title: Mg2+ confers DNA binding specificity to the EcoRV restriction endonuclease.
abstract: The EcoRV mutant D90A which carries an amino acid substitution in its active center does not cleave DNA. Therefore, it is possible to perform DNA binding experiments with the EcoRV-D90A mutant both in the absence and in the presence of Mg2+. Like wild-type EcoRV [Taylor et al. (1991) Biochemistry 30, 8743-8753], it does not show a pronounced specificity for binding to its recognition site in the absence of Mg2+ as judged by the appearance of multiple shifted bands in an electrophoretic mobility shift assay with a 377-bp DNA fragment carrying a single EcoRV recognition sequence. In the presence of Mg2+, however, only one band corresponding to a 1:1 complex appears even with a high excess of protein over DNA. This complex most likely is the specific one, because its formation is suppressed much more effectively by a 13-bp oligodeoxynucleotide with an EcoRV site than by a corresponding oligodeoxynucleotide without an EcoRV site. The preferential interaction of the EcoRV-D90A mutant with specific DNA in the presence of Mg2+ was also demonstrated directly: a 20-bp oligodeoxynucleotide with an EcoRV site is bound with KAss = 4 x 10(8) M-1, while a corresponding oligodeoxynucleotide without an EcoRV site is bound with KAss less than or equal to 1 x 10(5) M-1. From these data it appears that Mg2+ confers DNA binding specificity to this mutant by lowering the affinity to nonspecific sites and raising the affinity to specific sites as compared to binding in the absence of Mg2+. It is concluded that this is also true for wild-type EcoRV.
authors.affiliation: Zentrum Biochemie, Medizinische Hochschule Hannover, Germany.
authors.forename: V
authors.initials: V
authors.lastname: Thielking
authors.forename: U
authors.initials: U
authors.lastname: Selent
authors.forename: E
authors.initials: E
authors.lastname: Köhler
authors.forename: A
authors.initials: A
authors.lastname: Landgraf
authors.forename: H
authors.initials: H
authors.lastname: Wolfes
authors.forename: J
authors.initials: J
authors.lastname: Alves
authors.forename: A
authors.initials: A
authors.lastname: Pingoud
mesh_terms: D001483:Base Sequence; D002413:Cations, Divalent; D004269:DNA, Bacterial / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008274:Magnesium / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity
chemical_list: D002413:Cations, Divalent; D004269:DNA, Bacterial; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases; D008274:Magnesium