DNA Recombination:- Site-Specific Recombination Results in Precise DNA Rearrangements
Homologous genetic recombination can involve any two homologous sequences. The second general type of recombination, site-specific recombination, is a very different type of process: recombination is limited to specific sequences. Recombination reactions of this type occur in virtually every cell, filling specialized roles that vary greatly from one species to another. Examples include regulation of the expression of certain genes and promotion of programmed DNA rearrangements in embryonic development or in the replication cycles of some viral and plasmid DNAs. Each site-specific recombination system consists of an enzyme called a recombinase and a short (20 to 200 bp), unique DNA sequence where the recombinase acts (the recombination site). One or more auxiliary proteins may regulate the timing or outcome of the reaction. In vitro studies of many site-specific recombination systems have elucidated some general principles, in cluding the fundamental reaction pathway (Fig. 1a). A separate recombinase recognizes and binds to each of two recombination sites on two different DNA molecules or within the same DNA. One DNA strand in each site is cleaved at a specific point within the site, and the recombinase becomes covalently linked to the DNA at the cleavage site through a phosphotyrosine (or phosphoserine) bond (step 1). The transient protein-DNA linkage preserves the phosphodiester bond that is lost in cleaving the DNA, so high-energy cofactors such as ATP are unnecessary in subsequent steps. The cleaved DNA strands are rejoined to new partners to form a Holliday intermediate, with new phosphodiester bonds created at the expense of the protein-DNA linkage (step2). To complete the reaction, the process must be re peated at a second point within each of the two recombination sites (steps 3 and 4). In some systems, both strands of each recombination site are cut concurrently and rejoined to new partners without the Holliday inter mediate. The exchange is always reciprocal and precise, regenerating the recombination sites when the reaction is complete. We can view a recombinase as a site-specific endonuclease and ligase in one package. The sequences of the recombination sites recognized by site-specific recombinases are partially asymmetric (nonpalindromic), and the two recombining sites align in the same orientation during the recombinase reaction. The outcome depends on the location and orientation of the recombination sites (Fig. 2). If the two sites are on the same DNA molecule, the reaction either inverts or deletes the intervening DNA, determined by whether the recombination sites have the opposite or the same orientation, respectively. If the sites are on different DNAs, the recombination is intermolecular; if one or both DNAs are circular, the result is an insertion. Some recombinase systems are highly specific for one of these reaction types and act only on sites with particular orientations.
The first site-specific recombination system studied in vitro was that encoded by bacteriophage λ. When λ phage DNA enters an E. coli cell, a complex series of regulatory events commits the DNA to one of two fates.
The λ DNA either replicates and produces more bacteriophages (destroying the host cell) or integrates into the host chromosome, replicating passively along with the chromosome for many cell generations. Integration is accomplished by a phage-encoded recombinase (λ integrase) that acts at recombination sites on the phage and bacterial DNAs—at attachment sites attP and attB, respectively (Fig. 3).
FIGURE 1 A site-specific recombination reaction. (a) The reaction shown here is for a common class of site-specific recombinases called integrase-class recombinases (named after bacteriophage integrase, the first recombinase characterized). The reaction is carried out within a tetramer of identical subunits. Recombinase subunits bind to a specific sequence, often called simply the recombination site. 1One strand in each DNA is cleaved at particular points within the sequence. The nucleophile is the OH group of an active-site Tyr residue, and the product is a covalent phosphotyrosine link between protein and DNA. 2 The cleaved strands join to new partners, producing a Holliday intermediate. Steps 3 and 4 complete the re action by a process similar to the first two steps. The original sequence of the recombination site is regenerated after recombining the DNA flanking the site. These steps occur within a complex of multiple recombinase subunits that sometimes includes other proteins not shown here. (b) A surface contour model of a four-subunit integrase class recombinase called the Cre recombinase, bound to a Holliday intermediate (shown with light blue and dark blue helix strands). The protein has been rendered transparent so that the bound DNA is visible (derived from PDB ID 3CRX).
FIGURE 2 Effects of site-specific recombination. The outcome of site-specific recombination depends on the location and orientation of the recombination sites (red and green) in a double-stranded DNA molecule. Orientation here (shown by arrowheads) refers to the order of nucleotides in the recombination site, not the 5n3 direction.
- Recombination sites with opposite orientation in the same DNA molecule. The result is an inversion. (b) Recombination sites with the same orientation, either on one DNA molecule, producing a deletion, or on two DNA molecules, producing an insertion.
FIGURE 3 Integration and excision of bacteriophage DNA at the chromosomal target site. The attachment site on the phage DNA (attP) shares only 15 bp of complete homology with the bacterial site (attB) in the region of the crossover. The reaction generates two new attachment sites (attR and attL) flanking the integrated phage DNA.
The recombinase is the λ integrase (or INT protein). Integration and excision use different attachment sites and different auxiliary proteins. Excision uses the proteins XIS, encoded by the bacteriophage, and FIS, encoded by the bacterium. Both reactions require the protein IHF (integration host factor), encoded by the bacterium.
