Characterization of the DNA-binding domain of beta protein, a component of phage lambda red-pathway, by UV catalyzed cross-linking.
beta protein, a key component of Red-pathway of phage lambda 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 beta protein and purified it to homogeneity. A chemical cross-linking reagent, glutaraldehyde, was used to stabilize the physical association of beta 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 beta protein/5 nucleotide residues. Electrophoretic gel mobility shift assays suggested that beta protein formed stable nucleoprotein complexes with 36-mer, but not with 27- or 17-mer DNA. Interestingly, the interaction of beta protein with DNA and the stability of nucleoprotein complexes was dependent on the presence of MgCl2, and the binding was abolished by 250 mM NaCl. The Kd of beta protein binding to 36-mer DNA was on the order of 1.8 x 10(-6) M. Photochemical cross-linking of native beta 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 beta protein.
Mythili
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Department of Biochemistry, Indian Institute of Science, Bangalore, India.
Kumar
K A
KA
Muniyappa
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D010582:Bacteriophage lambda / Q000737:chemistry*; D002918:Chymotrypsin; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005976:Glutaral / Q000378:metabolism; D009698:Nucleoproteins / Q000378:metabolism; D009994:Osmolar Concentration; D011487:Protein Conformation; D011995:Recombination, Genetic / Q000235:genetics; D012965:Sodium Chloride / Q000494:pharmacology; D014466:Ultraviolet Rays; D014764:Viral Proteins / Q000737:chemistry / Q000302:isolation & purification
D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009698:Nucleoproteins; D014764:Viral Proteins; D012965:Sodium Chloride; D002918:Chymotrypsin; D005976:Glutaral