Abstract
beta protein from bacteriophage lambda promotes a single-strand annealing reaction that is central to Red-mediated recombination at double-strand DNA breaks and chromosomal ends. beta 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 beta 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 beta protein are sensitive to proteases in both the presence and absence of DNA. We probed the DNA binding regions of beta protein further using biotinylation of lysine residues and mass spectrometry. Several lysine residues within the first 177 residues of beta 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 beta 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 beta protein maintains normal binding to sequentially added complementary oligonucleotides and has significantly enhanced binding to single-strand DNA.
Highlights
Encodes the 29-kDa  protein, which binds ssDNA and promotes annealing of complementary strands [5,6]
Limited Proteolysis of  Protein Alone and in Complex with DNA—We first verified that our preparation of  protein, which has the extra N-terminal residues GSH, has the expected DNA binding properties
In the assay [14],  protein is first incubated with a 32P-end-labeled 33-mer (33ϩ) oligonucleotide, and as the complementary oligonucleotide (33-) is titrated in, the resulting  protein-DNA complex is shifted to the top of a 15% polyacrylamide gel
Summary
Expression and Purification of  Protein—The bet gene encoding  protein was PCR-amplified from phage genomic DNA (New England Biolabs) and cloned into pET-14b (Novagen) between the NdeI and BamH1 sites. The protein was loaded onto the Ni2ϩ column and the flow-through was dialyzed into 20 mM Tris, pH 8.0, loaded onto a 25-ml HiTrap Q HP anion exchange column (Amersham Biosciences), and eluted with a 180-ml linear gradient from 0 to 1 M NaCl. Pooled fractions were dialyzed into 20 mM Tris, pH 8.0, 1 mM DTT, concentrated to 47 mg/ml, and stored at Ϫ80 °C in small aliquots. Purification of 1–188 proteins with the K11A, K36A, K69A, and K172A mutations followed this same procedure, except the heparin chromatography step was omitted, and the final buffer included 100 mM NaCl. Oligonucleotides—Two complementary 33-mer oligonucleotides, 33ϩ (ACA GCA CCA GAT TCA GCA ATT AAG CTC TAA GCC) and 33Ϫ (GGC TTA GAG CTT AAT TGC TGA ATC TGG TGC TGT), which are derived from a sequence present in M13 phage DNA [14], were purchased from Integrated DNA Technologies and purified by ion exchange HPLC. All other peptide responses were normalized to the response of this peptide
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