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Molecular and Cellular Biology, October 2004, p. 9239-9247, Vol. 24, No. 20
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.20.9239-9247.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Mutational Analysis of the N-Terminal DNA-Binding Domain of Sleeping Beauty Transposase: Critical Residues for DNA Binding and Hyperactivity in Mammalian Cells

Stephen R. Yant, Julie Park, Yong Huang, Jacob Giehm Mikkelsen, and Mark A. Kay*

Departments of Pediatrics and Genetics, Stanford University School of Medicine, Stanford, California

Received 13 May 2004/ Returned for modification 6 July 2004/ Accepted 21 July 2004

The N-terminal domain of the Sleeping Beauty (SB) transposase mediates transposon DNA binding, subunit multimerization, and nuclear translocation in vertebrate cells. For this report, we studied the relative contributions of 95 different residues within this multifunctional domain by large-scale mutational analysis. We found that each of four amino acids (leucine 25, arginine 36, isoleucine 42, and glycine 59) contributes to DNA binding in the context of the N-terminal 123 amino acids of SB transposase, as indicated by electrophoretic mobility shift analysis, and to functional activity of the full-length transposase, as determined by a quantitative HeLa cell-based transposition assay. Moreover, we show that amino acid substitutions within either the putative oligomerization domain (L11A, L18A, L25A, and L32A) or the nuclear localization signal (K104A and R105A) severely impair its ability to mediate DNA transposition in mammalian cells. In contrast, each of 10 single amino acid changes within the bipartite DNA-binding domain is shown to greatly enhance SB's transpositional activity in mammalian cells. These hyperactive mutations functioned synergistically when combined and are shown to significantly improve transposase affinity for transposon end sequences. Finally, we show that enhanced DNA-binding activity results in improved cleavage kinetics, increased SB element mobilization from host cell chromosomes, and dramatically improved gene transfer capabilities of SB in vivo in mice. These studies provide important insights into vertebrate transposon biology and indicate that Sleeping Beauty can be readily improved for enhanced genetic research applications in mammals.

* Corresponding author. Mailing address: Stanford University, Department of Pediatrics, 300 Pasteur Dr., Room G305, Stanford, CA 94305-5208. Phone: (650) 498-6531. Fax: (650) 498-6540. E-mail: markay{at}stanford.edu.

Molecular and Cellular Biology, October 2004, p. 9239-9247, Vol. 24, No. 20
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.20.9239-9247.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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