Single-molecule analysis of πc31 integrase-mediated site-specific recombination by tethered particle motion

Abstract

Serine and tyrosine site-specific recombinases (SRs and YRs, respectively) provide templates for understanding the chemical mechanisms and conformational dynamics of strand cleavage/exchange between DNA partners. Current evidence suggests a rather intriguing mechanism for serine recombination, in which one half of the cleaved synaptic complex undergoes a 180° rotation relative to the other. The 'small' and 'large' SRs contain a compact aminoterminal catalytic domain, but differ conspicuously in their carboxyl-Terminal domains. So far, only one serine recombinase has been analyzed using single substrate molecules. We now utilized singlemolecule tethered particle motion (TPM) to follow step-by-step recombination catalyzed by a large SR, phage πC31 integrase. The integrase promotes unidirectional DNA exchange between attB and attP sites to integrate the phage genome into the host chromosome. The recombination directionality factor (RDF; πC31 gp3) activates the excision reaction (attL x attR). From integrase-induced changes in TPM in the presence or absence of gp3, we delineated the individual steps of recombination and their kinetic features. The gp3 protein appears to regulate recombination directionality by selectively promoting or excluding active conformations of the synapse formed by specific att site partners. Our results support a 'gated rotation' of the synaptic complex between DNA cleavage and joining.