Bellamy Stuart R W, Milsom Susan E, Kovacheva Yana S, Sessions Richard B, Halford Stephen E
The DNA-Protein Interactions Unit, Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK.
J Mol Biol. 2007 Nov 9;373(5):1169-83. doi: 10.1016/j.jmb.2007.08.030. Epub 2007 Aug 21.
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.
虽然许多II型限制酶是二聚体，亚基之间有一个单一的DNA结合裂隙，但SfiI是由相同亚基组成的四聚体。它的两个亚基（一个二聚体单元）形成一个DNA结合裂隙，另外两个亚基在蛋白质的另一侧形成第二个裂隙。这两个裂隙协同结合特定的DNA，形成一个具有两个识别位点的SfiI复合物。这个复合物基本上负责SfiI的所有DNA切割反应：实际上，几乎没有反应是由具有一个位点的复合物引起的。通过破坏二聚体单元之间原本疏水界面中极少数的极性相互作用之一来研究DNA结合裂隙之间的通讯：通过将酪氨酸羟基突变为苯丙氨酸来去除。突变蛋白在溶液中仍保持四聚体状态，并且可以结合两个DNA位点。但是，与野生型SfiI不同，它不是通过结合两个位点而被激活，而是在结合单个位点时表现出最大活性，在结合两个位点时活性较低。因此，在野生型SfiI中，这种跨二聚体界面的相互作用在两个二聚体中强制实现非活性状态和活性状态之间的协同转变，但是在突变蛋白中没有这种相互作用时，单个二聚体可以发生转变，形成一个具有一个非活性二聚体和一个活性二聚体的稳定中间体。
