BanI restriction endonuclease binds in the major groove of DNA: An inhibition kinetic study using substrates with mismatch basepairs

Structural information on BanI-DNA interaction was obtained from simple inh ibition kinetic assays using altered substrates. Self-complementary 13-mer oligodeoxynucleotides with or without mismatch basepairs in the BanI recogn ition sequence (GGPy-PuCC) were synthesized. UV melting curves and CD spect ra indicated double-stranded B-DNA structure for all the oligomers. Among t he seven oligomers with recognition sequences, GGTACC, GGTGCC, GGTATC, GGCA CC, GGAGCC, GGTAAC, and GGATCC, only the first two were cleaved with BanI. Kinetics of BanI cleavage of the native substrate was inhibited competitive ly by all of the other oligomers except the one with sequence GGCACC. From inhibition kinetic analysis in presence of a fixed concentration of the inh ibitor, apparent K-m and K-I were determined. The data were analyzed in the context of alterations made in the hydrogen bonding potential in the major and minor groove of DNA within the recognition sequence due to basepair mi smatches. Such analyses led to the conclusion that BanI, like BamHI, binds in the major groove and the central thymines make important contact with th e protein. (C) 2000 Academic Press.