Looney M C, Moran L S, Jack W E, Feehery G R, Benner J S, Slatko B E, Wilson G G
New England Biolabs, Inc., Beverly, MA 01915.
Gene. 1989 Aug 15;80(2):193-208. doi: 10.1016/0378-1119(89)90284-9.
The genes for FokI, a type-IIS restriction-modification system from Flavobacterium okeanokoites (asymmetric recognition sequence: 5'-GGATG/3'-CCTAC), were cloned into Escherichia coli. Recombinants carrying the fokIR and fokIM genes were found to modify their DNA completely, and to restrict lambdoid phages weakly. The nt sequences of the genes were determined, and the probable start codons were confirmed by aa sequencing. The FokI endonuclease (R.FokI) and methyltransferase (M.FokI) are encoded by single, adjacent genes, aligned in the same orientation, in the order M then R. The genes are large by the standards of type-II systems, 1.9 kb for the M gene, and 1.7 kb for the R gene. Preceding each gene is a pair of FokI recognition sites; it is conceivable that interactions between the sites and the FokI proteins could regulate expression of the genes. The aa sequences of the N- and C-terminal halves of M.FokI are similar to one another, and to certain other DNA-adenine methyltransferases, suggesting that the enzyme has a 'tandem' structure, such as could have arisen by the fusion of a pair of adjacent, ancestral M genes. Truncated derivatives of M. FokI were constructed by deleting the 5'- or 3'-ends of the fokIM gene. Deleting most of the C-terminus of M.FokI produced derivatives that methylated only the top (GGATG) strand of the recognition sequence. Conversely, deleting most of the N-terminus produced derivatives that methylated only the bottom (CATCC) strand of the recognition sequence. These results indicate that the domains in M.FokI for methylating the two strands of the recognition sequence are largely separate.
将来自大洋黄杆菌的IIS型限制修饰系统FokI（不对称识别序列：5'-GGATG/3'-CCTAC）的基因克隆到大肠杆菌中。发现携带fokIR和fokIM基因的重组体可完全修饰其DNA，并对类λ噬菌体有较弱的限制作用。测定了这些基因的核苷酸序列，并通过氨基酸测序确认了可能的起始密码子。FokI核酸内切酶（R.FokI）和甲基转移酶（M.FokI）由单个相邻基因编码，以相同方向排列，顺序为M然后是R。按照II型系统的标准，这些基因很大，M基因1.9 kb，R基因1.7 kb。每个基因之前都有一对FokI识别位点；可以想象，这些位点与FokI蛋白之间的相互作用可能会调节基因的表达。M.FokI的N端和C端的氨基酸序列彼此相似，并且与某些其他DNA腺嘌呤甲基转移酶相似，这表明该酶具有“串联”结构，例如可能是由一对相邻的祖先M基因融合产生的。通过删除fokIM基因的5'端或3'端构建了M.FokI的截短衍生物。删除M.FokI的大部分C端产生的衍生物仅甲基化识别序列的顶部（GGATG）链。相反，删除大部分N端产生的衍生物仅甲基化识别序列的底部（CATCC）链。这些结果表明，M.FokI中用于甲基化识别序列两条链的结构域在很大程度上是分开的。
