Altering the DNA-binding specificity of Mu transposase in vitro
Open Access
- 1 August 1998
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 26 (15), 3521-3527
- https://doi.org/10.1093/nar/26.15.3521
Abstract
We describe the isolation of a variant of Mu transposase (MuA protein) which can recognize altered att sites at the ends of Mu DNA. No prior knowledge of the structure of the DNA binding domain or its mode of interaction with att DNA was necessary to obtain this variant. Protein secondary structure programs initially helped target mutations to predicted helical regions within a subdomain of MuA demonstrated to harbor att DNA binding activity. Of the 54 mutant positions examined, only two showed decreased affinity for att DNA, while eight others affected assembly of the Mu transpososome. A variant impaired in DNA binding [MuA(R146V)], and predicted to be in the recognition helix of an HTH motif, was challenged with altered att sites created from degenerate oligonucleotides to select for novel DNA binding specificity. DNA sequences bound to MuA(R146V) were detected by gel-retardation, and following several steps of PCR amplification/enrichment, were identified by cloning and sequencing. The strategy allowed recovery of an altered att site for which MuA(R146V) showed higher affinity than for the wild-type site, although this site was bound by wild-type MuA as well. The altered association between MuA(R146V) and an altered att site target was competent in transposition. We discuss the strengths and limitations of this methodology, which has applications in dissecting the functional role of specific protein-DNA associations.Keywords
This publication has 35 references indexed in Scilit:
- Mutational analysis of domain IIβ of bacteriophage Mu transposase: domains IIα and IIβ belongs to different catalytic complementation groupsJournal of Molecular Biology, 1998
- Solution structure of the Iγ subdomain of the Mu end DNA-binding domain of phage Mu transposaseJournal of Molecular Biology, 1997
- DNA transposition: Assembly of a jumping gene machineCurrent Biology, 1996
- Mu Transpositional Recombination: Donor DNA Cleavage and Strand Transfer in trans by the Mu TransposaseCell, 1996
- Assembly and Orientation of Flp Recombinase Active Sites on Two-, Three- and Four-armed DNA Substrates: Implications for a Recombination MechanismJournal of Molecular Biology, 1996
- DNA-Protein Cooperativity in the Assembly and Stabilization of Mu Strand Transfer Complex: Relevance of DNA Phasing and att Site CleavageJournal of Molecular Biology, 1994
- Division of labor among monomers within the Mu transposase tetramerCell, 1993
- Efficient Mu transposition requires interaction of transposase with a DNA sequence at the Mu operator: Implications for regulationCell, 1989
- Interaction of distinct domains in Mu transposase with Mu DNA ends and an internal transpositional enhancerNature, 1989
- Transpososomes: Stable protein-DNA complexes involved in the in vitro transposition of bacteriophage Mu DNACell, 1987