Symmetric DNA sites are functionally asymmetric within Flp and Cre site-specific DNA recombination synapses.
Flp and Cre-mediated recombination on symmetrized FRT and loxP sites, respectively, in circular plasmid substrates yield both DNA inversion and deletion. However, upon sequestering three negative supercoils outside the recombination complex using the resII-resIII synapse formed by Tn3 resolvase and the LER synapse formed by phage Mu transposase in the case of Flp and Cre, respectively, the reactions are channeled towards inversion at the expense of deletion. The inversion product is a trefoil, its unique topology being conferred by the external resolvase or LER synapse. Thus, Flp and Cre assign their symmetrized substrates a strictly antiparallel orientation with respect to strand cleavage and exchange. These conclusions are supported by the product profiles from tethered parallel and antiparallel native FRT sites in dilution and competition assays. Furthermore, the observed recombination bias favoring deletion over inversion in a nicked circular substrate containing two symmetrized FRT sites is consistent with the predictions from Monte Carlo simulations based on antiparallel synapsis of the DNA partners.
Grainge
Ian
I
Section of Molecular Genetics and Microbiology and the Institute of Cell and Molecular Biology, University of Texas at Austin, 78712, USA.
Pathania
Shailja
S
Vologodskii
Alexander
A
Harshey
Rasika M
RM
Jayaram
Makkuni
M
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004278:DNA, Superhelical / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019426:Integrases / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009010:Monte Carlo Method; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism*
D004278:DNA, Superhelical; D014764:Viral Proteins; D004247:DNA; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D019426:Integrases