Light scattering studies of the recA protein of Escherichia coli: relationship between free recA filaments and the recA X ssDNA complex.
Light scattering has been used to monitor and distinguish between two types of aggregation reactions observed with the recA protein of Escherichia coli. These are (1) the cooperative binding of recA protein to ssDNA in a pathway leading to DNA strand exchange and (2) the formation of free filaments by recA protein in the absence of DNA. Free filament formation requires Mg2+, is very sensitive to ionic strength, and occurs in the absence of single-stranded DNA and RNA. Turbidity measurements indicate that free recA filaments exhibit properties consistent with rigid rods which are 1 micron or more in length. A kinetically distinct nucleation step in free filament formation is observed under some conditions and becomes rate limiting at high pH. Ninety-degree light scattering was employed to measure binding of recA protein to ssDNA under conditions that either favor or block free filament formation. recA protein saturates ssDNA at a stoichiometric ratio of approximately four nucleotide residues per recA monomer. When free filament formation is blocked by various means, the apparent dissociation constant of the recA X ssDNA complex is approximately 10 nM. Under conditions in which free recA filaments form readily, however, the apparent dissociation constant increases to approximately 1 microM. This dramatic decrease in the observed affinity of recA protein for ssDNA under conditions that permit free filament formation does not reflect a change in the intrinsic affinity of recA protein for ssDNA. Instead, it provides evidence that free filament formation and ssDNA binding by recA protein are competing reactions.(ABSTRACT TRUNCATED AT 250 WORDS)
D004277:DNA, Single-Stranded / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008274:Magnesium / Q000494:pharmacology; D009391:Nephelometry and Turbidimetry / Q000379:methods; D009994:Osmolar Concentration; D011485:Protein Binding; D011938:Rec A Recombinases / Q000302:isolation & purification / Q000378:metabolism*; D012542:Scattering, Radiation
D004277:DNA, Single-Stranded; D011938:Rec A Recombinases; D008274:Magnesium