Domain III function of Mu transposase analysed by directed placement of subunits within the transpososome |
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Authors: | Susana Mariconda Soon-Young Namgoong Ki-Hoon Yoon Hong Jiang Rasika M Harshey |
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Institution: | (1) Department of Molecular Genetics and Microbiology and Institute of Cellular and Molecular Biology, University of Texas at Austin, 78712 Austin, TX, USA |
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Abstract: | 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. |
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Keywords: | DNA transposition MuA transposase phage Mu transpososome assembly |
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