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Mol Ther. 2016 Aug;24(8):1369-77. doi: 10.1038/mt.2016.110. Epub 2016 Jun 6.

Structural Determinants of Sleeping Beauty Transposase Activity.

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MRC Human Genetics Unit, Institute of Genetics & Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, UK.
Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary.
Department of Pediatrics and Genetics, Stanford University School of Medicine, Stanford, California, USA.
Present address: Gilead Sciences Inc., Foster City, California, USA.
Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
Instistute of Genetics, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary.
Max Delbrück Center for Molecular Medicine, Berlin, Germany.
European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany.
Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany.


Transposases are important tools in genome engineering, and there is considerable interest in engineering more efficient ones. Here, we seek to understand the factors determining their activity using the Sleeping Beauty transposase. Recent work suggests that protein coevolutionary information can be used to classify groups of physically connected, coevolving residues into elements called "sectors", which have proven useful for understanding the folding, allosteric interactions, and enzymatic activity of proteins. Using extensive mutagenesis data, protein modeling and analysis of folding energies, we show that (i) The Sleeping Beauty transposase contains two sectors, which span across conserved domains, and are enriched in DNA-binding residues, indicating that the DNA binding and endonuclease functions of the transposase coevolve; (ii) Sector residues are highly sensitive to mutations, and most mutations of these residues strongly reduce transposition rate; (iii) Mutations with a strong effect on free energy of folding in the DDE domain of the transposase significantly reduce transposition rate. (iv) Mutations that influence DNA and protein-protein interactions generally reduce transposition rate, although most hyperactive mutants are also located on the protein surface, including residues with protein-protein interactions. This suggests that hyperactivity results from the modification of protein interactions, rather than the stabilization of protein fold.

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