Kim S C, Skowron P M, Szybalski W
Department of Biological Sciences, Korea Advanced Institute of Sciences and Technology, Yusong-Gu Taejon, Korea.
J Mol Biol. 1996 May 17;258(4):638-49. doi: 10.1006/jmbi.1996.0275.
The FokI restriction endonuclease recognizes the double-stranded (ds) 5'-GGATG-3' site and cuts at the 9th and 13th nucleotides downstream from the 5'-3' and 3'-5' strands, respectively. To elucidate the interaction between FokI and DNA, and the effect of Mg2+ on this interaction, we used FokI with various combinations of dsDNA, single-stranded (ss) DNA and oligodeoxyribonucleotides (oligos) containing a double-stranded hairpin carrying the FokI recognition site. Oligo- and dsDNA-FokI interactions showed that for fully effective recognition, two or more base-pairs were required outside the 5'-GGATG-3' site. When using FokI with ssDNA and oligos, precise cutting with no observable byproducts was observed at the 9th or 13th nucleotide. This was independent of whether the region between the recognition and cut sites was perfectly complementary or whether there were up to four mismatches in this region, or a single mismatch within the cut site. Moreover, FokI cleavage, when followed by step-wise filling-in of FokI cohesive ends in the dsDNA, allowed FokI to recleave such sites when two or more nucleotides were added, releasing 2-mer, 3-mer, or 4-mer single-stranded chains. Electrophoretic mobility shift assays showed that the DNA helix was bent when complexed with FokI (without Mg2+. Such a complex, when formed in the absence of Mg2+, did not accept the subsequently added Mg2+ for several minutes. This suggests a tight, diffusion-resistant contact between the enzyme and the cognate DNA sequence. In the presence of Mg2+, the half-life of the complex FokI and dsDNA was 12 minutes at 22 degrees C. In the absence of Mg2+, such a complex, possessing a terminally located 5'-GGATG-3' site, had a half-life of 1.5 to 2 minutes. However, if magnesium ions were present, this complex had a stability similar to that of a complex formed with dsDNA containing a centrally located 5'-GGATG-3' site.
FokI限制性内切核酸酶识别双链（ds）5'-GGATG-3'位点，并分别在5'-3'链和3'-5'链下游的第9和第13个核苷酸处切割。为了阐明FokI与DNA之间的相互作用以及Mg2+对这种相互作用的影响，我们使用了FokI与双链DNA、单链（ss）DNA和含有携带FokI识别位点的双链发夹的寡脱氧核糖核苷酸（oligos）的各种组合。寡核苷酸和双链DNA与FokI的相互作用表明，为了实现完全有效的识别，在5'-GGATG-3'位点之外需要两个或更多碱基对。当将FokI与单链DNA和寡核苷酸一起使用时，在第9或第13个核苷酸处观察到精确切割且无明显副产物。这与识别位点和切割位点之间的区域是否完全互补无关，也与该区域中是否存在多达四个错配或切割位点内是否存在单个错配无关。此外，当在双链DNA中逐步填充FokI粘性末端后进行FokI切割时，当添加两个或更多核苷酸时，FokI能够再次切割此类位点，释放出2聚体、3聚体或4聚体单链。电泳迁移率变动分析表明，与FokI（无Mg2+）复合时DNA螺旋发生弯曲。在没有Mg2+的情况下形成的这种复合物，在几分钟内不接受随后添加的Mg2+。这表明酶与同源DNA序列之间存在紧密的、抗扩散的接触。在Mg2+存在的情况下，FokI与双链DNA复合物在22℃下的半衰期为12分钟。在没有Mg2+的情况下，这种具有末端定位的5'-GGATG-3'位点的复合物的半衰期为1.5至2分钟。然而，如果存在镁离子，这种复合物的稳定性与由含有位于中心的5'-GGATG-3'位点的双链DNA形成的复合物相似。
