New insights into protein-DNA interactions obtained by electron microscopy.
The electron microscopic study of DNA-protein complexes can yield valuable information that is often not easily available by other methods. In this article we give a number of examples that were chosen to illustrate the utility of electron microscopy. Along with the strategy used are protocols that allow such experiments to be carried out. The first example employs the following strategy. Points of close proximity between nucleic acid and protein within a bacteriophage or virus are made permanent by crosslinking. Bacteriophage or virus are then partially disrupted so that individual components can be visualized. With bacteriophages, such experiments show which DNA end first enters the host on infection and therefore can in principle indicate which phage genes would be first available for transcription. This type of experiment can also show which DNA end is first to be encapsulated during formation of the bacteriophage. Information on direction of encapsulation and indirectly, direction of replication of the rolling circles that lead to concatermeric DNA to be encapsulated, can also be derived. Such experiments can additionally accurately define the degree of DNA permutation, if present, within a bacteriophage population. Finally, examples are shown for in vitro reactions involving DNA, RecA, RecO, RecF, RecR, and SSB that lead to a further understanding of recombinational repair. Additionally antibody-gold labeling is used to locate various proteins in such complexes.
Schnos
M
M
Institute of Molecular Virology, University of Wisconsin, Madison 53706, USA. mschnos@facstaff.wisc.edu
Inman
R B
RB
D000327:Adsorption; D001426:Bacterial Proteins / Q000737:chemistry / Q000648:ultrastructure; D003574:Cytochrome c Group; D004247:DNA / Q000737:chemistry* / Q000648:ultrastructure*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000648:ultrastructure; D008854:Microscopy, Electron / Q000379:methods; D016253:Microscopy, Immunoelectron / Q000379:methods; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D011938:Rec A Recombinases / Q000737:chemistry / Q000648:ultrastructure; D011995:Recombination, Genetic
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D004268:DNA-Binding Proteins; D011506:Proteins; D004247:DNA; D011938:Rec A Recombinases