Poster abstracts
Poster number 39 submitted by Kye Stachowski
Stepwise DNA bending accommodates assembly of Cre recombination complexes
Kye Stachowski (Department of Chemistry and Biochemistry), Andrew Norris (Department of Chemistry and Biochemistry), Devante Potter (Department of Microbiology), Vicky Wysocki (Department of Chemistry and Biochemistry), Mark Foster (Department of Chemistry and Biochemistry)
Abstract:
Gene editing technologies are a hot topic due to the rise of the CRISPR-Cas system, but due to off-target effects and unwanted DNA double strand breaks (DSBs), less error prone systems should be explored. Cre recombinase is a site-specific, tyrosine recombinase that catalytically recombines DNA without creating DSBs, utilizes no cofactors, and functions with single-nucleotide precision.1 An activated, tetrameric complex forms when two Cre protomers bind to one, 34-base pair, loxP DNA sequence that contains two Cre binding sites, followed by antiparallel assembly of two such Cre2-loxP complexes.2 Crystallographic studies of tetrameric structures of Cre show that the loxP substrates are bent at ~ 108°, and the complexes feature many interprotomer protein-protein contacts, hallmarks of an activated complex.3,4 Structural details regarding assembly and activation of Cre pre-tetrameric intermediate structures are unknown.
Here we used protein engineering to isolate Cre-loxP and Cre2-loxP complexes. Then we determined their structures along with a tetrameric complex using cryo-EM to resolutions of 5.1Å, 4.5Å and 3.2Å, respectively. We found that as Cre assembles into an activated complex, the bend of the loxP site becomes more pronounced as each intermediate is reached. The progressive DNA bending is accompanied by increased protein-protein interactions. Our work shows how tetramerization is required for Cre to become activated to recombine DNA. We also used 3D variability analysis to uncover motion in the tetramer that shows how the protein-protein interface plasticity is important for activation of Cre. These new insights could prove useful in design of new Cre variants with engineered site-specificity and improved recombination efficiency.
References:
1. Abremski, K. & Hoess, R. Bacteriophage P1 site-specific recombination. Purification and properties of the Cre recombinase protein. J. Biol. Chem. 259, 1509–14 (1984).
2. Ringrose, L. et al. Comparative kinetic analysis of FLP and cre recombinases: mathematical models for DNA binding and recombination. J. Mol. Biol. 284, 363–384 (1998).
3. Guo, F., Gopaul, D. N. & Van Duyne, G. D. Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse. Nature 389, 40–46 (1997).
4. Ghosh, K., Guo, F. & Van Duyne, G. D. Synapsis of loxP sites by cre recombinase. J. Biol. Chem. 282, 24004–24016 (2007).
Keywords: Cryo-EM, DNA Bending, Cre Recombinase