Site‐specific Recombination


Site‐specific recombination is defined as the rearrangement of two DNA partner molecules by specific enzymes performing recombination at their cognate pairs of sequences or target sites. Site‐specific recombination, in contrast to homologous recombination, requires very little DNA homology between partner DNA molecules, is RecA‐independent, and does not involve DNA replication at any stage.

Keywords: integrase; resolvase; invertase; gene expression; chromosome segregation; lysogeny; gene targeting

Figure 1.

A chromosome is shown with two recombination target sites (the target sites shown are symmetrical). If the recombination sites are arranged in inverse orientation with respect to each other, they will recombine to create inversions of the genetic material between them. If the recombination sites are arranged in direct order, they will recombine to generate a deletion of the intervening material; this is also called resolution. When a recombination target site is present on each of two independent chromosomes (e.g. the chromosome of a host bacterium and the chromosome of a phage), recombination will result in the cointegration (the union) of the two chromosomes. When one of the genomes is much smaller, it is said to integrate into the other. In nature, all recombination sites have a specific orientation – that is, either inversion or resolution is allowed but not both.

Figure 2.

Types of recombination target sites. (a) The simplest recombination sites, for example the lox sites of bacteriophage P1, only have binding sites for the recombinase protein (in this case, Cre) flanking a short intervening region. The recombinase binding sites are placed as inverse repeats. (b) Somewhat more complex recombination sites are exemplified by the res site of the γδ resolvase protein. Within the res site, the recombinase binding sites are again present as inverse repeats, but three such pairs of binding sites are necessary for the full function of the site. The pair labelled I flanks the region where strand exchange occurs; the recombinases bound here are the only ones that mediate catalysis. The pairs of sites labelled II and III also bind resolvase, but at these sites the recombinases fulfil an architectural rather than a catalytic role. Thus, resolvase is its own accessory protein. (c) The recombination sites of bacteriophages are significantly more complicated. Temperate phage genomes (the sites of bacteriophage λ are shown) carry sites known as attP (P for phage), which recombine with sites in the host chromosome known as attB (B for bacterium). Both of these sites contain inversely repeated binding sites for the recombinase protein (in this case, Int or integrase) in the region labelled ‘core’. The recombination event occurs between these central core binding sites for Int. The recombination event is conservative – no DNA is gained or lost during the event – and creates hybrid sites known as attL and attR which flank the integrated phage genome, now called a prophage, on the left and right. The attP site is considerably more complex and roughly 10 times longer than the attB site, and contains binding sites for several accessory proteins (in the case of phage λ, the host accessory factors integration host factor (IHF) and factor for inversion stimulation (FIS), and the phage‐encoded Xis), as well as a second type of binding site, known as an arm site, for the Int protein. In the complex, Int forms bridges between the core sites and the arm sites either within a single att site or between two att sites. This property is important in pairing the two partner att sites prior to the recombination event.

Figure 3.

The pairing of some recombination sites, for example the res sites of transposons such as Tn3 or γδ, generates a very particular topological arrangement. This arrangement will only be productive (i.e. lead to recombination) if the sites start out on the same DNA molecule as direct repeats. If the sites are arranged as inverse repeats, the alignment occurs but is not productive.

Figure 4.

The recombination mechanisms of two site‐specific recombinase families. (a) The mechanism of tyrosine recombinases begins with the pairing of two recombination targets, followed by cleavage of a single DNA strand of each substrate. A tyrosine residue on the recombinase attacks a specific DNA phosphodiester bond and forms a transient covalent intermediate in which the recombinase is covalently attached to the 3′ end of the DNA. The strands with free 5′‐OH groups exchange between the two targets and the 5′‐OH mediates a nucleophilic attack on the protein–DNA covalent bond, causing release of the protein and ligating the nick. This results in formation of a Holliday junction intermediate; this type of intermediate is also seen in homologous recombination. The same series of steps is then repeated on the second set of strands of each DNA target. At the end of the reaction, two recombinant DNA molecules result, each of which contains a short region in which one strand comes from one of the parental DNA molecules while the second strand comes from the other parental molecule. Recombination is only productive if this short region is the same (homologous) in the two parental DNA molecules. (b) The mechanism of serine recombinases is different in that both strands of each DNA target are cut simultaneously. The catalytic residue is a serine rather than a tyrosine. This serine also makes a transient covalent bond to the DNA backbone, but at the 5′ end of the DNA strand rather than at the 3′ end. Strands are exchanged between the two DNA targets and the free 3′‐OH groups mediate nucleophilic attacks on the protein–DNA covalent bonds, releasing the proteins and resealing the nicks, thereby creating two recombinant DNA molecules. Note that the serine recombinases do not generate a Holliday junction intermediate during the reaction.


Further Reading

Craig NL (1988) The mechanism of conservative site‐specific recombination. Annual Review of Genetics 22: 77–105.

Gopaul DN and van Duyne GD (1999) Structure and mechanism in site‐specific recombination. Current Opinion in Structural Biology 9: 14–20.

Nash HA (1996) Site‐specific recombination: integration, excision, resolution, and inversion of defined DNA segments. In: Neidhardt FC (ed.) Escherichia coli and Salmonella: Cellular and Molecular Biology, pp. 2363–2376. Washington, DC: ASM Press.

Sherratt DJ and Wigley DB (1998) Conserved themes but novel activities in recombinases and topoisomerases. Cell 93: 149–152.

Stark WM, Boocock MR and Sherratt DJ (1992) Catalysis by site‐specific recombinases. Trends in Genetics 8: 432–439.

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Segall, Anca(May 2001) Site‐specific Recombination. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1038/npg.els.0001058]