Domain structure and DNA binding regions of β protein from bacteriophage λ

Abstract

β protein from bacteriophage λ promotes a single-strand annealing reaction that is central to Red-mediated recombination at double-strand DNA breaks and chromosomal ends. β protein binds most tightly to an intermediate of annealing formed by the sequential addition of two complementary oligonucleotides. Here we have characterized the domain structure of β protein in the presence and absence of DNA using limited proteolysis. Residues 1-130 form an N-terminal "core" domain that is resistant to proteases in the absence of DNA, residues 131-177 form a central region with enhanced resistance to proteases upon DNA complex formation, and the C-terminal residues 178-261 of β protein are sensitive to proteases in both the presence and absence of DNA. We probed the DNA binding regions of β protein further using biotinylation of lysine residues and mass spectrometry. Several lysine residues within the first 177 residues of β protein are protected from biotinylation in the DNA complex, whereas none of the lysine residues in the C-terminal portion are protected. The results lead to a model for the domain structure and DNA binding of β protein in which a stable N-terminal core and a more flexible central domain come together to bind DNA, whereas a C-terminal tail remains disordered. A fragment consisting of residues 1-177 of β protein maintains normal binding to sequentially added complementary oligonucleotides and has significantly enhanced binding to single-strand DNA. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.