This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yu, A Articles by Haggård-Ljungquist, E Search for Related Content PubMed PubMed Citation Articles by Yu, A Articles by Haggård-Ljungquist, E

research-article

Characterization of the binding sites of two proteins involved in the bacteriophage P2 site-specific recombination system.

Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT

Integration of the bacteriophage P2 genome into the Escherichia coli host chromosome occurs by site-specific recombination between the phage attP and E. coli attB sites. The phage-encoded 38-kDa protein, integrase, is known to be necessary for both phage integration as well as excision. In order to begin the molecular characterization of this recombination event, we have cloned the int gene and overproduced and partially purified the Int protein and an N-terminal truncated form of Int. Both the wild-type Int protein and the integration host factor (IHF) of E. coli were required to mediate integrative recombination in vitro between a supercoiled attP plasmid and a linear attB substrate. Footprint experiments revealed one Int-protected region on both of the attP arms, each containing direct repeats of the consensus sequence TGTGGACA. The common core sequences at attP and attB were also protected by Int from nuclease digestion, and these contained a different consensus sequence, AA T/A T/A C/A T/G CCC, arranged as inverted repeats at each core. A single IHF-protected site was located on the P (left) arm, placed between the core- and P arm-binding site for Int. Cooperative binding by Int and IHF to the attP region was demonstrated with band-shift assays and footprinting studies. Our data support the existence of two DNA-binding domains on Int, having unrelated sequence specificities. We propose that P2 Int, IHF, attP, and attB assemble in a higher-order complex, or intasome, prior to site-specific integrative recombination analogous to that formed during lambda integration.

This article has been cited by other articles:

• Pelludat, C., Mirold, S., Hardt, W.-D. (2003). The SopE{Phi} Phage Integrates into the ssrA Gene of Salmonella enterica Serovar Typhimurium A36 and Is Closely Related to the Fels-2 Prophage. J. Bacteriol. 185: 5182-5191 [Abstract] [Full Text]  
• Raynal, A., Friedmann, A., Tuphile, K., Guerineau, M., Pernodet, J.-L. (2002). Characterization of the attP site of the integrative element pSAM2 from Streptomyces ambofaciens. Microbiology 148: 61-67 [Abstract] [Full Text]  
• Eriksson, J. M., Haggård-Ljungquist, E. (2000). The Multifunctional Bacteriophage P2 Cox Protein Requires Oligomerization for Biological Activity. J. Bacteriol. 182: 6714-6723 [Abstract] [Full Text]  
• Liu, T., Haggård-Ljungquist, E. (1999). The Transcriptional Switch of Bacteriophage WPhi , a P2-Related but Heteroimmune Coliphage. J. Virol. 73: 9816-9826 [Abstract] [Full Text]  
• Brøndsted, L., Hammer, K. (1999). Use of the Integration Elements Encoded by the Temperate Lactococcal Bacteriophage TP901-1 To Obtain Chromosomal Single-Copy Transcriptional Fusions in Lactococcus lactis. Appl. Environ. Microbiol. 65: 752-758 [Abstract] [Full Text]  
• Peña, C. E. A., Kahlenberg, J. M., Hatfull, G. F. (1999). Protein-DNA Complexes in Mycobacteriophage L5 Integrative Recombination. J. Bacteriol. 181: 454-461 [Abstract] [Full Text]  
• Esposito, D., Scocca, J. J. (1997). Purification and Characterization of HP1 Cox and Definition of Its Role in Controlling the Direction of Site-specific Recombination. J. Biol. Chem. 272: 8660-8670 [Abstract] [Full Text]  
• Pedulla, M. L., Lee, M. H., Lever, D. C., Hatfull, G. F. (1996). A novel host factor for integration of mycobacteriophage L5. Proc. Natl. Acad. Sci. USA 93: 15411-15416 [Abstract] [Full Text]