Dissecting nucleic acid-protein interactions using challenge phage

The bacteriophage P22-based challenge system is a sophisticated genetic tool for the characterization of sequence-specific recognition of DNA and RNA in vivo. The construction of challenge phage follows simple phage lysate preparations and detection of constructs by positive selection methods for plaques on selective strains. The challenge phage system is a powerful tool for the characterization of protein-DNA and protein-RNA interactions in vivo. The challenge phage has been further developed to characterize the interactions of multiple proteins in heteromultimeric complexes that are required for DNA binding. Under appropriate conditions, expression of the ant gene determines the lysis-lysogeny decision of P22. This provides a positive selection for and against DNA binding: repression of ant can be selected by requiring growth of lysogens, and mutants that cannot repress ant can be selected by requiring lytic growth of the phage. Thus, placing ant gene expression under the control of a specific DNA-protein interaction provides very strong genetic selections for regulatory mutations in the DNA-binding protein and DNA-binding site that either increase or decrease the apparent strength of a DNA-protein interaction in vivo. Furthermore, the challenge phage contains a kanamycin-resistance element that can be used to either directly select for lysogeny or to determine the frequency of lysogeny for a given protein-DNA interaction to measure the efficiency of DNA binding in vivo. Selection for lysogeny can be used to isolate DNA-binding proteins with altered or enhanced DNA-binding specificities. The challenge phage selection provides a general method for identifying critical residues involved in DNA-protein interactions. Challenge phage selections have been used to genetically dissect many different prokaryotic and eukaryotic DNA-binding interactions.