COORDINATION AND INTERNAL EXCHANGE OF 2 DNA-MOLECULES IN A RECA FILAMENT IN THE PRESENCE OF HYDROLYZING ATP - INFORMATION ON ATP-RECA-DNA STRUCTURE FROM LINEAR DICHROISM SPECTROSCOPY

Solution structure of complexes between DNA and recombinase RecA from Escherchia coli, in the presence of the physiological cofactor ATP, is probed by flow linear dichroism (LD) spectroscopy. A problem of ADP accumulation which promotes dissociation of DNA-RecA is circumvented by using an ATP-regenerating system. The LD features indicate that the local structure of the complex is very similar to that found in the presence of the non-hydrolysable analog of ATP, adenosine-5'-O-[gamma-thio]triphosphate (ATP[gammaS]); the DNA bases are oriented with their planes preferentially perpendicular to the long axis of the filament, while the indole chromophores of the two tryptophan residues of RecA are rather parallel to this reference direction. A much smaller overall amplitude of the LD spectrum, compared to ATP[gammaS], is interpreted as a result of fast dissociation of RecA due to hydrolysis of ATP, producing transiently naked DNA regions which act like flexible joints, diminishing the macroscopic orientation of the RecA filaments. However, the ATP hydrolysis is not found to prevent simultaneous accommodation of two non-complementary DNA molecules in the RecA complex, as judged from the LD behaviour upon successive addition of two different polynucleotides or modified DNA strands. A notable difference from corresponding complexes formed with ATP[gammaS] is that, in the presence of ATP hydrolysis, the order in which the two DNA molecules have been added is insignificant as judged from virtually identical resulting structures; this observation indicates that exchange of DNA occurs between the two DNA accommodation sites within the RecA filament