Synapsis and DNA cleavage in fC31 integrase- mediated site-speci®c recombination

The Streptomyces phage fC31 encodes an integrase belonging to the serine recombinase family of sitespeci®c recombinases. The well studied serine recombinases, the resolvase/invertases, bring two recombination sites together in a synapse, and then catalyse a concerted four-strand staggered break in the DNA substrates whilst forming transient covalent attachments with the recessed 5¢ ends. Rotation of one pair of half sites by 180 relative to the other pair occurs, to form the recombinant con®guration followed by ligation of the DNA backbone. Here we address the nature of the recombination intermediates formed by fC31 integrase when acting on its substrates attP and attB. We have identi®ed intermediates containing integrase covalently attached to cleaved DNA substrates, attB or attP, by analysis of complexes in gels and after treatment of these complexes with proteinases. Using a catalytically inactive integrase mutant, S12A, the synaptic complexes containing integrase, attP and attB were identi®ed. Furthermore, we have shown that attB mutants containing insertions or deletions are blocked in recombination at the stage of strand cleavage. Thus, there is a strict spacing requirement within attB, possibly for correct positioning of the catalytic serine relative to the scissile phosphate in the active site. Finally, using integrase S12A we con®rmed the inability of attL and attR or other combinations of sites to form a stable synapse, indicating that the directionality of integrative recombination is determined at synapsis.