Fidelity of DNA sequence recognition by the SfiI restriction endonuclease is determined by communications between its two DNA-binding sites.
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.
The DNA-Protein Interactions Unit, Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK.
Stuart R W
SR
Bellamy
Petros
P
Mina
Susan E
SE
Retter
Stephen E
SE
Halford
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D004247:DNA; C041749:endodeoxyribonuclease SfiI; D015252:Deoxyribonucleases, Type II Site-Specific