In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family

Abstract

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...

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