TY - JOUR
T1 - DNA topology and geometry in Flp and Cre recombination
AU - Vetcher, Alexandre A.
AU - Lushnikov, Alexander Y.
AU - Navarra-Madsen, Junalyn
AU - Scharein, Robert G.
AU - Lyubchenko, Yuri L.
AU - Darcy, Isabel K.
AU - Levene, Stephen D.
PY - 2006/4/7
Y1 - 2006/4/7
N2 - The Flp recombinase of yeast and the Cre recombinase of bacteriophage P1 both belong to the λ-integrase (Int) family of site-specific recombinases. These recombination systems recognize recombination-target sequences that consist of two 13 bp inverted repeats flanking a 6 or 8 bp 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.
AB - The Flp recombinase of yeast and the Cre recombinase of bacteriophage P1 both belong to the λ-integrase (Int) family of site-specific recombinases. These recombination systems recognize recombination-target sequences that consist of two 13 bp inverted repeats flanking a 6 or 8 bp 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.
KW - DNA looping
KW - DNA topology
KW - Protein-DNA interactions
KW - Site-specific recombination
KW - Tangle analysis
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U2 - 10.1016/j.jmb.2006.01.037
DO - 10.1016/j.jmb.2006.01.037
M3 - Article
C2 - 16483600
AN - SCOPUS:33644832221
VL - 357
SP - 1089
EP - 1104
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 4
ER -