Luetke K H, Sadowski P D
Department of Medical Genetics and Microbiology, Medical Sciences Building, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Nucleic Acids Res. 1998 Mar 15;26(6):1401-7. doi: 10.1093/nar/26.6.1401.
The Flp site-specific recombinase from Saccharomyces cerevisiae induces DNA bending upon interaction with the Flp recognition target (FRT) site. The minimal FRT site is comprised of two inverted binding elements which flank a central core region. Binding of a single monomer of Flp to DNA induces a DNA bend of 60 degrees. The position of this bend differed depending on whether the substrate contained a single binding element or a two-element FRT site. In the present work we tested and disproved a model in which a single Flp monomer interacts with both symmetry elements of a single FRT site. Likewise, we showed that a model in which a Flp monomer dissociates from a singly occupied FRT site and reassociates with the unbound element of another singly occupied FRT site during electrophoresis, does not account for the apparent shift in the position of the bend centre. It seems that the movement of a Flp monomer between the a and b elements of one FRT site during electrophoresis accounts for this anomaly. The position of the DNA bend resulting from the association of a Flp monomer with the FRT site is also influenced by the DNA sequences flanking the site. We conclude that attempts to measure the bend centre of a complex of one Flp molecule bound to a DNA containing two binding elements give misleading results. The position of the bend is more accurately measured in the presence of a single binding element.
来自酿酒酵母的Flp位点特异性重组酶与Flp识别靶标（FRT）位点相互作用时会诱导DNA弯曲。最小的FRT位点由两个反向结合元件组成，它们位于一个中央核心区域两侧。单个Flp单体与DNA结合会诱导60度的DNA弯曲。这种弯曲的位置因底物包含单个结合元件还是双元件FRT位点而有所不同。在本研究中，我们测试并否定了一个模型，即单个Flp单体与单个FRT位点的两个对称元件相互作用。同样，我们表明，在电泳过程中，一个Flp单体从单个占据的FRT位点解离并与另一个单个占据的FRT位点的未结合元件重新结合的模型，无法解释弯曲中心位置的明显移动。似乎电泳过程中Flp单体在一个FRT位点的a和b元件之间的移动解释了这种异常现象。Flp单体与FRT位点结合导致的DNA弯曲位置也受到该位点侧翼DNA序列的影响。我们得出结论，试图测量与包含两个结合元件的DNA结合的一个Flp分子复合物的弯曲中心会产生误导性结果。在存在单个结合元件的情况下，弯曲位置的测量更为准确。
