Site-specific recombination
Site-specific recombination systems are highly specific, fast, and efficient, even when faced with complex eukaryotic genomes.
[4] They are employed naturally in a variety of cellular processes, including bacterial genome replication, differentiation and pathogenesis, and movement of mobile genetic elements.
The names stem from the conserved nucleophilic amino acid residue present in each class of recombinase which is used to attack the DNA and which becomes covalently linked to it during strand exchange.
The earliest identified members of the serine recombinase family were known as resolvases or DNA invertases, while the founding member of the tyrosine recombinases, lambda phage integrase (using attP/B recognition sites), differs from the now well-known enzymes such as Cre (from the P1 phage) and FLP (from the yeast Saccharomyces cerevisiae).
It is within this synaptic complex that the strand exchange takes place, as the DNA is cleaved and rejoined by controlled transesterification reactions.
This phosphodiester bond between the hydroxyl group of the nucleophilic serine or tyrosine residue conserves the energy that was expended in cleaving the DNA.
The now classical members gamma-delta and Tn3 resolvase, but also new additions like φC31-, Bxb1-, and R4 integrases, cut all four DNA strands simultaneously at points that are staggered by 2 bp (Fig.
What the outcome of the reaction will be is dictated mainly by the relative locations and orientations of the sites that are to be recombined, but also by the innate specificity of the site-specific system in question.
Most site-specific systems are highly specialised, catalysing only one of these different types of reaction, and have evolved to ignore the sites that are in the "wrong" orientation.
Top: Traditional view including strand-exchange followed by branch-migration (proofreading). The mechanism occurs in the framework of a synaptic complex (1) including both DNA sites in parallel orientation. While branch-migration explains the specific homology requirements and the reversibility of the process in a straightforward manner, it cannot be reconciled with the motions recombinase subunits have to undergo in three dimensions.
Bottom: Current view. Two simultaneous strand-swaps, each depending on the complementarity of three successive bases at (or close to) the edges of the 8-bp spacer (dashed lines indicate base-pairing). Didactic complications arise from the fact that, in this model, the synaptic complex must accommodate both substrates in an anti-parallel orientation.
This synaptic complex (1) arises from the association of two individual recombinase subunits ("protomers"; gray ovals) with the respective target site. Its formation depends on inter-protomer contacts and DNA bending, which in turn define the subunits (green) with an active role during the first crossover reaction. Both representations illustrate only one half of the respective pathway. These parts are separated by a Holliday junction/isomerization step before the product (3) can be released.
Contrary to Tyr-recombinases, the four participating DNA strands are cut in synchrony at points staggered by only 2 bp (leaving little room for proofreading). Subunit-rotation (180°) permits the exchange of strands while covalently linked to the protein partner. The intermediate exposure of double-strand breaks bears risks of triggering illegitimate recombination and thereby secondary reactions.
Here, the synaptic complex arises from the association of pre-formed recombinase dimers with the respective target sites (CTD/NTD, C-/N-terminal domain). Like for Tyr-recombinases, each site contains two arms, each accommodating one protomer. As both arms are structured slightly differently, the subunits become conformationally tuned and thereby prepared for their respective role in the recombination cycle. Contrary to members of the Tyr-class the recombination pathway converts two different substrate sites (attP and attB) to site-hybrids (attL and attR) . This explains the irreversible nature of this particular recombination pathway, which can only be overcome by auxiliary "recombination directionality factors" (RDFs).
