Bellamy Stuart R W, Mina Petros, Retter Susan E, 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. 2008 Dec 19;384(3):557-63. doi: 10.1016/j.jmb.2008.09.057. Epub 2008 Oct 2.
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.
SfiI限制性内切酶是一种四聚体，其中两个亚基形成一个二聚体单元，该单元包含一个DNA结合裂隙，另外两个亚基在蛋白质的另一侧包含第二个裂隙。完整的活性要求两个裂隙都被其识别序列填满：SfiI与一个位点结合时活性较低。SfiI切割与真实位点相差一个碱基对的非同源位点的能力，最初是在缺乏特定位点但含有一个或多个非同源位点的底物上进行测试的。未检测到含有一个非同源位点的DNA被切割，而含有多个位点的DNA中有一小部分被切口。然而，当DNA还携带SfiI的识别位点或由SfiI产生的末端时，但替代序列在两条链中都被切割，尽管切割水平较低。进一步的测试使用了一种在二聚体界面发生改变的SfiI突变体，已知该突变体在与单个位点结合时比野生型SfiI更具活性。该突变体同样未能切割含有一个非同源位点的DNA，但比天然酶更容易切割含有多个非同源位点的底物。因此，为了切割额外的位点，SfiI需要同时与两个非同源位点或一个非同源位点和一个同源位点（或其末端）相互作用，然而，除非两个DNA结合裂隙之间的通讯途径被破坏，这种排列仍然无法切割替代位点。
