Phoebe Rice is a Professor in the Department of Biochemisty & Molecular Biology at the University of Chicago
Title: Large serine recombinases: how do they know which way to go?
Abstract: Large serine recombinases are promising genetic tools because they can catalyze very large DNA insertions, inversions, and deletions in an extremely tidy fashion, leaving no broken phosphodiester bonds and requiring no host DNA repair. Efficient, unidirectional insertion requires only a tetramer of integrase and two DNA sites: an ~40-46 bp attB and an ~50-54 bp attP site. Expression of the phage-encoded RDF (recombination directionality factor) protein, which binds the integrase itself, triggers the reverse (excision) reaction and inhibits integration.
Our 8 cryoEM structures of a large serine recombinase, in complex with DNA substrates and products and with and without its cognate RDF, reveal how these powerful genetic tools work in 3D. These structures answer a long-standing thermodynamic puzzle: the net number of high-energy bonds in the product is the same as that in the substrate, so how do these enzymes drive their reactions to near completion? Furthermore, how do they “decide” which pairs of sites to pair as substrates and in what relative orientation, and how does the RDF flip the reaction direction?
We are also interested in expanding the toolkit of verified integrase – RDF pairs for use as synthetic biology tools. Surprisingly, although the RDFs encoded by a wide variety of phages are all predicted to bind to the same region of their cognate integrase proteins, they share no universally conserved sequence or predicted structural motifs, nor do their genes show consistent synteny with integrase genes. Finding RDFs by sequence alone has therefore been a bottleneck in the field. We found that AlphaFold2-multimer can be used to perform “virtual pulldowns” to identify putative RDFs. Wet lab testing of these predictions shows a high rate of true positives.
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