Analysis of two distinct single-stranded DNA binding sites on the recA nucleoprotein filament.

摘要: The binding stoichiometry of Escherichia coli recA protein to single-stranded DNA (ssDNA) determined by two separate assays differs a factor 2.2-2.4. Using the fluorescence etheno-DNA (epsilon DNA), chemically modified ssDNA, was found be 7.0 +/- 0.6 bases/recA monomer in nucleo-protein filament. competition assay, similar stoichiometry, 7.5 bases/recA, is for unmodified poly(dT). DNA-dependent ATPase recA, which monitors bound rather than DNA, we find that each needs bind only 3.1 0.5 bases fully activate ATPase. difference site size indicates there are sites with differential effects on activation. When mixed ssDNA at ratio 7 or greater, complex forms contains and acts as kinetic trap ssDNA. Upon further addition protein, no additional activity observed. If, other hand, initially excess (at 3-3.5 less) twice high. Analysis curves suggests first strand binds form filament bases/protein monomer. completely active this complex. A second can then yielding final approximately presence neither enhances nor inhibits ATP hydrolysis. This ternary may mimic structures formed searching homologous sequences and/or exchange reaction.