Dynamic light scattering investigations of RecA self-assembly and interactions with single strand DNA.
Dynamic light scattering (DLS) measurements were performed on self-assembled solutions of RecA as a function of assembly time under strand exchange ionic strength conditions (10 mM MgCl2, 65 mM NaCl, 10 mM Tris-HCl, pH = 7.5, 1 mM DTT, 3-4 microM RecA) in the absence of ATP. These measurements yield distributions of the translational diffusion coefficients of the changing populations of assembling protein species. Interpretations of results of DLS measurements are made in terms of model hydrodynamic calculations that indicate, under the solution conditions employed, the smallest fundamental quaternary subunit of RecA is a hexamer in a toroidal or lock-washer configuration. Interactions of M13mp19 circular single strand DNA (ssDNA) with RecA assembled to different stages were also investigated. Additions of ssDNA to self-assembled solutions of RecA acts to dissociate the associated structures into hexamer subunits. However, the effect of ssDNA on assembled RecA is highly dependent on the RecA self-assembly state. The longer the assembly time, the less reversible the self-assembled structures of RecA become. Binding isotherms of titrated mixtures of ssDNA with RecA self-assembled to various stages were also determined. Evaluated dissociation constants of RecA/ssDNA complexes were found to increase with increases of the associated state of RecA. These results strongly suggest that, under the solvent conditions employed, the active ssDNA binding form of RecA is a hexamer.
Benight
A S
AS
Department of Chemistry, University of Illinois, Chicago 60680.
Wilson
D H
DH
Budzynski
D M
DM
Goldstein
R F
RF
D000255:Adenosine Triphosphate / Q000494:pharmacology; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004270:DNA, Circular / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004926:Escherichia coli / Q000032:analysis; D007700:Kinetics; D008027:Light; D046911:Macromolecular Substances; D009994:Osmolar Concentration; D011938:Rec A Recombinases / Q000737:chemistry / Q000378:metabolism*; D012542:Scattering, Radiation; D012996:Solutions
D004270:DNA, Circular; D004277:DNA, Single-Stranded; D046911:Macromolecular Substances; D012996:Solutions; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases