Prophages Inserted by Reciprocal Site Specific Recombination

l is one of the many phages that insert their DNA into the host chromosome by reciprocal recombination between the chromosome and a circular form of the phage DNA. The overall reaction comprises cointegration of the two circles. The recombination takes place between a unique site on the phage DNA and a unique site on the E. coli

Table 7-1 Modes of Prophage Maintenance

Mode

Example

Insertion By site-specific recombination

By transposition Plasmid formation

Coliphage l Salmonella phage P22 Mycobacterial phage L5 Vibrio phage CTX Phi Streptomyces phage fC31 Mu-1 D108

Coliphages P1, P7 Salmonella phage D6

chromosome (figure 7-1). Host and phage DNA are identical in sequence for 15 bp at the crossover point. Fifteen base pairs is not enough to allow recombination by the general recombinase of the host (rec) or of the phage (red). Phage l encodes a protein (integrase) that efficiently mediates reciprocal recombination at these sites. Integrase is made in greatest abundance in cells that are recovering from infection. After lysogeny is established, the integrase gene is turned off.

Figure 7-1 Integration by site-specific recombination. Integration occurs between the attP site of the phage and the attB site of the host. The two circular molecules form a cointegrate, with junction points attL (left prophage end) and attR (right prophage end). Integration is carried out by a phage-coded protein, integrase. In many phages (including l), the reverse reaction (excision) frequently requires a second phage-coded protein, excisonase. Host proteins contributing to the reaction are not shown.

Figure 7-1 Integration by site-specific recombination. Integration occurs between the attP site of the phage and the attB site of the host. The two circular molecules form a cointegrate, with junction points attL (left prophage end) and attR (right prophage end). Integration is carried out by a phage-coded protein, integrase. In many phages (including l), the reverse reaction (excision) frequently requires a second phage-coded protein, excisonase. Host proteins contributing to the reaction are not shown.

This is precisely what is expected of a mechanism to promote lysogeny: it should go on after infection, so that every surviving cell has an inserted prophage. It is unnecessary in an established lysogen, where insertion has already occurred. Finally, if lysogeny is disrupted and the cell switches back to the lytic cycle (as occasionally happens spontaneously and can be induced), the prophage should be excised from the chromosome. For reasons that are not completely understood at a biochemical level, the insertion reaction is not directly reversible. To excise the prophage, another phage protein, excisionase, is needed. Following induction, the two proteins are produced coordinately.

The integrase/excisionase system puts the phage in charge of the timing and direction of site-specific recombination. If the phage and bacterium had a longer stretch of homology and depended on general recombinases, insertion and excision would happen rarely and haphazardly.

The effective irreversibility of the insertion reaction in the absence of excisionase has suggested that it might be adapted to insert DNA into specific sites of the human genome for gene therapy (7). The idea is to promote stable insertion into some human sequence resembling the bacterial site. The integration system of Streptomyces phage f C31 has been used for this purpose.

l inserts in intergenic DNA, but many other temperate phages, including some natural relatives of l, insert at sites that are within structural genes (21) or tRNA genes (P22). What these sites have in common is an interrupted (frequently imperfect) dyad symmetry centered on the crossover point. In tRNA genes, this configuration is found in the anticodon loop, which is used not only by the l-related phage P22 but also by coliphage 186, Haemophilus phage HP1 and mycobacterium phage L5.

The l integrase reaction proceeds through two successive strand exchanges placed 7 bp apart. The symmetry of the DNA site allows equivalent recognition at the two exchange points. The sites used by some phages have no obvious symmetry. The satellite coliphages P4 and its relatives insert into tRNA genes, but in theTCC loop near the 3' end, with no DNA symmetry centered on the insertion point. P4 uses an integrase of the same superfamily as l integrase, but the two integrases are barely related. Streptomyces phage f C31 inserts into a site with no apparent symmetry, using a site-specific recombinase from a different superfamily. Thus the site-specific recombination systems used for insertion/excision employ various mechanisms, but all produce the same final result.

Insertion by site-specific recombinases requires a double-stranded circle of phage DNA (figure 7-1). As packaged in the virion, the DNAs of most temperate phages are not double-stranded circles. They are generally converted to that form early in phage development. l, for example, has linear DNA with projecting complementary single-stranded 5' ends, which pair following infection, allowing ligation to a double-stranded circle. The cholera phage CTX Phi has

Figure 7-2 Some pathways to the double-stranded, circular integration substrate. See text for details.

Figure 7.3 Formation of specialized transducing phages by bacteriophage l. The central column shows normal excision of the phage genome, mediated by integrase and excisionase. For every such excision, there are about 10~5 abnormal excisions, where either gal or bio is effectively cloned into l. These are rare because they require breakage and joining of heterologous DNA. The reciprocal product (a deleted chromosome), which may or may not be formed in the same event, is not shown. The presence of the cos site allows the excised DNA to be packaged into virions and injected into other cells. Since l is cut at cos during packaging, part of cos occurs at each end of the packaged DNA.

Figure 7-2 Some pathways to the double-stranded, circular integration substrate. See text for details.

single-stranded circles in the virion, but like most phages, it replicates as a double-stranded circle. Phage P22 has headful packaging, where the DNA molecule injected from the virion has a direct repetition (different among virions) of double-stranded DNA at the ends. Within the cell, homology-dependent recombination produces a circle (the same for all virions). Some of these pathways to the circular integration substrate are illustrated in figure 7-2. In all these cases, the circular form is an intermediate in lytic development as well as in integration.

Although site-specific recombinases of the integrase family occur in eukaryotes, they are not known to be used in viral integration. The dependent parvoviruses (so-called adeno-associated viruses) insert with high preference in a specific small human chromosomal segment, but there is no indication that the mechanism has much in common with phage insertion (8).

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