Abstract
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.
Highlights
The DNA target sequence for the Cre protein is called loxP (Fig. 1b) and consists of two identical 13-bp inverted symmetry elements surrounding an 8-bp asymmetric sequence [18]
We find that Cre induces asymmetric bends in the loxP site, and the position of the bends is dictated by the scissile base pairs: the binding of one Cre molecule1 induces a bend near the margin of the spacer region adjacent to the scissile G nucleotide, whereas the binding of two Cre molecules induces a bend near the center of the spacer region with a slight bias toward the scissile A
Guo and colleagues [25] have proposed that the position and/or direction of the asymmetric DNA bend in the lox spacer region revealed in the crystal structure of the Cre-lox synaptic complex determine the site of initial strand exchange
Summary
The DNA target sequence for the Cre protein is called loxP (Fig. 1b) and consists of two identical 13-bp inverted symmetry elements surrounding an 8-bp asymmetric sequence [18]. The Cre-induced DNA bend in lox4, like that in loxP, was asymmetrically positioned, because the cII phasing curve for “lox4-R” was shifted by almost Ϫ1.5 bp relative to “lox4-F” (Fig. 3d). Single Symmetry Elements and cI: Wild Type Cre Bends loxP and lox4 Near the Scissile G Base Pair in cI—We wished to examine whether the asymmetric lox spacer sequence influences the Cre-induced bend in cI.
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