Abstract
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.
Highlights
The immunolabeling was specific for the higher order complexes since no colloidal gold was visible when any of the required proteins was omitted or when a plasmid with one of the Mu ends in an inverted orientation was used
Using an immunoelectron microscopic approach, we have probed the HU protein content of synaptic complexes involved in Mu DNA transposition
NaCl but was observed in greater than 90% of complexes fixed under reaction conditions (140 mM NaCl)
Summary
IZeugents-Sources were as follows: glutaraldehyde (8% in water), Polysciences Inc.; Sepharose 6B, Pharmacia LKB Biotechnology Inc.; DEAE-Affi-Gel blue, Bio-Rad; Staphylococcus aurem protein A-colloidal gold conjugate, 15-nm particle size, E. Disruption of the Type 2 complex with SDS liberates the protein-free strand-transferred product or 0 structure (Miller and Chaconas, 198e Craigie and Mizuuchi, 1985). 150 ng of cross-linked complexes, 0.6-0.9 pg of the desired antibody, and 0.25 @g/ml bovine serum albumin in Buffer A (140 mM NaCl); reactions were incubated for 30 min. A-colloidal gold particles (0.15 pg) were added, and the incubation continued a further 30 min followed by glutaraldehyde cross-linking and spin-gel filtration as described above. Fractions containing the DNA were pooled and subsequently desalted on 0.5.ml spin-gel filtration columns (Sepharose 6B in Buffer A containing 0.01% bovine serum albumin). Type 1 Complex to the Strandtransferred Product-The conversion of preformed Type 1 complexes into strand-transferred product was performed by adding target DNA (pBR322), Mu B protein, ATP, and M$+ to salt-treated The reactions were incubated for 15 min at 30 ‘C. The extent of conversion of Type 1 complex to Type 2 complex was determined by densitometric analysis of stained agarose gels as described (Surette and Chaconas, 1989)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
