Structure of helical RecA-DNA complexes. II. Local conformational changes visualized in bundles of RecA-ATP gamma S filaments.
Complexes of RecA-DNA filaments, formed in the presence of a non-hydrolyzable ATP analog, ATP gamma S, aggregate together into regular bundles in the presence of Mg2+. Electron micrographs of several different forms of RecA-double-stranded DNA bundles have been analyzed: bundles of six supercoiled filaments at two different concentrations of Mg2+, and bundles of three supercoiled filaments at a single concentration of Mg2+. The bundles are all characterized by a regular left-handed supercoiling of the component filaments arising from the non-integral number of RecA subunits per turn of the RecA helix in these aggregates, about 6.15 units/turn. When single-stranded DNA is used instead of double-stranded DNA, regular aggregates composed of many filaments are formed. These aggregates do not supercoil, consistent with a symmetry of the component filaments of close to 6.0 units/turn. These different structures have provided a strong confirmation of the analysis of isolated RecA filaments. Since different RecA protomers within the component filaments of these aggregates are in different environments, they have provided a direct view of different conformations that RecA subunits may adopt within the same filament as a result of nonequivalent contacts. The conformational changes we have visualized are quite large, with apparent movements of mass over distances greater than 2 nm. The RecA-mediated strand exchange reaction is a highly dynamic process, which involves both the unwinding and stretching of DNA, in addition to the physical movement of DNA strands. It is quite likely, therefore, that the different conformations of RecA subunits seen in these aggregates represent different states of RecA during its enzymatic strand exchange activity.
Egelman
E H
EH
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511.
Stasiak
A
A
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D004269:DNA, Bacterial; D046911:Macromolecular Substances; D008274:Magnesium; D008854:Microscopy, Electron; D008954:Models, Biological; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D011938:Rec A Recombinases
D004269:DNA, Bacterial; D046911:Macromolecular Substances; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D011938:Rec A Recombinases; D008274:Magnesium