Characterization of the DNA-binding domain of β protein, a component of phage λ Red-pathway, by UV catalyzed cross-linking

Abstract

β protein, a key component of Red-pathway of phage λ is necessary for its growth and general genetic recombination in recombination-deficient mutants of Escherichia coli To facilitate studies on structure-function relationships, we overexpressed β protein and purified it to homogeneity. A chemical cross-linking reagent, glutaraldehyde, was used to stabilize the physical association of β protein in solution. A 67-kDa band, corresponding to homodimer, was identified after separation by SDS-polyacrylamide gel electrophoresis. Stoichiometric measurements indicated a site-size of 1 monomer of β protein/5 nucleotide residues. Electrophoretic gel mobility shift assays suggested that β protein formed stable nucleoprotein complexes with 36-mer, but not with 27-or 17-mer DNA. Interestingly, the interaction of β protein with DNA and the stability of nucleoprotein complexes was dependent on the presence of MgC1₂, and the binding was abolished by 250 mM NaCl. The K_d of β protein binding to 36-mer DNA was on the order of 1.8 × 10^-6M. Photochemical cross-linking of native β protein or its fragments, generated by chymotrypsin, to 36-mer DNA was performed to identify its DNA-binding domain. Characterization of the cross-linked peptide disclosed that amino acids required for DNA-binding specificity resided within a 20-kDa peptide at the N-terminal end. These findings provide a basis for further understanding of the structure and function of β protein.