Interactions between λ Int molecules bound to sites in the region of strand exchange are required for efficient Holliday junction resolution

Abstract

lambda Site-specific recombination proceeds via two sequential single-strand exchanges that first generate and then resolve a Holliday recombination intermediate. The resolution of artificial Holliday junctions (chi-forms) is well suited to studying the mechanisms involved in reciprocal strand exchange because the linear products of this reaction are stable and easily quantitated. To study the interactions between Int molecules bound at the sites of strand exchange, artificial Holliday junctions containing only the seven base-pair overlap region and the four core-type Int binding sites were used as a model system. In vitro resolution of these structures yields products of both top- and bottom-strand exchange. An abortive product resulting from simultaneous cleavage of the top and bottom strands also occurs at low frequency. Inactivation of one of the four Int binding sites by multiple base substitutions does not significantly affect the efficiency of resolution but has a dramatic effect on the directionality, i.e. the choice of top- or bottom-strand exchange. When any two of the four core-type sites are similarly inactivated, strand exchange is very inefficient and the amount of aberrant cleavage is somewhat greater than for the Holliday junction with four intact Int binding sites. Analysis of the resolution products of Holliday junctions with various combinations of defective Int binding sites leads to the following conclusions: (1) three functional core-type Int binding sites are necessary and sufficient for a strand exchange; (2) the Int molecules that are partners in a strand exchange interact with Int bound to a "cross-core" site that is not directly involved in carrying out the reaction; (3) Int molecules bound to the core-type sites interact in a way that reduces the occurrence of abortive double-strand cleavage events.