BPS2026 – Autoinhibition enables efficient DNA scanning and site recognition by Cre recombinase

Site-specific DNA recombinases are promising gene editing tools with the ability to manipulate DNA without producing double-strand DNA breaks, in contrast to nuclease-based methods (e.g., Cas9). The enzyme Cre (causes recombination) has been a widely used DNA recombinase in the study of human health and disease, which functions by selectively recombining DNA at cognate loxP sequences with high specificity. Its versatility is demonstrated through production of conditional knockout mouse models and targeted insertion of genes through recombinase-mediated cassette exchange technologies (RMCE). While mutant Cre enzymes have been iteratively engineered to recombine DNA at non-canonical sites, its potential as a therapeutic in human health is hindered by a lack of understanding of the site-selection process. In the absence of DNA, Cre adopts an autoinhibited conformation in which the C terminus of the protein docks in cis over the DNA binding interface. This mechanism is proposed to enhance the rate of DNA scanning by weakening interactions with DNA. Using single-molecule fluorescence microscopy in combination with optical trapping, we have measured the rates at which Cre undergoes one-dimensional translocation on long DNA substrates. We found that autoinhibition enhances the rate of DNA scanning by enabling a fast-scanning conformation on noncognate DNA. Additionally, autoinhibition has an antagonistic effect on site recognition, facilitating a two-state recognition process with short- and long-lifetime conformations at cognate sites. These findings further our understanding of how Cre locates and recognizes target sequences in a genomic context and highlights the role of the C terminus in that process.