Lagunavicius Arunas, Sasnauskas Giedrius, Halford Stephen E, Siksnys Virginijus
Institute of Biotechnology, Graiciuno 8, Vilnius 2028, Lithuania.
J Mol Biol. 2003 Feb 28;326(4):1051-64. doi: 10.1016/s0022-2836(03)00020-2.
BfiI is a novel type IIs restriction endonuclease that, unlike all other restriction enzymes characterised to date, cleaves DNA in the absence of Mg(2+). The amino acid sequence of the N-terminal part of BfiI has some similarities to Nuc of Salmonella typhimurium, an EDTA-resistant nuclease akin to phospholipase D. The dimeric form of Nuc contains a single active site composed of residues from both subunits. To examine the roles of the amino acid residues of BfiI that align with the catalytic residues in Nuc, a set of alanine replacement mutants was generated by site-directed mutagenesis. The mutationally altered forms of BfiI were all catalytically inactive but were still able to bind DNA specifically. The active site of BfiI is thus likely to be similar to that of Nuc. BfiI was also found by gel-filtration to be a dimer in solution. Both gel-shift and pull-down assays indicated that the dimeric form of BfiI binds two copies of its recognition sequence. In reactions on plasmids with either one or two copies of its recognition sequence, BfiI cleaved the DNA with two sites more rapidly than that with one site. Yet, when bound to two copies of its recognition sequence, the BfiI dimer cleaved only one phosphodiester bond at a time. The dimer thus seems to contain two DNA-binding domains but only one active site.
BfiI是一种新型的IIS型限制性内切酶，与迄今已鉴定的所有其他限制性酶不同，它在没有Mg(2+)的情况下切割DNA。BfiI N端部分的氨基酸序列与鼠伤寒沙门氏菌的Nuc有一些相似之处，Nuc是一种类似于磷脂酶D的耐EDTA核酸酶。Nuc的二聚体形式包含一个由两个亚基的残基组成的单一活性位点。为了研究BfiI中与Nuc催化残基对齐的氨基酸残基的作用，通过定点诱变产生了一组丙氨酸替代突变体。BfiI的突变形式均无催化活性，但仍能特异性结合DNA。因此，BfiI的活性位点可能与Nuc的相似。通过凝胶过滤还发现BfiI在溶液中是二聚体。凝胶迁移和下拉试验均表明，BfiI的二聚体形式结合其识别序列的两个拷贝。在具有一个或两个识别序列拷贝的质粒反应中，BfiI切割具有两个位点的DNA比切割具有一个位点的DNA更快。然而，当与两个识别序列拷贝结合时，BfiI二聚体一次仅切割一个磷酸二酯键。因此，二聚体似乎包含两个DNA结合结构域，但只有一个活性位点。
