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

摘要: The genome of the broad host range Streptomyces temperate phage, φC31, is known to integrate into chromosome via an enzyme that a member resolvase/invertase family site-specific recombinases. recombination properties this novel integrase on phage and ambofaciens attachment sites, attP attB, respectively, were investigated in heterologous host, Escherichia coli, vitro assay by using purified integrase. products attP/B recombination, i.e., attL attR, identical those obtained after integration prophage S. ambofaciens. In only buffer, integrase, DNAs encoding attB required. Recombination occurred irrespective whether substrates supercoiled or linear. A mutant containing S12F mutation was completely defective both E. coli vitro. No observed between attB/attB, attP/attP, attL/R, any combination with suggesting excision (attL/R recombination) requires additional phage- Streptomyces-encoded factor. could occur intramolecularly cause deletion appropriately orientated sites. results show directionality φC31 strictly controlled nonidentical sites no requirement form topologically defined structures are more typical resolvases/invertases.