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 inhibition kinetic assays using altered substrates. Self-complementary 13-mer oligodeoxynucleotides with or without mismatch basepairs in the BanI recognition sequence (GGPyPuCC) were synthesized. UV melting curves and CD spectra indicated double-stranded B-DNA structure for all the oligomers. Among the seven oligomers with recognition sequences, GGTACC, GGTGCC, GGTATC, GGCACC, GGAGCC, GGTAAC, and GGATCC, only the first two were cleaved with BanI. Kinetics of BanI cleavage of the native substrate was inhibited competitively by all of the other oligomers except the one with sequence GGCACC. From inhibition kinetic analysis in presence of a fixed concentration of the inhibitor, 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 mismatches. Such analyses led to the conclusion that BanI, like BamHI, binds in the major groove and the central thymines make important contact with the protein.
Advani
S
S
Centre for Biotechnology, Jawaharlal Nehru University, New Delhi, 110067, India.
Roy
K B
KB
D020137:Base Pair Mismatch; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013941:Thymine / Q000737:chemistry
D009838:Oligodeoxyribonucleotides; D004247:DNA; C051612:endodeoxyribonuclease BanI; D015252:Deoxyribonucleases, Type II Site-Specific; D013941:Thymine