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Key Terms
  • Site-specific recombination (Specialized recombination) occurs between two specific sequences, as in phage integration/excision or resolution of cointegrate structures during transposition.
  • Prophage is a phage genome covalently integrated as a linear part of the bacterial chromosome.
  • Integration of viral or another DNA sequence describes its insertion into a host genome as a region covalently linked on either side to the host sequences.
  • The excision step in an excision-repair system consists of removing a single-stranded stretch of DNA by the action of a 5′-3′ exonuclease.
  • att sites are the loci on a lambda phage and the bacterial chromosome at which recombination integrates the phage into, or excises it from, the bacterial chromosome.
  • A secondary attachment site is a locus on the bacterial chromosome into which phage lambda integrate inefficiently because the site resembles the att site.
  • The core sequence is the segment of DNA that is common to the attachment sites on both the phage lambda and bacterial genomes. It is the location of the recombination event that allows phage lambda to integrate.
  • The arms of a lambda phage attachment site are the sequences flanking the core region where the recombination event occurs.
Key Terms
  • Specialized recombination involves reaction between specific sites that are not necessarily homologous.
  • Phage lambda integrates into the bacterial chromosome by recombination between a site on the phage and the att 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 codes for an integrase that catalyzes the integration reaction.

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Specialized recombination involves a reaction between two specific sites. The target sites are short, typically in a 14-50 bp length range. 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 (see Chromosomal segregation may require site-specific recombination). In this case the recombining sequences are identical.

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The enzymes that catalyze site-specific recombination are generally called recombinases, and >100 of them are now known (2921). 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 prototype Int from phage lambda, Cre from phage P1, and the yeast FLP enzyme (which catalyzes a chromosomal inversion) (for review see 2922).

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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 event. The pattern of gene expression is regulated as described in Phage strategies. And the physical condition of the DNA is different in the lysogenic and lytic states:

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  • 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 prophage).

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Transition between these states involves site-specific recombination:

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  • 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.

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Integration and excision occur by recombination at specific loci on the bacterial and phage DNAs called attachment (att) sites. The attachment site on the bacterial chromosome is called attλ 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 attλ 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 <0.1% of the frequency of integration at attλ. This inefficient integration occurs at secondary attachment sites, which resemble the authentic att sequences.

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Figure 15.25  
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.

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For describing the integration/excision reactions, the bacterial attachment site (attλ) is called attB, consisting of the sequence components BOB′. The attachment site on the phage, attP, consists of the components POP′. Figure 15.25 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. Because the phage DNA is circular, 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.

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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; while excision requires recognition between attL and attR. The directional character of site-specific recombination is controlled by the identity of the recombining sites.

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Although the recombination event is reversible, different conditions prevail for each direction of the reaction. This is an important feature in the life of the phage, since it offers a means to ensure that an integration event is not immediately reversed by an excision, and vice versa.

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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:

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  • Integration (attB×attP) requires the product of the phage gene int, which codes for 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.

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So 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.

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A similar system, but with somewhat simpler requirements for both sequence and protein components, is found in the bacteriophage P1. The Cre recombinase coded by the phage catalyzes a recombination between two target sequences. Unlike phage lambda, where 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. Because 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 site-specific recombination (2923).

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Last Revised on April 19, 2004

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reviews
  • 2921 Nunes-Duby, S. E., Kwon, H. J., Tirumalai, R. S., Ellenberger, T., and Landy, A. (1998).  Similarities and differences among 105 members of the Int family of site-specific recombinases.  Nucleic Acids Res. 26, 391-406.  PubMed   Journal
  • 2923 Metzger, D., Clifford, J., Chiba, H., and Chambon, P. (1995).  Conditional site-specific recombination in mammalian cells using a ligand-dependent chimeric Cre recombinase.  Proc. Natl. Acad. Sci. USA 92, 6991-6995.  PubMed  

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