Viral integration and excision: structure of the lambda att sites

Site-specific recombination is an important genetic pathway in both prokaryotes and eukaryotes. The range of phenomena associated with this genomic transaction in prokaryotes includes the generation and integration of episomal factors, the highly mobile antibiotic resistance elements, the influence on gene expression by small DNA insertion sequences (IS-elements), and the transition between cytoplasmic vegetative phage genomes and host-integrated prophage. Although less is known about eukaryotic systems, recent biochemical experiments and a renewed appreciation of older genetic experiments are beginning to suggest that site-specific recombination, in addition to being important in the integration of viral genomes, may also be relevant in cellular regulation or developmental pathways (or both) (1). An up-to-date collection of recent work in this area is presented in (2). One of the most thoroughly characterized examples of site-specific recombination, and also the subject of this article, is the integration and excision reaction of bacteriophage lambda (λ) (3). According to the now well-documented model proposed by Campbell for λ integration, the infecting phage genome is first circularized by means of its cohesive termini and then undergoes a single reciprocal recombination event at specific loci (att sites) on the bacterial and viral chromosomes (4) (Fig. 1). The product of the phage gene int, which maps immediately to the right of att, is required for both integration and excision of the λ prophage (5). The excision reaction, in addition, requires the product of a second phage gene, xis, which maps immediately to the right of the int gene (6). Although no other phage-specific proteins are required (7), there are at least one or more host-specified proteins that are also involved in the integration-excision pathway (8). The enzymology of the integration-excision reaction has only recently yielded to direct biochemical analysis (9, 10), and information crucial to the formulation of a complete model will undoubtedly be forthcoming in the near future. Not included within the 1400 base-pair att-int-xis cluster are the regulatory genes cII and c-III, whose products greatly enhance the expression of the int gene, but not of xis, by mechanisms which remain to be established (11, 12). Fig. 1 The λ integration-excision pathway and the origin of transducing phage carrying different att sites. Integration of the circularized phage genome requires the phage-encoded protein int and proceeds via site-specific recombination between the phage ... In current nomenclature the phage (attP) and bacterial (attB) att sites are designated POP’ and BOB’, respectively, and the prophage att sites are designated BOP’ (attL) and POB’ (attR) (Fig. 1). Transducing phage carrying attL (λgal) or attR (λbio) are generated from a prophage which has excised from the host chromosome by a rare int-independent recombination which deletes phage DNA from one end of the prophage and adds bacterial DNA to the other (4). A phage carrying the bacterial att site, BOB’, is obtained as a product of int-promoted recombination between a gal (BOP’) and a bio (POB’) transducing phage which is capable of transducing gal and bio together (13). The analysis of int-dependent recombination between phage bearing different att sites lead to the proposal by Guerrini (14) that the arms of the att sites, P, P’, B, and B’, differ from each other in nucleotide sequence, and that the extent of int-dependent recombination between different att sites does not depend on the degree of homology between them. Furthermore, all four arms of a given pair of att sites are somehow important together in determining the results of a cross (13, 15), as are the temperature and the activity of xis protein (16). Electron microscopy of appropriate heteroduplex molecules has yielded estimates for the maximum size of the postulated recognition elements, for example, less than 20 base pairs for element B (17); however, such conclusions are necessarily qualified because of the number of possible models for att site structure (18). In addition to the unique character of each att site, the genetic results of Shulman and Gottesman (19) suggested that there is a common region, O, which is shared by all of the λ att sites. Mutants defective in att function can be crossed from one att site into another; and, since some of the progeny of a mutant by wild-type cross are heterozygous, they have postulated that the actual crossover might be made by staggered nicks in the two strands of the common core region (19, 20). Failure to find evidence of this type of homology by electron microscope heteroduplex analysis yields an upper limit of 20 to 50 base pairs for a common core region (17, 21). When Escherichia coli carries a deletion of the gal-bio region, that is, of the primary bacterial att site BOB’, int-dependent integration of λ can be detected at numerous loci (secondary bacterial att sites) on the E. coli chromosome (22). This integration always involves the phage att site (POP’) (23) and is thus very similar to the behavior of IS-elements (24, 25). The secondary prophage att sites are given the general designation ΔOP’ and POΔ’ and they differ from BOP’ and POB’ and from each other in their biological properties as determined by int- and xis -dependent recombination frequencies with various att sites (26). We have undertaken a study of the structures involved in site-specific integrative and excisive recombination in bacteriophage λ. In this article, we report the DNA sequences for each of the four primary att sites, POP’, POB’, BOP’, and BOB’. Our results show that the phage att site and bacterial att site have in common a sequence 15 nucleotide pairs in length. The crossover event of integrative recombination must be within the limits (or at the boundaries) of this “common core” sequence since it occurs unaltered in both the leftward and rightward prophage att sites. The four sets of sequences, adjacent to the common core region, are different from one another and hereafter will be referred to as “arms” (P arm, B’ arm, and so forth) without any implication as to their functional significance. Several features of the att site structures are discussed in terms of possible models for the site-specific recombination.