Talk Abstracts

Talk on Tuesday 04:15-04:30pm submitted by Aparna Unnikrishnan

Conformational switch in the C-terminal α-helix of Cre upon binding to loxP DNA

Aparna Unnikrishnan (The Ohio State University), Carlos Amero (The Ohio State University )

Abstract:
Cre recombinase catalyzes site-specific recombination of DNA. This well-studied member of the Tyrosine Recombinase family of phage-derived enzymes does not require auxiliary proteins, high-energy co-factors or downstream repair mechanisms to bind a pair of loxP DNA sequences and to catalyze strand exchange, insertion or excision. For these reasons, Cre recombinase has been widely used as a gene-editing tool. To initiate recombination, two Cre molecules cooperatively bind to a loxP site and further assemble with another Cre₂-loxP to form a tetrameric synaptic Cre₄-loxP₂ complex. The need to coordinate a series of events including DNA bending, selective strand cleavage, strand exchange and re-ligation implicates protein conformational changes (i.e., dynamics) in the progression of the Cre-loxP recombination reaction. We characterized the motions, timescales and structural basis for protein dynamics in the Cre-loxP system at atomic resolution using solution NMR spectroscopy and other biophysical techniques. Backbone ¹⁵N T₁, T_1ρ and {¹H}-¹⁵N hetNOE NMR relaxation measurements on the catalytic domain of Cre in the absence and presence of the DNA substrate identified regions that exhibit dynamics on the ps-ns timescales. These regions map to those observed in the crystal structures to mediate significant protein-protein or protein-DNA contacts in the tetrameric state. Moreover, NMR structural studies provide new insights into a cis to trans docking molecular switch of the C-terminal α-helix upon binding to loxP, aiding in the site selection and cooperative binding mechanisms.

Keywords: Cre-loxP, Solution NMR, Protein dynamics