A switch in the mechanism of communication between the two DNA-binding sites in the SfiI restriction endonuclease.
While many Type II restriction enzymes are dimers with a single DNA-binding cleft between the subunits, SfiI is a tetramer of identical subunits. Two of its subunits (a dimeric unit) create one DNA-binding cleft, and the other two create a second cleft on the opposite side of the protein. The two clefts bind specific DNA cooperatively to give a complex of SfiI with two recognition sites. This complex is responsible for essentially all of the DNA-cleavage reactions by SfiI: virtually none is due to the complex with one site. The communication between the DNA-binding clefts was examined by disrupting one of the very few polar interactions in the otherwise hydrophobic interface between the dimeric units: a tyrosine hydroxyl was removed by mutation to phenylalanine. The mutant protein remained tetrameric in solution and could bind two DNA sites. But instead of being activated by binding two sites, like wild-type SfiI, it showed maximal activity when bound to a single site and had a lower activity when bound to two sites. This interaction across the dimer interface thus enforces in wild-type SfiI a cooperative transition between inactive and active states in both dimers, but without this interaction as in the mutant protein, a single dimer can undergo the transition to give a stable intermediate with one inactive dimer and one active dimer.
Bellamy
Stuart R W
SR
The DNA-Protein Interactions Unit, Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK. stuart.bellamy@bristol.ac.uk
Milsom
Susan E
SE
Kovacheva
Yana S
YS
Sessions
Richard B
RB
Halford
Stephen E
SE
D019943:Amino Acid Substitution; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D007700:Kinetics; D011487:Protein Conformation; D021122:Protein Subunits
D021122:Protein Subunits; D004247:DNA; C041749:endodeoxyribonuclease SfiI; D015252:Deoxyribonucleases, Type II Site-Specific