Domain III function of Mu transposase analysed by directed placement of subunits within the transpososome.
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 (IIIalpha and IIIbeta, respectively). Although the central domain II (the 'DDE' domain) of MuA harbours the known catalytic DDE residues, a 26 amino acid peptide within IIIalpha 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, IIIalpha or IIIbeta domains. From the ability of these oriented tetramers to carry out DNA cleavage and strand transfer we conclude that domain IIIalpha or IIIbeta 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.
Mariconda
S
S
Department of Molecular Genetics and Microbiology and Institute of Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
Namgoong
S Y
SY
Yoon
K H
KH
Jiang
H
H
Harshey
R M
RM
D000596:Amino Acids / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D004247:DNA / Q000378:metabolism; D008954:Models, Biological; D008956:Models, Chemical; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009698:Nucleoproteins / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019895:Transposases / Q000737:chemistry*
D000596:Amino Acids; D009698:Nucleoproteins; D004247:DNA; C114697:mu transposase; D019895:Transposases