DNA-BINDING SPECIFICITY OF THE ECORV RESTRICTION-ENDONUCLEASE IS INCREASED BY MG2-ION BINDING-SITE DISTINCT FROM THE CATALYTIC CENTER OF THE ENZYME( BINDING TO A METAL)

In contrast to many other type II restriction endonucleases, EcoRV bin ds specifically to DNA only in the presence Mg2+. According to the co- crystal structure of an EcoRV-DNA complex, Mg2+ ion(s) bind to the act ive site of EcoRV liganded by Glu(45), Asp(74) and Asp(90). Here we pr esent experimental evidence suggesting that the EcoRV-DNA complex also interacts with Mg2+ ions at other sites: (i) We have prepared an EcoR V triple mutant, in which all acidic amino acids in the catalytic cent er are replaced by alanine. This mutant is catalytically inactive: It binds nonspecifically to DNA in the absence of Mg2+, whereas it binds specifically to DNA in the presence of Mg2+. This means that Mg2+ indu ces specific DNA binding in this mutant, although all Mg2+ ligands in the catalytic center are removed. Therefore, additional interactions b etween Mg2+ End the EcoRV-DNA complex probably occur at sites distinct from the catalytic center. (ii) We have measured the specific and non specific DNA binding constants of EcoRV and of the triple mutant in th e presence and absence of Mg2+. Mg2+ reduces nonspecific binding by 3- 4 orders of magnitude, presumably because Mg2+ ions bound to the DNA h ave to be released upon complex formation, In contrast, the specific b inding of the wild-type enzyme and the triple mutant is increased in t he presence of Mg2+. This result can only be explained if a Mg2+ ion b inds to the specific EcoRV-DNA complex probably at a site distinct fro m the catalytic center. (iii) To locate additional metal ion binding s ites in the EcoRV-DNA complex, we have determined the cleavage rates o f several undecadeoxynucleotides which contain single phosphorothioate linkages in the presence of Mg2+ and Mn2+. It turned out that an olig odeoxynucleotide in which the first phosphate group within the GpATATC sequence (p(3)) is replaced by an R(p), phosphorothioate is cleaved b y: a factor of 50 more readily in the presence of Mn2+ than with Mg2+. This result is interpreted to mean that p(3), which is far away from the active site of EcoRV, interacts with a Mg2+ ion. (iv) We have prod uced the EcoRV Y219C mutant whose DNA cleavage activity compared to wi ld-type EcoRV is reduced by 3 orders of magnitude. It binds nonspecifi cally to DNA in the absence of Mg2+ but not detectably in the presence of Mg2+ Although the distinct role of Tyr(219) is unclear at present, it must be pointed out that this amino acid is far away from the acti ve site of EcoRV but located in close proximity to amino acid residues vis a vis p(3). Hence, the behavior of this mutant also supports the conclusion that an important interaction between Mg2+ and the EcoRV-DN A complex occurs at a site distinct from the catalytic center.