DNA cleavage at two recognition sites by the SfiI restriction endonuclease: salt dependence of cis and trans interactions between distant DNA sites.
At low ionic strength, the SfiI restriction enzyme cleaved at similar rates both supercoiled and linear DNA with two SfiI sites and linear DNA with one SfiI site. For the substrates with two sites, the majority of the DNA was converted directly to products cut at both sites; the enzyme appears to bind to two sites before catalyzing its reactions, looping out the intervening DNA. At high ionic strength, linear DNA with one SfiI site was not cut at all, linear DNA with two sites was cleaved slowly while supercoiled DNA with two sites was cleaved rapidly, though only half of the DNA with two sites was cut at both sites; the DNA that had been cut at one site was not cleaved again at the remaining site. The singly cut product must therefore have been generated by a reaction incorporating both sites. All DNA cleavage reactions by SfiI thus involve the tetrameric enzyme bound to two copies of its recognition sequence, but weakened DNA-protein interactions at high ionic strength can cause this complex to dissociate before cleaving both sites. Intramolecular interactions between distant DNA sites are generally thought to be enhanced by supercoiling and to be more stable than intermolecular interactions. The preference of SfiI at high ionic strength for substrates with two sites over substrates with one site and, in the former case, for supercoiled over linear DNA, validates this view. At low ionic strength, the similar rates with the different substrates may be due to rate-limiting product dissociation.
Nobbs
T J
TJ
Department of Biochemistry, Centre for Molecular Recognition, School of Medical Sciences, University of Bristol, UK.
Halford
S E
SE
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010957:Plasmids / Q000378:metabolism; D012965:Sodium Chloride; D013379:Substrate Specificity
D004278:DNA, Superhelical; D012965:Sodium Chloride; D004247:DNA; C041749:endodeoxyribonuclease SfiI; D015252:Deoxyribonucleases, Type II Site-Specific