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

Nucleic Acids Research
Tiina S RasilaHarri Savilahti

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 ge...Continue Reading

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Methods Mentioned

BETA
PCR
electrophoresis
NMR
X-ray

Software Mentioned

AnalySIS
UCSF Chimera
DSSP
Himar1
PyMOL
GeneCust
PyMOL Molecular Graphics System
PiggyBac
Mos1

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