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Nucleic Acids Res. 2018 May 18;46(9):4649-4661. doi: 10.1093/nar/gkx1281.

Mu transpososome activity-profiling yields hyperactive MuA variants for highly efficient genetic and genome engineering.

Author information

1
Division of Genetics and Physiology, Department of Biology, FI-20014 University of Turku, Turku, Finland.
2
Institute of Biotechnology, Viikki Biocenter, P. O. Box 56, FI-00014 University of Helsinki, Helsinki, Finland.
3
Division of Biochemistry and Biotechnology, Department of Biosciences, FI-00014 University of Helsinki, Helsinki, Finland.
4
Department of Biochemistry, FI-20014 University of Turku, Turku, Finland.
5
Department of Experimental Medical Science, Lund University, SE-221 84, Lund, Sweden.
6
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA.

Abstract

The phage Mu DNA transposition system provides a versatile species non-specific tool for molecular biology, genetic engineering and genome modification applications. Mu transposition is catalyzed by MuA transposase, with DNA cleavage and integration reactions ultimately attaching the transposon DNA to target DNA. To improve the activity of the Mu DNA transposition machinery, we mutagenized MuA protein and screened for hyperactivity-causing substitutions using an in vivo assay. The individual activity-enhancing substitutions were mapped onto the MuA-DNA complex structure, containing a tetramer of MuA transposase, two Mu end segments and a target DNA. This analysis, combined with the varying effect of the mutations in different assays, implied that the mutations exert their effects in several ways, including optimizing protein-protein and protein-DNA contacts. Based on these insights, we engineered highly hyperactive versions of MuA, by combining several synergistically acting substitutions located in different subdomains of the protein. Purified hyperactive MuA variants are now ready for use as second-generation tools in a variety of Mu-based DNA transposition applications. These variants will also widen the scope of Mu-based gene transfer technologies toward medical applications such as human gene therapy. Moreover, the work provides a platform for further design of custom transposases.

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