Characterization of bacteriophage lambda excisionase mutants defective in DNA binding

The bacteriophage lambda excisionase (Xis) is a sequence-specific DNA bindi ng protein required for excisive recombination. Xis binds cooperatively to two DNA sites arranged as direct repeats on the phage DNA. Efficient excisi on is achieved through a cooperative interaction between Xis and the host-e ncoded factor for inversion stimulation as well as a cooperative interactio n between Xis and integrase. The secondary structure of the Xis protein was predicted to contain a typical amphipathic helix that spans residues 18 to 28. Several mutants, defective in promoting excision in vivo, were isolate d with mutations at positions encoding polar amino acids in the putative he lix (T. E. Numrych, R. I. Gumport, and J. F. Gardner, EMBO J. 11:3797-3806, 1992). We substituted alanines for the polar amino acids in this region. M utant proteins with substitutions for polar amino acids in the amino-termin al region of the putative helix exhibited decreased excision in vivo and we re defective in DNA binding. In addition, an alanine substitution at glutam ic acid 40 also resulted in altered DNA binding. This indicates that the hy drophilic face of the alpha-helix and the region containing glutamic acid 4 0 may form the DNA binding surfaces of the Xis protein.