Specialized Recombination Involves Specific Sites

KEY CONCEPTS
- Specialized recombination involves reaction between specific sites that are not necessarily homologous.
- Phage lambda integrates into the bacterial chromosome by recombination between the attP site on the phage and the attB site on the E. coli chromosome.
- The phage is excised from the chromosome by recombination between the sites at the end of the linear prophage.
- Phage lambda int encodes an integrase that catalyzes the integration reaction.
Specialized recombination involves a reaction between two specific sites. The lengths of target sites are short and are typically in a range of 14 to 50 bp. In some cases the two sites have the same sequence, but in other cases they are nonhomologous. The reaction is used to insert a free phage DNA into the bacterial chromosome or to excise an integrated phage DNA from the chromosome, and in this case the two recombining sequences are different from one another. It is also used before division to regenerate monomeric circular chromosomes from a dimer that has been created by a generalized recombination event . In this case the recombining sequences are identical.
The enzymes that catalyze site-specific recombination are generally called recombinases, and more than 100 of them are now known. Those involved in phage integration or related to these enzymes are also known as the integrase family. Prominent members of the integrase family are the prototypical Int from phage lambda, Cre from phage P1, and the yeast FLP enzyme (which catalyzes a chromosomal inversion).
The classic model for site-specific recombination is illustrated by phage lambda. The conversion of lambda DNA between its different life forms involves two types of events. The pattern of gene expression is regulated as described in the chapter titled Phage Strategies. The physical condition of the DNA is different in the lysogenic and lytic states:
- In the lytic lifestyle, lambda DNA exists as an independent, circular molecule in the infected bacterium.
- In the lysogenic state, the phage DNA is an integral part of the bacterial chromosome (called the prophage).
Transition between these states involves site-specific recombination:
- To enter the lysogenic condition, free lambda DNA must be inserted into the host DNA. This is called integration.
- To be released from lysogeny into the lytic cycle, prophage DNA must be released from the chromosome. This is called excision.
Integration and excision occur by recombination at specific loci on the bacterial and phage DNAs called attachment (att) sites. The attB attachment site on the bacterial chromosome is formally called att^λ in bacterial genetics. The locus is defined by mutations that prevent integration of lambda; it is occupied by prophage λ in lysogenic strains. When the att site is deleted from the E. coli chromosome, an infecting lambda phage can establish lysogeny by integrating elsewhere, although the efficiency of the reaction is less than 0.1% of the frequency of integration at att^λ . This inefficient integration occurs at secondary attachment sites, which resemble the authentic att sequences.
For describing the integration/excision reactions, the bacterial attachment site (att^λ ) is called attB, consisting of the sequence components BOB′. The attachment site on the phage, attP, consists of the components POP′. FIGURE 1. outlines the recombination reaction between these sites. The sequence O is common to attB and attP. It is called the core sequence, and the recombination event occurs within it. The flanking regions B, B′ and P, P′ are referred to as the arms; each is distinct in sequence. The phage DNA is circular, so the recombination event inserts it into the bacterial chromosome as a linear sequence. The prophage is bounded by two new att sites (the products of the recombination) called attL and attR.

FIGURE 1. Circular phage DNA is converted to an integrated prophage by a reciprocal recombination between attP and attB; the prophage is excised by reciprocal recombination between attL and attR.
An important consequence of the constitution of the att sites is that the integration and excision reactions do not involve the same pair of reacting sequences. Integration requires recognition between attP and attB, whereas excision requires recognition between attL and attR. The directional character of site-specific recombination is controlled by the identity of the recombining sites.
The recombination event is reversible, but different conditions prevail for each direction of the reaction. This is an important feature in the life of the phage, because it offers a means to ensure that an integration event is not immediately reversed by an excision, and vice versa.
The difference in the pairs of sites reacting at integration and excision is reflected by a difference in the proteins that mediate the two reactions:
- Integration (attB × attP) requires the product of the phage gene int, which encodes an integrase enzyme, and a bacterial protein called integration host factor (IHF).
- Excision (attL × attR) requires the product of phage gene xis, in addition to Int and IHF.
Thus, Int and IHF are required for both reactions. Xis plays an important role in controlling the direction; it is required for excision, but inhibits integration.
A similar system, but with somewhat simpler requirements for both sequence and protein components, is found in the bacteriophage P1. The Cre recombinase encoded by the phage catalyzes a recombination between two target sequences. Unlike phage lambda, for which the recombining sequences are different, in phage P1 they are identical. Each consists of a 34-bp-long sequence called loxP. The Cre recombinase is sufficient for the reaction; no accessory proteins are required. As a result of its simplicity and its efficiency, what is now known as the Cre/lox system has been adapted for use in eukaryotic cells, where it has become one of the standard techniques for undertaking sitespecific recombination.