DNA duplexes containing altered sugar residues as probes of EcoRII and MvaI endonuclease interactions with sugar-phosphate backbone

Oligonucleotides containing 1-(beta-D-2'-deoxy-threo-pentofuranosyl)cytosin e (dCx) and/or 1-(beta-D-2'-deoxy-threo-pentofuranosyl)thymine (dT(x)) in p lace of dC and dT residues in the EcoRII and MvaI recognition site CCA/(T)G G were synthesized in order to investigate specific recognition of the DNA sugar-phosphate backbone by EcoRII and MvaI restriction endonucleases. In 2 '-deoxyxylosyl moieties of dC(x) and dT(x), 3'-hydroxyl groups were inverte d, which perturbs the related individual phosphates. Introduction of a sing le 2'-deoxyxylosyl moiety into a dC.dG pair resulted in a minor destabiliza tion of double-stranded DNA structure. In the case of a dA.dT pair the effe ct of a 2'-deoxyxylose incorporation was much more pronounced. Multiple dC( x) modifications and their combination with dT(x) did not enhance the desta bilization effect. Hydrolysis of dC(x)-containing DNA duplexes by EcoRII en donuclease was blocked and binding affinity was strongly depended on the lo cation of an altered sugar. A DNA duplex containing a dT, residue was cleav ed by the enzyme, but k(cat)/K-M was slightly reduced. In contrast, MvaI en donuclease efficiently cleaved both types of sugar-altered substrate analog s. However it did not cleave conformationally perturbed scissile bonds, whe n the corresponding unmodified bonds were perfectly hydrolyzed in the same DNA duplexes. Based on these data the possible contributions of individual phosphates in the recognition site to substrate recognition and catalysis b y EcoRII were proposed. We observed strikingly non-equivalent inputs for di fferent phosphates with respect to their effect on EcoRII-DNA complex forma tion.