The genome of the broad host range Streptomyces temperate phage, C31, is known to integrate into the host chromosome via an enzyme that is a member of the resolvase͞invertase family of site-specific recombinases. The recombination properties of this novel integrase on the phage and Streptomyces ambofaciens attachment sites, attP and attB, respectively, were investigated in the heterologous host, Escherichia coli, and in an in vitro assay by using purified integrase. The products of attP͞B recombination, i.e., attL and attR, were identical to those obtained after integration of the prophage in S. ambofaciens. In the in vitro assay only buffer, purified integrase, and DNAs encoding attP and attB were required. Recombination occurred irrespective of whether the substrates were supercoiled or linear. A mutant integrase containing an S12F mutation was completely defective in recombination both in E. coli and in vitro. No recombination was observed between attB͞attB, attP͞attP, attL͞R, or any combination of attB or attP with attL or attR, suggesting that excision of the prophage (attL͞R recombination) requires an additional phage-or Streptomyces-encoded factor. Recombination could occur intramolecularly to cause deletion between appropriately orientated attP and attB sites. The results show that directionality in C31 integrase is strictly controlled by nonidentical recombination sites with no requirement to form the topologically defined structures that are more typical of the resolvases͞invertases.In site-specific recombination a recombinase interacts with a specific site in the DNA, brings the sites together in a synapse, and then catalyzes strand exchange so that the DNA is cleaved and religated to opposite partners (1, 2). The reaction can result in integration, inversion, or resolution͞excision depending on the position and orientation of the recombination sites, their interactions with recombinase, and the presence or absence of accessory factors or sites. Site-specific recombinases in bacteria fall into one of two very distinct families, the integrase-like enzymes and the resolvase͞invertases, on the basis of amino acid sequence similarities and their different mechanisms of catalysis (1-3). Recombination by members of the integrase family (e.g., integrase, P1 Cre-loxP) is well understood and involves the formation and resolution of a Holliday junction intermediate during which the DNA is transiently attached to the enzyme through a phosphotyrosine linkage (4-6). The resolvase͞invertase family of enzymes (e.g., Tn3 or ␥␦ resolvases, Mu Gin invertase) act via a concerted, four-strand staggered break and rejoining mechanism during which a phosphoserine linkage is formed between the enzyme and the DNA (2, 7). The crystal structure of ␥␦ resolvase bound to a cleavage site reveals a unique arrangement of the catalytic and DNA-binding domains in that they bind to different faces of the helix (8). Although two models have been proposed (9-11), the structure of the synapse and the changes in the conformation of...

Summary Most site‐specific recombinases fall into one of two families, based on evolutionary and mechanistic relatedness. These are the tyrosine recombinases or λ integrase family and the serine recombinases or resolvase/invertase family. The tyrosine recombinases are structurally diverse and functionally versatile and include integrases, resolvases, invertases and transposases. Recent studies have revealed that the serine recombinase family is equally versatile and members have a variety of structural forms. The archetypal resolvase/invertases are highly regulated, only affect resolution or inversion and they have an N‐terminal catalytic domain and a C‐terminal DNA binding domain. Phage‐encoded serine recombinases (e.g. φC31 integrase) cause integration and excision with strictly controlled directionality, and have an N‐terminal catalytic domain but much longer C‐terminal domains compared with the resolvase/invertases. This high molecular weight group also contains transposases (e.g. TnpX from Tn4451). Other transposases, which belong to a third structurally different group, are similar in size to the resolvase/invertases but have the DNA binding domain N‐terminal to the catalytic domain (e.g. IS607 transposase). These three structural groups represented by the resolvase/invertases, the large serine recombinases and relatives of IS607 transposase correlate with three major groupings seen in a phylogeny of the catalytic domains. These observations indicate that the serine recombinases are modular and that fusion of the catalytic domain to unrelated sequences has generated structural and functional diversity.

SummaryThe genome of the Streptomyces temperate phage fC31 integrates into the host chromosome via a recombinase belonging to a novel group of phage integrases related to the resolvase/invertase enzymes. Previously, it was demonstrated that, in an in vitro recombination assay, fC31 integrase catalyses integration (attP/attB recombination) but not excision (attL/attR). The mechanism responsible for this recombination site selectivity was therefore investigated. Purified integrase was shown to bind with similar apparent binding affinities to between 46 bp and 54 bp of DNA at each of the attachment sites, attP, attB, attL and attR. Assays using recombination sites of 50 bp and 51 bp for attP and attB, respectively, showed that these fragments were functional in attP/attB recombination and maintained strict site selectivity, i.e. no recombination between non-permissive sites, such as attP/attP, attB/attL, etc., was observed. Using bandshifts and supershift assays in which permissive and non-permissive combinations of att sites were used in the presence of integrase, only the attP/attB combination could generate supershifts. Recombination products were isolated from the supershifted complexes. It was concluded that these supershifted complexes contained the recombination synapse and that site specificity, and therefore directionality, is determined at the level of stable synapse formation.