Cre induces an asymmetric DNA bend in its target loxP site.
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.
Lee
Linda
L
Department of Medical Genetics and Microbiology, University of Toronto, Toronto M5S 1A8, Canada.
Chu
Linda C H
LC
Sadowski
Paul D
PD
D019943:Amino Acid Substitution; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D019175:DNA Methylation; D019426:Integrases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D011995:Recombination, Genetic; D015183:Restriction Mapping; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014764:Viral Proteins; D004247:DNA; C045073:Cre recombinase; D019426:Integrases