Effect of mutations in the Mu-host junction region on transpososome assembly.
Mu transposition occurs through a series of higher-order nucleoprotein complexes called transpososomes. The region where the Mu DNA joins the host DNA plays an integral role in the assembly of these transpososomes. We have created a series of point mutations at the Mu-host junction and characterized their effect on the Mu in vitro strand transfer reaction. Analysis of these mutant constructs revealed an inhibition in transpososome assembly at the point in the reaction pathway when the junction region is engaged by the transposase active site (i.e. the transition from LER to type 0). We found that the degree of inhibition was dependent upon the particular base-pair change at each position and whether the substitution occurred at the left or right transposon end. The MuB transposition protein, an allosteric effector of MuA, was shown to suppress all of the inhibitory Mu-host junction mutants. Most of the mutant constructs were also suppressed, to varying degrees, by the substitution of Mg(2+) with Mn(2+). Analysis of the mutant constructs has revealed hierarchical nucleotide preferences at positions -1 through +3 for transpososome assembly and suggests the possibility that specific metal ion-DNA base interactions are involved in DNA recognition and transpososome assembly.
Coros
C J
CJ
The Department of Biochemistry, University of Western Ontario, London, Ontario, N6A 5C1, Canada.
Chaconas
G
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D001287:Attachment Sites, Microbiological / Q000235:genetics*; D001426:Bacterial Proteins / Q000378:metabolism; D010583:Bacteriophage mu / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D034982:Integration Host Factors; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D009154:Mutation / Q000235:genetics*; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary / Q000187:drug effects; D021122:Protein Subunits; D011995:Recombination, Genetic / Q000235:genetics; D019895:Transposases / Q000737:chemistry* / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D034982:Integration Host Factors; D046911:Macromolecular Substances; C069215:MuB protein, Enterobacteria phage Mu; D021122:Protein Subunits; D014764:Viral Proteins; C040330:histone-like protein HU, bacteria; D008345:Manganese; D019895:Transposases; D008274:Magnesium