Definitions

cleavage site

Site-specific recombination

Site-specific recombination

In site-specific-recombination, DNA strand exchange takes place between segments possessing only a limited degree of sequence homology (Kolb 2002; Coates et al., 2005). Site-specific recombinases perform rearrangements of DNA segments by recognising and binding to short DNA sequences (sites), at which they: cleave the DNA backbone, exchange the two DNA helices involved and rejoin the DNA strands.

While in some site-specific recombination systems having just a recombinase enzyme together with the recombination sites is perfectly adequate to be able to perform all these reactions, in some other systems a number of accessory proteins and accessory sites are also needed.

The recombination sites are typically between 30 and 200 nucleotides in length and consist of two motifs with a partial inverted-repeat symmetry, to which the recombinase binds, and which flank a central crossover sequence at which the recombination takes place. The pairs of sites between which the recombination occurs are usually identical, but there are exceptions e.g. attP and attB of λ integrase (Landy 1989)(see lambda phage).

The reaction catalysed by the recombinase may lead to the excision of the DNA segment between flanked by the two sites, but also to the integration or inversion of the orientation of the flanked DNA segment. 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. In nature site-specific recombination systems are highly specific, fast and efficient, even when faced with complex eukaryotic genomes (Bode et al., 2000; Sauer 1998). As such, they are employed in number of processes such as: bacterial genome replication, differentiation and pathogenesis, movement of genetic elements such as transposons, plasmids, phages and integrons (Nash 1996) and present an attractive starting material for development of potential genetic engineering tools (Akopian and Stark 2005).

Classification of site-specific recombinases: tyrosine vs. serine recombinases

Based on amino acid sequence homology and mechanistic relatedness most site-specific recombinases are grouped into one of two families: the tyrosine recombinase family or the serine recombinase family. The names stem from the conserved nucleophilic amino acid residue that they use to attack the DNA and which becomes covalently linked to it during strand exchange. Serine recombinase family is also sometimes known as resolvase/invertase family, while tyrosine recombinases are known as the integrase family, which reflects the types of reaction that most known members in each family have evolved to catalyse. Typical examples of tyrosine recombinases are the well known enzymes such as Cre (from the P1 phage), FLP (from yeast S. cerevisiae) and λ integrase (lambda phage) while famous serine recombinases include enzymes such as: gamma-delta resolvase (from the Tn1000 transposon), Tn3 resolvase (from the Tn3 transposon) and φC31 integrase (from the φC31 phage) (Nash 1996; Stark and Boocock 1995). Although the individual members of the two recombinase families can perform reactions with same practical outcomes, the two families are unrelated to each other, having different protein structures and reaction mechanisms. Unlike tyrosine recombinases, serine recombinases are highly modular as was first hinted by the biochemnical studies (Abdel-Meguid et al., 1984) and later shown by crystallographic structures (Yang and Steitz, 1995; Li et al., 2005); a fact which could prove useful when attempting to reengineer these proteins as tools for genetic manipulation.

Mechanism and the chemistry of the site-specific DNA recombination reaction

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. As stated previously, 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 the recombination 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. Left-left or right-right half-site recombinants would contain mismatched base pairs (Stark and Boocock 1995). 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 the time at points that are staggered by 6-8bp, linking 3’ end of DNA to the hydroxyl group of the tyrosine nucleophile (Van Duyne 2002). Strand exchange than proceeds via a crossed strand intermediate analogous to the Holliday junction (Holliday 1964; Grainge and Jayaram 1999) in which only one pair of strands has been exchanged. Conversely, serine recombinases like gamma-delta and Tn3 resolvase cut all four DNA strands simultaneously at points that are staggered by 2bp (Stark et al., 1992). 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 (Reed and Grindley 1981; Reed and Moser 1984). 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° (Stark et al., 1989; Stark and Boocock 1994). Two current models can account for this, namely the subunit rotation model and the domain swapping model (Sarkis et al., 2001). In both of these models DNA duplexes are situated outside of the protein complex, and large movement of protein is needed to achieve the strand exchange. This is in stark contrast to the mechanism employed by the tyrosine recombinases.

