Visualization of DNA and DNA-protein complexes by TEM
A large class of DNA and DNA-protein complexes of great interest to modern molecular biologists lie in a realm of size and complexity that it is too large for structural approaches using X ray diffraction or NMR techniques. Such complexes are usually highly irregular so that EM methods employing the formation of 2 dimensional crystals or dense-packing followed by image averaging are not usable. Examples of such complexes include topoisomerase II heterodimers bound to supercoiled DNA, plasmid DNAs being replicated by the battery of nearly 20 different proteins that initiate and carry out replication in E. coli, complexes of reverse transcriptase enzyme degrading the RNA strand of an RNA/DNA hybrid duplex, and many of the recombinational intermediates of DNA strand exchanges. In the latter complexes large protein scaffolds built of hundreds of RecA protein monomers form filaments with in which the events of DNA strand exchange occur.To visualize such large and complex structures the demands of the biochemical reactions must be given first prior ity: is ATP, glycerol, or salt required for the on going reactions? Do the reactions occur at room temperature or only at 37 degrees? Efforts in this laboratory have been focused on determining which of the various routes for visualizing macromolecules provides the best general approach to obtaining useful structural information that can be directly related to the biochemical processes.