA site d(GATATC). The endonuclease requires magnesium as a cofactor and cleaves both strands of the DNA between the central d(TA) base pairs. The methylase transfers a methyl group from S-adenosylmethionine to the first deoxyadenosine (dA) in each strand of this recognition sequence. Methylation at this site prevents the endonuclease action and thus protects host DNA from cleavage.
The nature of the interactions governing the specificity of EcoRV methylase for the EcoRV site in DNA have been probed. The first approach used base analogues incorporated into synthetic oligonucleotide substrates. The unmodified oligonucleotide substrate had the sequence d(GACGATATCGTC)(EcoRV site underlined). Base analogues of deoxyguanosine (dG) and deoxycytidine (dC), each differing from the unmodified base in one site of potential contact with the enyzme, have been individually incorporated into the EcoRV site of the substrate dodecamer and the effect upon the rate of methylation studied. The dG/dC base analogue studies have indicated the following groups of the dG/dC bases in the EcoRV site as probably being important sites of contact to EcoRV methylase: The ring 7-N group in the DNA major groove and the exocyclic 2-NH2 group in the minor groove of dG and the 5-H group in the major groove of dC. Functional groups of the dG/dC bases in the recognition site that were not important for recognition by the EcoRV methylase included the 6-keto O in the major groove and the ring 3-N in the minor groove of dG, and the exocyclic 4-NH2 group of dC present in the major groove.
The second approach in the study of the specific contacts between EcoRV methylase and its recognition site, has been the mutagenesis of amino acid residues in the methylase.
University of Southampton
Jones, Helen
c33f6a5f-e87f-4b8d-8962-d95f345cc19e
1993
Jones, Helen
c33f6a5f-e87f-4b8d-8962-d95f345cc19e
Jones, Helen
(1993)
Studies on EcoRV methylase.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
EcoRV is a type II restriction/modification system consisting of a restriction endonuclease and a modification methylase. Both these enzymes specifically recognise the double stranded DNA site d(GATATC). The endonuclease requires magnesium as a cofactor and cleaves both strands of the DNA between the central d(TA) base pairs. The methylase transfers a methyl group from S-adenosylmethionine to the first deoxyadenosine (dA) in each strand of this recognition sequence. Methylation at this site prevents the endonuclease action and thus protects host DNA from cleavage.
The nature of the interactions governing the specificity of EcoRV methylase for the EcoRV site in DNA have been probed. The first approach used base analogues incorporated into synthetic oligonucleotide substrates. The unmodified oligonucleotide substrate had the sequence d(GACGATATCGTC)(EcoRV site underlined). Base analogues of deoxyguanosine (dG) and deoxycytidine (dC), each differing from the unmodified base in one site of potential contact with the enyzme, have been individually incorporated into the EcoRV site of the substrate dodecamer and the effect upon the rate of methylation studied. The dG/dC base analogue studies have indicated the following groups of the dG/dC bases in the EcoRV site as probably being important sites of contact to EcoRV methylase: The ring 7-N group in the DNA major groove and the exocyclic 2-NH2 group in the minor groove of dG and the 5-H group in the major groove of dC. Functional groups of the dG/dC bases in the recognition site that were not important for recognition by the EcoRV methylase included the 6-keto O in the major groove and the ring 3-N in the minor groove of dG, and the exocyclic 4-NH2 group of dC present in the major groove.
The second approach in the study of the specific contacts between EcoRV methylase and its recognition site, has been the mutagenesis of amino acid residues in the methylase.
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Published date: 1993
Identifiers
Local EPrints ID: 462262
URI: http://eprints.soton.ac.uk/id/eprint/462262
PURE UUID: 40334d69-c469-43af-8781-3241cc69c68e
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Date deposited: 04 Jul 2022 19:04
Last modified: 04 Jul 2022 19:04
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Author:
Helen Jones
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