Domain III function of Mu transposase analysed by directed placement of subunits within the transpososome
- 43 Downloads
Assembly of the functional tetrameric form of Mu transposase (MuA protein) at the two att ends of Mu depends on interaction of MuA with multiple att and enhancer sites on supercoiled DNA, and is stimulated by MuB protein. The N-terminal domain I of MuA harbours distinct regions for interaction with the att ends and enhancer; the C-terminal domain III contains separate regions essential for tetramer assembly and interaction with MuB protein (IIIα and IIIβ, respectively). Although the central domain II (the ‘DDE’ domain) of MuA harbours the known catalytic DDE residues, a 26 amino acid peptide within IIIα also has a non-specific DNA binding and nuclease activity which has been implicated in catalysis. One model proposes that active sites for Mu transposition are assembled by sharing structural/catalytic residues between domains II and III present on separate MuA monomers within the MuA tetramer. We have used substrates with altered att sites and mixtures of MuA proteins with either wild-type or altered att DNA binding specificities, to create tetrameric arrangements wherein specific MuA subunits are nonfunctional in II, IIIα or IIIβ domains. From the ability of these oriented tetramers to carry out DNA cleavage and strand transfer we conclude that domain IIIα or IIIβ function is not unique to a specific subunit within the tetramer, indicative of a structural rather than a catalytic function for domain III in Mu transposition.
KeywordsDNA transposition MuA transposase phage Mu transpososome assembly
Heart muscle kinase
double-end strand transfer products
single-end strand transfer products
Unable to display preview. Download preview PDF.
- Chaconas G, Lavoie B D and Watson M A 1996 DNA Transposition—jumping gene machine, some assembly required;Curr.Biol. 7 817–820Google Scholar
- Kruklitis R, Welty D J and Nakai H 1996 ClpX protein ofEscherichia coli activates bacteriophage Mu transposase in the strand transfer complex for initiation of Mu DNA synthesis;EMBO J. 15 935–944Google Scholar
- Lavoie B D and Chaconas G 1995 Transposition of phage Mu DNA;Curr. Top. Microbiol. Immunol. 204 83–102Google Scholar
- Sarnovsky R J, May E W and Craig N L 1996 The Tn7 transposase is a heteromeric complex in which DNA breakage and joining activities are distributed between different gene products;EMBO J. 15 6348–6361Google Scholar
- Surette M G, Harkness T and Chaconas G 1991 Stimulation of the Mu A protein-mediated strand cleavage reaction by the Mu B protein, and the requirement of DNA nicking for stable type 1 transpososome formation.In vitro transposition characteristics of mini-Mu plasmids carrying terminal base pair mutations;J. Biol. Chem. 266 3118–3124PubMedGoogle Scholar