J Biol Chem, Vol. 273, Issue 22, 13886-13891, May 29, 1998

Recombinase Catalyzes Inversion and Resolution between Two Inversely Oriented six Sites on a Supercoiled DNA Substrate and Only Inversion on Relaxed or Linear Substrates

Inés Canosa, Rudi Lurz^§, Fernando Rojo, and Juan C. Alonso

From the  Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, C.S.I.C., Campus de la Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain and the ^§ Max-Planck-Institut für molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Federal Republic of Germany

The  recombinase, in the presence of a chromatin-associated protein such as Hbsu, catalyzes DNA resolution or DNA inversion on supercoiled substrates containing two directly or inversely oriented six sites. Hbsu stabilizes the formation of the recombination complex (Alonso, J. C., Weise, F., and Rojo, F. (1995) J. Biol. Chem. 270, 2938-2945). In this study we show that resolution by  recombinase strictly requires supercoiled DNA, but inversion does not. On a substrate with two inversely oriented six sites,  recombinase catalyzed both resolution and inversion if the DNA was supercoiled but only inversion if the substrate was relaxed or linear. Hbsu was critical for the formation of synaptic complexes; its concentration relative to that of the supercoiled DNA substrate determined whether resolution or inversion products were preferentially formed. The results suggest that the  recombinase forms unproductive short-lived synaptic complexes between two juxtaposed inversely oriented six sites; the presence of 3 to 13 Hbsu dimers per supercoiled DNA molecule would stabilize a synaptic complex with a relative geometry of the six sites allowing  recombinase preferentially to achieve resolution. Supercoiling probably helps to overcome an energetic barrier, since resolution does not occur in relaxed DNA. The presence of >30 Hbsu dimers per DNA molecule probably favors the formation of a recombination complex with a different geometry since the reaction is directed preferentially toward DNA inversion.