DNA Binding Induces a cis to trans Switch in Cre Recombinase to Enable Intasome Assembly

Mechanistic understanding of DNA recombination in the Cre-loxP system has largely been guided by crystallographic structures of tetrameric synaptic complexes. Those studies have suggested a role for protein conformational dynamics that has not been well characterized at the atomic level. We used solution NMR to discover the link between intrinsic flexibility and enzyme function in Cre recombinase. Remarkably, in the absence of DNA the C-terminal helix αN, implicated in assembly of synaptic complexes and regulation of DNA cleavage activity via trans protein-protein interactions, is found to adopt an apparent auto-inhibitory cis conformation. Binding to loxP DNA dislodges the C-terminus from this cis conformation, thereby enabling the trans protein-protein interactions required for assembly of recombinogenic Cre intasomes. These findings necessitate a re-examination of the mechanisms by which this widely utilized gene-editing tool selects target sites, avoids spurious DNA cleavage activity, and controls DNA recombination efficiency.