Mechanism of Inhibition of Site-specific Recombination by the Holliday Junction-trapping Peptide WKHYNY: Insights into Phage λ Integrase-mediated Strand Exchange

Abstract

Holliday junctions are central intermediates in site-specific recombination reactions mediated by tyrosine recombinases. Because these intermediates are extremely transient, only artificially assembled Holliday junctions have been available for study. We have recently identified hexapeptides that cause the accumulation of natural Holliday junctions of bacteriophage λ Integrase (Int)-mediated reactions. We now show that one of these peptides acts after the first DNA cleavage event to stabilize protein-bound junctions and to prevent their resolution. The peptide acts before the step affected by site affinity ( saf) mutations in the core region, in agreement with a model that the peptide stabilizes the products of strand exchange (i.e. Holliday junctions) while saf mutations reduce ligation of exchanged strands. Strand exchange events leading to Holliday junctions in phage λ integration and excision are asymmetric, presumably because interactions between Int and some of its core-binding sites determine the order of strand cleavage. We have compared the structure of Holliday junctions in one unidirectional and in two bidirectional Int-mediated pathways and show that the strand cleavage steps are much more symmetric in the bidirectional pathways. Thus Int–DNA interactions which determine the order of top and bottom strand cleavage and exchange are unique in each recombination pathway.