Identification of a defined epitope on the surface of the active RecA-DNA filament using a monoclonal antibody and three-dimensional reconstruction.
Studies of the Escherichia coli RecA protein are expected to illuminate mechanisms of DNA recombination and repair in bacteria, and in all higher organisms as well, due to the functional and structural homology with the eukaryotic Rad51 protein. The active form of the RecA protein is a helical filament formed on DNA in the presence of ATP or ATP analogs, and this has been studied at low-resolution by electron microscopy. An atomic model of the protein comes from an X-ray crystallographic study of a filament formed in the absence of DNA and ATP. This filament is believed to be an inactive, storage form of the protein. A key step in generating an atomic model of the active filament, and a detailed model for function, is to understand the large conformational changes that occur between these two states. Towards this end, we have decorated active RecA-DNA filaments with monoclonal antibodies (ARM191) against a known epitope (residues 285 to 320) to determine the position of this epitope in the low-resolution structure. Electron microscopy and three-dimensional reconstruction of the RecA-antibody complex reveal that the lobe containing the epitope is very disordered on the surface of the filament, but in a position similar to that in the inactive crystal filament. The antibody binding also induces a significant conformational change in the RecA filament. This study shows that the basic orientation of the subunit is likely to be similar within the inactive and active filaments, and that the large movement of mass that occurs between these two states must involve other residues than the 285-320 region.
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Department of Cell Biology and Neuroanatomy, University of Minnesota Medical School, Minneapolis, MN, 55455, USA.
Shibata
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Egelman
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D000818:Animals; D000911:Antibodies, Monoclonal; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D000939:Epitopes / Q000737:chemistry / Q000648:ultrastructure; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D011938:Rec A Recombinases / Q000737:chemistry* / Q000276:immunology* / Q000648:ultrastructure; D011995:Recombination, Genetic
D000911:Antibodies, Monoclonal; D004269:DNA, Bacterial; D000939:Epitopes; D011938:Rec A Recombinases