LexA repressor forms stable dimers in solution. The role of specific dna in tightening protein-protein interactions.
Cooperativity in the interactions among proteins subunits and DNA is crucial for DNA recognition. LexA repressor was originally thought to bind DNA as a monomer, with cooperativity leading to tighter binding of the second monomer. The main support for this model was a high value of the dissociation constant for the LexA dimer (micromolar range). Here we show that the protein is a dimer at nanomolar concentrations under different conditions. The reversible dissociation of LexA dimer was investigated by the effects of hydrostatic pressure or urea, using fluorescence emission and polarization to monitor the dissociation process. The dissociation constant lies in the picomolar range (lower than 20 pM). LexA monomers associate with an unusual large volume change (340 ml/mol), indicating the burial of a large surface area upon dimerization. Whereas nonspecific DNA has no stabilizing effect, specific DNA induces tightening of the dimer and a 750-fold decrease in the K(d). In contrast to the previous model, a tight dimer rather than a monomer is the functional repressor. Accordingly, the LexA dimer only loses its ability to recognize a specific DNA sequence by RecA-induced autoproteolysis. Our work provides insights into the linkage between protein-protein interactions, DNA recognition, and DNA repair.
Mohana-Borges
R
R
Departamento de Bioquímica Médica-ICB, Centro Nacional de Ressonância Magnética Nuclear de Macromoléculas and Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, RJ, Brazil.
Pacheco
A B
AB
Sousa
F J
FJ
Foguel
D
D
Almeida
D F
DF
Silva
J L
JL
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D004247:DNA; D012697:Serine Endopeptidases; D012996:Solutions; D000099160:LexA Repressor Protein