Binding stoichiometry and structure of RecA-DNA complexes studied by flow linear dichroism and fluorescence spectroscopy. Evidence for multiple heterogeneous DNA co-ordination

Abstract

The interaction between RecA and DNA (in the form of unmodified single-stranded DNA, fluorescent single-stranded DNA and double-stranded DNA) is studied with linear dichroism and fluorescence spectroscopy. RecA is found to form a complex with single-stranded DNA with a binding stoichiometry of about four nucleotides per RecA monomer, in which the DNA bases appear to have a random orientation. Addition of ATP gamma S (a non-hydrolyzable analog of ATP) reduces the stoichiometry to about three nucleotides per RecA and causes the DNA bases to adopt an orientation preferentially perpendicular to the fiber axis. This complex can incorporate an additional strand of single-stranded DNA or double-stranded DNA, yielding a total stoichiometry of six nucleotides or three nucleotides and three base-pairs, respectively, per RecA. RecA, in the presence of ATP gamma S, is also found to interact with double-stranded DNA, with a stoichiometry of about three base-pairs per RecA. In all studied complexes, the tryptophan residues in the RecA protein are oriented with their planes preferentially parallel to the fiber axis, whereas in complexes involving ATP gamma S the planes of the DNA bases are oriented preferentially perpendicular to the fiber. This virtually excludes the possibility that the tryptophan residues are intercalated in the DNA helix. On the basis of these results, a model for the research of homology in the RecA-mediated, strand-exchange reaction in the genetic recombination process is proposed.