Structure of the Holliday junction intermediate in Cre-loxP site-specific recombination.
We have determined the X-ray crystal structures of two DNA Holliday junctions (HJs) bound by Cre recombinase. The HJ is a four-way branched structure that occurs as an intermediate in genetic recombination pathways, including site-specific recombination by the lambda-integrase family. Cre recombinase is an integrase family member that recombines 34 bp loxP sites in the absence of accessory proteins or auxiliary DNA sequences. The 2.7 A structure of Cre recombinase bound to an immobile HJ and the 2.5 A structure of Cre recombinase bound to a symmetric, nicked HJ reveal a nearly planar, twofold-symmetric DNA intermediate that shares features with both the stacked-X and the square conformations of the HJ that exist in the unbound state. The structures support a protein-mediated crossover isomerization of the junction that acts as the switch responsible for activation and deactivation of recombinase active sites. In this model, a subtle isomerization of the Cre recombinase-HJ quaternary structure dictates which strands are cleaved during resolution of the junction via a mechanism that involves neither branch migration nor helical restacking.
Department of Biochemistry and Biophysics and Johnson Research Foundation, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
D N
DN
Gopaul
F
F
Guo
G D
GD
Van Duyne
D019943:Amino Acid Substitution; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry*; D019426:Integrases / Q000737:chemistry*; D007536:Isomerism; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D011487:Protein Conformation; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D014764:Viral Proteins
D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins; D004247:DNA; C045073:Cre recombinase; D019426:Integrases