Immunoelectron microscopic analysis of the A, B, and HU protein content of bacteriophage Mu transpososomes.
Stable protein-DNA complexes or transpososomes mediate the Mu DNA strand transfer reaction in vitro (Surette, M. G., Buch, S. J., and Chaconas, G. (1987) Cell 49, 253-262; Craigie, R., and Mizuuchi, K. (1987) Cell 51, 493-501). Formation of the Type 1 complex, an intermediate in the strand transfer reaction, requires the Mu A and Escherichia coli HU proteins. Generation of the Type 2 complex, in which the Mu ends have been covalently linked to the target DNA, requires the Mu B protein, ATP, and target DNA in addition to A and HU. The protein content of these higher order synaptic complexes has been studied by immunoelectron microscopy using protein A-colloidal gold conjugates to visualize antibody-bound complexes. Under our in vitro transposition conditions, Type 1 complexes were found to contain A and HU; in addition, Type 2 complexes contained Mu B. However, both the HU and the Mu B protein were found to be loosely associated and could be quantitatively removed from the nucleoprotein core of both complexes by incubation in 0.5 M NaCl. Depletion of HU from the Type 1 complex did not affect the ability of this complex to be converted into the strand-transferred product. Hence, the indispensable role of the HU protein in the Mu DNA strand transfer reaction is limited to the formation of the Type 1 transpososome.
Lavoie
B D
BD
Department of Biochemistry, University of Western Ontario, London, Canada.
Chaconas
G
G
D001426:Bacterial Proteins / Q000378:metabolism; D010583:Bacteriophage mu / Q000032:analysis* / Q000648:ultrastructure; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000378:metabolism; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D009698:Nucleoproteins / Q000378:metabolism / Q000648:ultrastructure; D014764:Viral Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D009698:Nucleoproteins; D014764:Viral Proteins; C040330:histone-like protein HU, bacteria