References

1. Kolb, A. F. (2002). Genome engineering using site-specific recombinases. Cloning Stem Cells. 4, 65-80

2. Coates, C. J., Kaminski, J. M., Summers, J. B., Segal, D. J., Miller, A. D., Kolb, A. F. (2005). Site-directed genome modification: derivatives of DNA-modifying enzymes as targeting tools. Trends in Biotech. 23, 407-419

3. Landy, A. (1989). Dynamic, structural, and regulatory aspects of lambda site-specific recombination. Annu. Rev. Biochem. 58, 913-949

4. Bode, J., Schlake, T., Iber, M., Schubeler, D., Seibler, J., Snezhkov, E. and Nikolaev, L. (2000). The transgeneticist’s toolbox: novel methods for the targeted modification of eukaryotic genomes. Biol. Chem. 381, 801-813

5. Sauer, B. (1998). Inducible gene targeting in mice using the Cre/lox system. Methods: a Companion Methods Enzymol. 14, 381-392

6. Akopian, A. and Stark, W. M. (2005). Site-specific DNA Recombinases as instruments for genomic surgery. Adv. in Genet. 55, 1-23

7. Stark, W.M. and Boocock, M.R. (1995). Topological selectivity in site-specific recombination. In Mobile Genetic Elements, Sherratt, D.J. (ed.), pp 101-129, Oxford University Press

8. Abdel-Meguid, S.S., Grindley, N.D., Templeton, N.S. and Steitz, T.A. (1984). Cleavage of the site-specific recombination protein gamma delta resolvase: the smaller of two fragments binds DNA specifically. Proc. Natl. Acad. Sci. USA. 81, 2001-2005

9. Yang, W. and Steitz, T.A. (1995). Crystal structure of the site-specific recombinase γδ resolvase complexed with a 34 bp cleavage site. Cell 82, 193-207.

10. Li, W., Kamtekar, S., Xiong, Y., Sarkis, G.J., Grindley, N.D.F. and Steitz, T.A. (2005). Structure of a synaptic γδ resolvase tetramer covalently linked to two cleaved DNAs. Science 309, 1210-1215.

11. Van Duyne, G.D. (2002). A structural view of tyrosine recombinase site-specific recombination. In Mobile DNA II (N. L. Craig, R. Craigie, M. Gellert, and A.M. Lambowitz, eds.), pp. 93-117. ASM Press, Washington, DC.

12. Holliday, R. (1964). A mechanism for gene conversion in fungi. Genet. Res. 5, 282-304 Grainge, I. and Jayaram, M. (1999). The integrase family of recombinase: organisation and function of the active site. Mol. Microbiol. 33, 449-456

13. Stark, W.M., Boocock M.R. and Sherratt, D.J. (1992). Catalysis by site-specific recombinases. Trends Genet. 8, 432-439.

14. Reed, R. R. and Grindley, N. D. (1981). Transposon-mediated site-specific recombination in vitro: DNA cleavage and protein-DNA linkage at the recombination site. Cell. 25, 721-728

15. Reed, R. R. and Moser, C. D. (1984). Resolvase-mediated recombination intermediates contain a serine residue covalently linked to DNA. Cold. Spring. Harb. Symp. Quant. Biol. 49, 245-249

16. Stark, W.M., Sherratt, D.J. and Boocock, M.R.(1989). Site-specific recombination by Tn3 resolvase: topological changes in the forward and reverse reaction. Cell. 58, 779-790

17. Stark, W.M. and Boocock, M.R. (1994). The linkage change of a knotting reaction catalysed by Tn3 resolvase. J. Mol. Biol. 239, 25-36.

18. Sarkis, G. J., Murley, L. L., Leschziner, A. E., Boocock, M. R., Stark, W.M. and Grindley,N. D. (2001). A model for the resolvase synaptic complex. Mol. Cell. 8, 623-631

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