DNA looping by the Sfi I restriction endonuclease.
The SfiI endonuclease has to interact with two copies of its recognition sequence before it can cleave DNA. To demonstrate that the reaction of SfiI on a DNA with two sites involves the formation of a DNA loop, and to characterise the looping interactions on supercoiled and linear DNA, a series of plasmids was constructed with lengths of DNA between two SfiI sites varying from 104 to 211 bp. Both supercoiled and linear forms of each DNA were tested as substrates for SfiI. The reactions were monitored from the rates of DNA cleavage and from the generation of partially cleaved products, the latter indicating loop disruption before cleavage of both sites. On both supercoiled and linear DNA, the stabilities of the complexes spanning two SfiI sites varied in sinusoidal fashion with the distance between the sites, in the manner characteristic of a process governed by the helical periodicity of DNA. In all cases, the looping interaction was stabilised by DNA supercoiling. The sinusoidal variation from SfiI reactions on supercoiled DNA at 50 degreesC yielded a helical repeat of about 11.5 base-pairs per turn.
Department of Biochemistry, School of Medical Sciences, Bristol, BS8 1TD, UK.
L M
LM
Wentzell
S E
SE
Halford
D004247:DNA / Q000737:chemistry*; D004278:DNA, Superhelical / Q000737:chemistry; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011487:Protein Conformation
D004278:DNA, Superhelical; D004247:DNA; C041749:endodeoxyribonuclease SfiI; D015252:Deoxyribonucleases, Type II Site-Specific