DNA topology and geometry in Flp and Cre recombination.
The Flp recombinase of yeast and the Cre recombinase of bacteriophage P1 both belong to the lambda-integrase (Int) family of site-specific recombinases. These recombination systems recognize recombination-target sequences that consist of two 13bp inverted repeats flanking a 6 or 8bp spacer sequence. Recombination reactions involve particular geometric and topological relationships between DNA target sites at synapsis, which we investigate using nicked-circular DNA molecules. Examination of the tertiary structure of synaptic complexes formed on nicked plasmid DNAs by atomic-force microscopy, in conjunction with detailed topological analysis using the mathematics of tangles, shows that only a limited number of recombination-site topologies are consistent with the global structures of plasmids bearing directly and inversely repeated sites. The tangle solutions imply that there is significant distortion of the Holliday-junction intermediate relative to the planar structure of the four-way DNA junction present in the Flp and Cre co-crystal structures. Based on simulations of nucleoprotein structures that connect the two-dimensional tangle solutions with three-dimensional models of the complexes, we propose a recombination mechanism in which the synaptic intermediate is characterized by a non-planar, possibly near-tetrahedral, Holliday-junction intermediate. Only modest conformational changes within this structure are needed to form the symmetric, planar DNA junction, which may be characteristic of shorter-lived intermediates along the recombination pathway.
Institute of Biomedical Sciences and Technology and Department of Molecular and Cell Biology, The University of Texas at Dallas, Richardson, TX 75083, USA.
Alexandre A
AA
Vetcher
Alexander Y
AY
Lushnikov
Junalyn
J
Navarra-Madsen
Robert G
RG
Scharein
Yuri L
YL
Lyubchenko
Isabel K
IK
Darcy
Stephen D
SD
Levene
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004254:DNA Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism; D019426:Integrases / Q000737:chemistry* / Q000378:metabolism; D046911:Macromolecular Substances; D008433:Mathematics; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
D046911:Macromolecular Substances; D014764:Viral Proteins; D004247:DNA; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D019426:Integrases