Site-specific Recombination - Mechanism

Mechanism

Recombination between two DNA sites begins by the recognition and binding of these sites by the recombinase protein. This is followed by the synapsis i.e. bringing the sites together to form the synaptic complex. It is within this synaptic complex that the strand exchange takes place, as the DNA is cleaved and rejoined by controlled transesterification reactions. During strand exchange, the DNA cut at fixed points within crossover region of the site releases a deoxyribose hydroxyl group, while recombinase protein forms a transient covalent bond to a DNA backbone phosphate. This phosphodiester bond between hydroxyl group of the nucleophilic, serine or tyrosine residue conserves the energy that was expended in cleaving the DNA. Energy stored in this bond is subsequently used for the rejoining of the DNA to the corresponding deoxyribose hydroxyl group on the other site. The entire process therefore goes through without the need for external energy rich cofactors such as ATP.

Although the basic chemical reaction is the same for both tyrosine and serine recombinases there are marked differences. Tyrosine recombinases, such as Cre or Flp, cleave one DNA strand at a time at points that are staggered by 6-8bp, linking 3’ end of DNA to the hydroxyl group of the tyrosine nucleophile (Fig. 1). Strand exchange then proceeds via a crossed strand intermediate analogous to the Holliday junction in which only one pair of strands has been exchanged.

The mechanism and control of serine recombinases is much less well understood. This group of enzymes was only discovered in the mid 1990es and is still relatively small. 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 2bp (Fig. 2). During cleavage protein-DNA bond is formed via transesterification reaction in which a phosphodiester bond is replaced by a phosphoserine bond between a 5’ phosphate at the cleavage site and hydroxyl group of the conserved serine residue (S10) in resolvase. It is still not entirely clear how the strand exchange occurs after the DNA has been cleaved. However, it has been shown that the strands are exchanged while covalently linked to the protein with a resulting net rotation of 180°. The most quoted (but not the only) model accounting for these facts is the "subunit rotation model" (Fig. 2). Independent of the model DNA duplexes are situated outside of the protein complex, and large movement of protein is needed to achieve the strand exchange. In this case the recombination sites are slightly asymmetric, which allows the enzyme to tell apart the left and right ends of the site. When generating products left ends are always joined to the right ends of their partner sites and vice versa. This causes different recombination hybrid sites to be reconstituted in the recombination products. Joining of left ends to left or right to right is avoided due to the asymmetric “overlap” sequence between the staggered points of top and bottom strand exchange, which is in stark contrast to the mechanism employed by the tyrosine recombinases

The reaction catalysed, for instance by Cre-recombinase may lead to excision of the DNA segment flanked by the two sites (Fig. 3A), but may also lead to integration or inversion of the orientation of the flanked DNA segment (Fig. 3B). What the outcome of the reaction will be, is dictated mainly by the relative location and the orientation of sites that are to be recombined, but also by the innate specificity of the site-specific system in question. Excisions and inversions occur if the recombination takes place between two sites that are found on the same molecule (intramolecular recombination), and if the sites are in the same (direct repeat) or in an opposite orientation (inverted repeat), respectively. Insertions on the other hand take place if the recombination occurs on sites that are situated on two different DNA molecules (intermolecular recombination), provided that at least one of these molecules is circular. 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.