A SYNTHETIC HOLLIDAY JUNCTION IS SANDWICHED BETWEEN 2 TETRAMERIC MYCOBACTERIUM-LEPRAE RUVA STRUCTURES IN SOLUTION - NEW INSIGHTS FROM NEUTRON-SCATTERING CONTRAST VARIATION AND MODELING

The interaction between homologous DNA molecules in recombination and DNA repair leads to the formation of crossover intermediates known as Holliday junctions. Their enzymatic processing by the RuvABC system in bacteria involves the formation of a complex between RuvA and the Hol liday junction. To study the solution structure of this complex, contr ast variation by neutron scattering was applied to Mycobacterium lepra e RuvA (MleRuvA), a synthetic analogue of a Holliday junction with 16 base-pairs in each arm, and their stable complex. Unbound MleRuvA was octameric in solution, and formed an octameric complex with the DNA ju nction. The radii of gyration at infinite contrast were determined to be 3.65 nm, 2.74 nm and 4.15 nm for MleRuvA, DNA junction and their co mplex, respectively, showing that the complex was structurally more ex tended than MleRuvA. No difference was observed in the presence or abs ence of Mg2+. The large difference in R-G values for the free and comp lexed protein in 65% (H2O)-H-2, where the DNA component is ''invisible '', showed that a substantial structural change had occurred in comple xed MleRuvA. The slopes of the Stuhrmann plots for MleRuvA and the com plex were 19 and 15 or less (x10(-5)), respectively, indicating that D NA passed through the centre of the complex. Automated constrained mol ecular modelling based on the Escherichia coli RuvA crystal structure demonstrated that the scattering curve of octameric MleRuvA in 65% and 100% (H2O)-H-2 is explained by a face-to-face association of two MleR uvA tetramers stabilised by salt-bridges. The corresponding modelling of the complex in 65% (H2O)-H-2 showed that the two tetramers are sepa rated by a void space of about 1-2 nm, which can accommodate the width of B-form DNA. Minor conformational changes between unbound and compl exed MleRuvA may occur. These observations show that RuvA plays a more complex role in homologous recombination than previously thought. (C) 1998 Academic Press.