title: Single-molecule analysis of ϕC31 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 amino-terminal 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 single-molecule tethered particle motion (TPM) to follow step-by-step recombination catalyzed by a large SR, phage ϕC31 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; ϕC31 gp3) activates the excision reaction (attL × 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.
authors.affiliation: Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, 112, Taiwan hffan2@ym.edu.tw.|Biophotonics and Molecular Imaging Research Center, National Yang-Ming University, 112, Taiwan.
authors.forename: Hsiu-Fang
authors.initials: HF
authors.lastname: Fan
authors.affiliation: Institute of Cellular and Organismic Biology Academia Sinica, 115, Taiwan.
authors.forename: Tao-Shih
authors.initials: TS
authors.lastname: Hsieh
authors.affiliation: Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712, USA.
authors.forename: Chien-Hui
authors.initials: CH
authors.lastname: Ma
authors.affiliation: Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712, USA.
authors.forename: Makkuni
authors.initials: M
authors.lastname: Jayaram
mesh_terms: D001287:Attachment Sites, Microbiological; D001435:Bacteriophages / Q000201:enzymology; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli; D019426:Integrases / Q000737:chemistry*; D007700:Kinetics; D011485:Protein Binding; D011995:Recombination, Genetic; D000072760:Single Molecule Imaging; D014764:Viral Proteins / Q000737:chemistry*
chemical_list: D004269:DNA, Bacterial; D014764:Viral Proteins; D019426:Integrases