Lee Linda, Chu Linda C H, Sadowski Paul D
Department of Medical Genetics and Microbiology, University of Toronto, Toronto M5S 1A8, Canada.
J Biol Chem. 2003 Jun 20;278(25):23118-29. doi: 10.1074/jbc.M302272200. Epub 2003 Apr 9.
Cre initiates recombination by preferentially exchanging the bottom strands of the loxP site to form a Holliday intermediate, which is then resolved on the top strands. We previously found that the scissile AT and GC base pairs immediately 5' to the scissile phosphodiester bonds are critical in determining this order of strand exchange. We report here that the scissile base pairs also influence the Cre-induced DNA bends, the position of which correlates with the initial site of strand exchange. The binding of one Cre molecule to a loxP site induces a approximately 35 degrees asymmetric bend adjacent to the scissile GC base pair. The binding of two Cre molecules to a loxP site induces a approximately 55 degrees asymmetric bend near the center of the spacer region with a slight bias toward the scissile A. Lys-86, which contacts the scissile nucleotides, is important for establishing the bend near the scissile GC base pair when one Cre molecule is bound but has little role in positioning the bend when two Cre molecules are bound to a loxP site. We present a model relating the position of the Cre-induced bends to the order of strand exchange in the Cre-catalyzed recombination reaction.
Cre通过优先交换loxP位点的底部链来启动重组，形成霍利迪中间体，然后该中间体在顶部链上被拆分。我们之前发现，位于可切割磷酸二酯键紧邻5'端的可切割AT和GC碱基对对于确定这种链交换顺序至关重要。我们在此报告，可切割碱基对还会影响Cre诱导的DNA弯曲，其位置与链交换的起始位点相关。一个Cre分子与一个loxP位点结合会在紧邻可切割GC碱基对处诱导大约35度的不对称弯曲。两个Cre分子与一个loxP位点结合会在间隔区中心附近诱导大约55度的不对称弯曲，且稍微偏向可切割A。与可切割核苷酸接触的Lys-86，在一个Cre分子结合时对于在可切割GC碱基对附近形成弯曲很重要，但在两个Cre分子与一个loxP位点结合时对弯曲的定位作用很小。我们提出了一个模型，将Cre诱导的弯曲位置与Cre催化的重组反应中的链交换顺序联系起来。
