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Tyrosine vs. Serine recombinases
Tyrosine vs. Serine recombinases
Site-specific recombinases have been classified as SRs and YRs, based on whether they utilize an active site serine or tyrosine as the nucleophile for DNA strand cleavage.
Site-specific recombinases have been classified as SRs and YRs, based on whether they utilize an active site serine or tyrosine as the nucleophile for DNA strand cleavage.
Tyrosine recombinase
Tyrosine recombinase
Tyrosine recombinases perform strand break and relegation one strand by one strand with a Holliday junction as the reaction intermediate.
Serine recombinase
Serine recombinase
Serine recombinases cut four strands in the att recognition sites simultaneously before strand exchange and relegation. Unlike tyrosine integrases, serine integrases mediate integration (attbB x attP) without accessory protein, and the attachment sites are relatively short sequences of <50 bp. A second phage-encoded protein, the recombination directionality factor (RDF) is required to activate excision whilst inhibit integration.
In this method, a specific length DNA molecule is bound between the coverglass surface and a nanometer-sized polystyrene bead. The tethered bead will perform Brownian motion and the movement will be recorded by the imaging system. The imaging method is DIC system, and the temporal resolution and spatial resolution are highly depending on the signal counts from polystyrene beads.
Experimental design
Experimental design
The ~1300 bp dsDNA with recognition sites on specific position is anchored on the coverglass by digoxigenin-antidigoxigenin interaction. The other end of DNA is tethered with streptavidin-labeled nanoparticles. The movement of NP is recorded under DIC system. The image is analyzed by a 2D Gaussian fitting program, and the DNA length change caused by recombinase-DNA interaction can be obtained by converting bead’s Brownian amplitude into corresponding DNA length using the estimated calibration curve (Brownian motion amplitude of tethered bead vs. Tethered DNA length). The whole process (synapsis, strand cleavage and exchange) can be recorded and studied.
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