Role of the A protein-binding sites in the in vitro transposition of mu DNA. A complex circuit of interactions involving the mu ends and the transpositional enhancer.
To investigate the role of the A protein-binding sites at the Mu ends in the DNA strand transfer reaction, we constructed mutant mini-Mu molecules in which these sites were deleted (L3 or R3) or substituted (L2 or R2) to conserve the spacing arrangements at the adjacent sites. The single site mutants are poor substrates for phosphodiester bond hydrolysis at the Mu ends in Type 1 reactions in the absence of Escherichia coli integration host factor (IHF). Addition of IHF to the reaction stimulates Type 1 cleavage more than 10 times for the delta-R3, delta-L3, S-L2 mutants and more than five times in the case of the S-R2 mutant under alternate conditions. The site of IHF stimulation resides within the transpositional enhancer which implicates the end-binding sites L2, L3, R2, and R3 in interactions with the enhancer. At least two of the L2, L3, and R3 sites are required for proficient reaction in the presence of IHF. By combining the single site mutants with O1 or O2 partially deleted enhancer elements, we have tentatively localized some of the interactions to each side of the functional enhancer revealing a complex circuit of end-enhancer interactions. The R3 site is suggested to be involved in interactions only with O2 and the L3 site only with O1. The data also suggest the possibility that L2 and R2 may be involved in interactions with both O1 and O2. Finally, our working model predicts that the L3-O1 and R3-O2 interactions may be required contacts for discriminating between the Mu left and right ends in transpososome formation.
Allison
R G
RG
Department of Biochemistry, University of Western Ontario, London, Canada.
Chaconas
G
G
D001426:Bacterial Proteins / Q000378:metabolism; D010583:Bacteriophage mu / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D004278:DNA, Superhelical / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D034982:Integration Host Factors; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D019895:Transposases
D001426:Bacterial Proteins; D004278:DNA, Superhelical; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D034982:Integration Host Factors; D009713:Nucleotidyltransferases; D019895:Transposases