The DNA-binding properties of the EcoRV restriction endonuclease and modification methlytransferase
The DNA-binding properties of the EcoRV restriction endonuclease and modification methlytransferase
The in vitro sequence specific DNA-binding properties of the EcoRV endonuclease (R.EcoRV) and methyltransferase (M.EcoRV) were investigated under equilibrium conditions using the electrophoretic band-shift assay, and for M.EcoRV, enzymic and chemical footprinting techniques. In the absence of the essential cofactor Mg2+, R.EcoRV binds all DNA sequences with the same affinity (Taylor et al., 1991). Sequence discrimination would appear to be due to the enzymic affinity for Mg2+; when bound at the EcoRV site, the complex has a high affinity for Mg2+, whilst complexes at all other sequences have a low affinity for Mg2+c. To investigate the possibility of a discriminatory role for divalent cations at the level of DNA-binding, a set of nucleolyticaly resistant oligonucleotide substrates were synthesized containing phosphate (phosphorothioate, 3'-thio-2'-deoxythymidine), furanose(4'-thio-2'-deoxythymidine) and base(7-deaza-2'-deoxyadenosine) modifications and tested against a control sequence containing a scrambled EcoRV site. The lack of any specific binding suggests that the modified DNA is essentially considered as non-specific, random DNA, and that nucleolytic resistance is probably a function of a vanishingly low metal ion affinity of the endonuclease when bound at the modified site.
M.EcoRV, on the other hand, binds DNA in sequence-specific manner in the presence of S-adenoxyl-L-methionine, the product S-adenosyl-L-homocysteine and the analogues sinefungin and N-methyladenosylmethionine. Binding in the complete absence of cofactor could not be recorded due to the copurification of S-adenosyl-L-methionine with M.EcoRV. Binding affinity is significantly enhanced in the presence of sinefungin or N-methyladenosylmethionine. M.EcoRV shows the highest binding affinity to DNA containing a hemimethylated EcoRV sequence (Kd= 11.1-13.0 nM), but binds less well to unmethylated DNA (Kd= 46.3 nM)
University of Southampton
1994
Szczelkun, Mark Dominik
(1994)
The DNA-binding properties of the EcoRV restriction endonuclease and modification methlytransferase.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The in vitro sequence specific DNA-binding properties of the EcoRV endonuclease (R.EcoRV) and methyltransferase (M.EcoRV) were investigated under equilibrium conditions using the electrophoretic band-shift assay, and for M.EcoRV, enzymic and chemical footprinting techniques. In the absence of the essential cofactor Mg2+, R.EcoRV binds all DNA sequences with the same affinity (Taylor et al., 1991). Sequence discrimination would appear to be due to the enzymic affinity for Mg2+; when bound at the EcoRV site, the complex has a high affinity for Mg2+, whilst complexes at all other sequences have a low affinity for Mg2+c. To investigate the possibility of a discriminatory role for divalent cations at the level of DNA-binding, a set of nucleolyticaly resistant oligonucleotide substrates were synthesized containing phosphate (phosphorothioate, 3'-thio-2'-deoxythymidine), furanose(4'-thio-2'-deoxythymidine) and base(7-deaza-2'-deoxyadenosine) modifications and tested against a control sequence containing a scrambled EcoRV site. The lack of any specific binding suggests that the modified DNA is essentially considered as non-specific, random DNA, and that nucleolytic resistance is probably a function of a vanishingly low metal ion affinity of the endonuclease when bound at the modified site.
M.EcoRV, on the other hand, binds DNA in sequence-specific manner in the presence of S-adenoxyl-L-methionine, the product S-adenosyl-L-homocysteine and the analogues sinefungin and N-methyladenosylmethionine. Binding in the complete absence of cofactor could not be recorded due to the copurification of S-adenosyl-L-methionine with M.EcoRV. Binding affinity is significantly enhanced in the presence of sinefungin or N-methyladenosylmethionine. M.EcoRV shows the highest binding affinity to DNA containing a hemimethylated EcoRV sequence (Kd= 11.1-13.0 nM), but binds less well to unmethylated DNA (Kd= 46.3 nM)
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Published date: 1994
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Local EPrints ID: 462624
URI: http://eprints.soton.ac.uk/id/eprint/462624
PURE UUID: b3c3736b-3fa8-4378-bb4d-5d16711aeafb
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Date deposited: 04 Jul 2022 19:33
Last modified: 04 Jul 2022 19:33
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Author:
Mark Dominik Szczelkun
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