Lee G, Saito I
Laboratory of Molecular Genetics, Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Gene. 1998 Aug 17;216(1):55-65. doi: 10.1016/s0378-1119(98)00325-4.
The Cre recombinase mediates precise site-specific recombination between a pair of loxP sequences through an intermediate containing Holiday junction. The recombination junction in the loxP sequence is located within the asymmetric 8-nucleotide spacer region. To examine the role of each nucleotide sequence of the spacer region in the recombination process, we synthesized a complete set of 24 loxP spacer mutants with single-base substitutions and 30 loxP spacer mutants with double-base substitutions. Each synthesized loxP mutant was ligated at both ends of a linear DNA or to one end of a DNA-containing wild-type loxP at the other end and their recombination efficiencies were analyzed with an in vitro system. The sequence identity of the right two nucleotides and left four nucleotides in the central six bases of the spacer region was found to be essential for formation and resolution, respectively, of the intermediate product. Furthermore, even when homology was maintained, the recombination efficiencies were lower than that of wild-type loxP and varied among mutants. Based on this knowledge, we identified two loxP mutants with double-base substitutions, mutants 5171 and 2272, which recombine efficiently with an identical mutant but not with the other mutant or wild-type loxP.
Cre重组酶通过包含霍利迪连接体的中间体介导一对loxP序列之间的精确位点特异性重组。loxP序列中的重组连接点位于不对称的8核苷酸间隔区。为了研究间隔区每个核苷酸序列在重组过程中的作用，我们合成了一套完整的24个单碱基替换的loxP间隔区突变体和30个双碱基替换的loxP间隔区突变体。每个合成的loxP突变体连接在线性DNA的两端或一端与另一端含野生型loxP的DNA相连，并使用体外系统分析它们的重组效率。发现间隔区中央六个碱基中右侧两个核苷酸和左侧四个核苷酸的序列一致性分别对于中间产物的形成和拆分至关重要。此外，即使保持同源性，重组效率也低于野生型loxP，并且在突变体之间有所不同。基于这一认识，我们鉴定出两个双碱基替换的loxP突变体，即突变体5171和2272，它们能与相同的突变体高效重组，但不能与其他突变体或野生型loxP重组。
