Fidelity of DNA Sequence Recognition by the SfiI Restriction Endonuclease Is Determined by Communications between Its Two DNA-Binding Sites

• Published in: Journal of molecular biology 2008-12, Vol.384 (3), p.557-563
• Main Authors: Bellamy, Stuart R.W., Mina, Petros, Retter, Susan E., Halford, Stephen E.
• Format: Article
• Language: English
• Subjects: Base Sequence; Binding Sites; Deoxyribonucleases, Type II Site-Specific - genetics; Deoxyribonucleases, Type II Site-Specific - metabolism; DNA - chemistry; DNA recognition; enzyme specificity; Escherichia coli - metabolism; Molecular Sequence Data; Mutation; Nucleic Acid Conformation; Plasmids - metabolism; Protein Binding; Protein Structure, Tertiary; recognition sequence; restriction enzyme; Sequence Analysis, DNA; Substrate Specificity; tetrameric protein
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2008.09.057

The SfiI restriction endonuclease is a tetramer in which two subunits form a dimeric unit that contains one DNA binding cleft and the other two subunits contain a second cleft on the opposite side of the protein. Full activity requires both clefts to be filled with its recognition sequence: SfiI has low activity when bound to one site. The ability of SfiI to cleave non-cognate sites, one base pair different from the true site, was initially tested on substrates that lacked specific sites but which contained either one or multiple non-cognate sites. No cleavage of the DNA with one non-cognate site was detected, while a small fraction of the DNA with multiple sites was nicked. The alternative sequences were, however, cleaved in both strands, albeit at low levels, when the DNA also carried either a recognition site for SfiI or the termini generated by SfiI. Further tests employed a mutant of SfiI, altered at the dimer interface, which was known to be more active than wild-type SfiI when bound to a single site. This mutant similarly failed to cleave DNA with one non-cognate site, but cleaved the substrates with multiple non-cognate sites more readily than did the native enzyme. To cleave additional sites, SfiI thus needs to interact concurrently with either two non-cognate sites or one non-cognate and one cognate site (or the termini thereof), yet this arrangement is still restrained from cleaving the alternative site unless the communication pathway between the two DNA-binding clefts is disrupted.