Structural aspects of a higher order nucleoprotein complex: induction of an altered DNA structure at the Mu-host junction of the Mu type 1 transpososome.
The Mu in vitro strand transfer reaction proceeds via two stable higher order nucleoprotein complexes, the Type 1 and Type 2 transpososomes. The Mu A protein is responsible for the structural and functional integrity of the Type 1 transpososome. We have investigated the quaternary structure of the Mu A protein within this complex by chemical cross-linking experiments and found that the basic structural unit is an A tetramer. Three Mu A binding sites in the transpososome are protected by DNase I footprinting: the outermost A binding sites L1 and R1, as well as R2. Genetic evidence is also presented which corroborates this result. Efficient formation of Type 1 complexes occurs in mini-Mus with the L3 or R3 sites deleted or when the L2 site has been substituted; but no reaction occurs in the absence of R2. The protection at the L1 and R1 sites extends 12-13 bp beyond the Mu-host junctions as seen by DNase I and methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] foot-printing, indicating Mu A contacts with the flanking host sequences in the transpososome but not on linear DNA; furthermore, hydroxyl radical footprinting shows an unprecedentedly large enhancement on the continuous strand, 2 bp beyond the nick site outside the Mu right end, which suggests that an altered DNA structure is induced upon Type 1 complex formation.
Lavoie
B D
BD
Department of Biochemistry, University of Western Ontario, London, Canada.
Chan
B S
BS
Allison
R G
RG
Chaconas
G
G
D001426:Bacterial Proteins / Q000235:genetics; D010583:Bacteriophage mu / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D016172:DNA Fingerprinting; D004269:DNA, Bacterial / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005814:Genes, Viral; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009698:Nucleoproteins / Q000235:genetics*; D009713:Nucleotidyltransferases / Q000235:genetics*; D019895:Transposases; D014764:Viral Proteins / Q000235:genetics*
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009698:Nucleoproteins; D014764:Viral Proteins; C040330:histone-like protein HU, bacteria; D009713:Nucleotidyltransferases; D019895:Transposases