Domain structure and DNA binding regions of beta protein from bacteriophage lambda.
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.
Wu
Zengru
Z
Division of Pharmaceutics, College of Pharmacy, Ohio State University, Columbus, Ohio 43210, USA.
Xing
Xu
X
Bohl
Casey E
CE
Wisler
James W
JW
Dalton
James T
JT
Bell
Charles E
CE
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000378:metabolism*; D019921:Biotinylation; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014764:Viral Proteins / Q000737:chemistry* / Q000502:physiology
D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D014764:Viral Proteins; C118730:beta protein, Bacteriophage lambda; D004247:DNA; D008239:Lysine; D000409:Alanine