A novel DNA binding and nuclease activity in domain III of Mu transposase: evidence for a catalytic region involved in donor cleavage.
The Mu A protein is a 75 kDa transposase organized into three structural domains. By severing the C-terminal region (domain III) from the remainder of the protein, we unmasked a novel non-specific DNA binding and nuclease activity in this region. Deletion analysis localized both activities to a 26 amino acid stretch (aa 575-600) which remarkably remained active in DNA binding and cleavage. The two activities were shown to be tightly linked by site-directed mutagenesis. To study the importance of these activities in the transposition process, an intact mutant transposase lacking the DNA binding and nuclease activity of domain III was constructed and purified. The mutant transposase was indistinguishable from wild-type Mu A in binding affinity for both the Mu ends and the enhancer, and in strand transfer activity when the cleavage step was bypassed. In contrast, the mutant transposase displayed defects in both synapsis and donor cleavage. Our results strongly suggest that the 26 amino acid region in domain III carries catalytic residues required for donor DNA cleavage by Mu A protein. Furthermore, our data suggest that an active site for donor cleavage activity in the Mu tetramer is assembled from domain II (metal ion binding) in one A monomer and domain III (DNA cleavage) in a separate A monomer. This proposal for active site assembly is in agreement with the recently proposed domain sharing model by Yang et al. (Yang, J.Y., Kim, K., Jayaram, M. and Harshey, R.M. [1995] EMBO J., 14, 2374-2384).
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Department of Biochemistry, University of Western Ontario, London, Canada.
Chaconas
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D000595:Amino Acid Sequence; D010583:Bacteriophage mu / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009713:Nucleotidyltransferases / Q000378:metabolism*; D017384:Sequence Deletion; D019895:Transposases
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009713:Nucleotidyltransferases; D019895:Transposases; D004706:Endodeoxyribonucleases