LexA-DNA bond strength by single molecule force spectroscopy.

The SOS system of Escherichia coli is coordinated by two proteins: LexA, a repressor protein of several unlinked genes, and the coprotease RecA. As known to date LexA controls 31 genes with slightly different DNA binding motifs allowing for a variable degree of repression from one gene to the other. Besides the SOS system LexA plays an important role in the regulation of transcription. The protein regulates transcription by using particular motifs to bind DNA, the helix-turn-helix motif. Here, we employed AFM-based single molecule force spectroscopy to characterize the interaction of LexA protein with two different DNA motifs: recA and yebG. We measured the dissociation rates to be 0.045 s(-1) for recA and 0.13 s(-1) for yebG, respectively, which is in accordance with the predicted higher affinity between LexA-recA compared to LexA-yebG. The widths of the binding potentials were determined to be 5.4 +/- 1 angstroms and 4.9 +/- 0.5 angstroms, respectively. This short-ranged potential is characteristic for a stiff hydrogen-bonding network between protein and DNA. The unbinding occurs in a breakup rather than a gradual sliding.

[1]  S. Harrison,et al.  DNA recognition by proteins with the helix-turn-helix motif. , 1990, Annual review of biochemistry.

[2]  P K Hansma,et al.  Stepwise unfolding of titin under force-clamp atomic force microscopy. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[3]  R. Merkel,et al.  Energy landscapes of receptor–ligand bonds explored with dynamic force spectroscopy , 1999, Nature.

[4]  M. Rief,et al.  Reversible unfolding of individual titin immunoglobulin domains by AFM. , 1997, Science.

[5]  H. Güntherodt,et al.  Dynamic force spectroscopy of single DNA molecules. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[6]  R. Waugh,et al.  A microcantilever device to assess the effect of force on the lifetime of selectin-carbohydrate bonds. , 2001, Biophysical journal.

[7]  A. Becker,et al.  Specific binding of the regulatory protein ExpG to promoter regions of the galactoglucan biosynthesis gene cluster of Sinorhizobium meliloti--a combined molecular biology and force spectroscopy investigation. , 2003, Journal of structural biology.

[8]  P. V. von Hippel,et al.  Selection of DNA binding sites by regulatory proteins. II. The binding specificity of cyclic AMP receptor protein to recognition sites. , 1988, Journal of molecular biology.

[9]  R. Knegtel,et al.  A model for the LexA repressor DNA complex , 1995, Proteins.

[10]  D. Lilley,et al.  DNA-protein: structural interactions , 1995 .

[11]  R. Kaptein,et al.  Solution structure of the LexA repressor DNA binding domain determined by 1H NMR spectroscopy. , 1994, The EMBO journal.

[12]  J. W. Little,et al.  Dimerization of a specific DNA-binding protein on the DNA. , 1992, Science.

[13]  U. Bockelmann,et al.  Unzipping DNA with optical tweezers: high sequence sensitivity and force flips. , 2002, Biophysical journal.

[14]  H. Gaub,et al.  Adhesion forces between individual ligand-receptor pairs. , 1994, Science.

[15]  S. Lowen.  The Biophysical Journal , 1960, Nature.

[16]  H. Gaub,et al.  Dynamic single-molecule force spectroscopy: bond rupture analysis with variable spacer length , 2003 .

[17]  D. Foguel,et al.  LexA Repressor Forms Stable Dimers in Solution , 2000, The Journal of Biological Chemistry.

[18]  K. Roland,et al.  Cleavage of LexA repressor. , 1994, Methods in enzymology.

[19]  R. Ebright,et al.  Structure of the LexA repressor-DNA complex probed by affinity cleavage and affinity photo-cross-linking. , 1996, Biochemistry.

[20]  D. Mount,et al.  The SOS regulatory system of Escherichia coli , 1982, Cell.

[21]  M Schnarr,et al.  DNA binding properties of the LexA repressor. , 1991, Biochimie.

[22]  U. Seifert,et al.  Rupture of multiple parallel molecular bonds under dynamic loading. , 2000, Physical review letters.

[23]  P. Tavan,et al.  Ligand Binding: Molecular Mechanics Calculation of the Streptavidin-Biotin Rupture Force , 1996, Science.

[24]  H. Gaub,et al.  Intermolecular forces and energies between ligands and receptors. , 1994, Science.

[25]  David A. Kidwell,et al.  Sensing Discrete Streptavidin-Biotin Interactions with Atomic Force Microscopy , 1994 .

[26]  R. Lévy,et al.  Measuring the spring constant of atomic force microscope cantilevers: thermal fluctuations and other methods , 2002 .

[27]  Wolfgang A. Linke,et al.  Reverse engineering of the giant muscle protein titin , 2002, Nature.

[28]  R. Sauer,et al.  Transcription factors: structural families and principles of DNA recognition. , 1992, Annual review of biochemistry.

[29]  Gil U. Lee,et al.  Direct measurement of the forces between complementary strands of DNA. , 1994, Science.

[30]  R. Woodgate,et al.  Identification of additional genes belonging to the LexA regulon in Escherichia coli , 2000, Molecular microbiology.

[31]  M. Viani,et al.  Small cantilevers for force spectroscopy of single molecules , 1999 .

[32]  R. Lloubès,et al.  Interaction of a regulatory protein with a DNA target containing two overlapping binding sites. , 1991, The Journal of biological chemistry.

[33]  M. Benoit.  Cell adhesion measured by force spectroscopy on living cells. , 2002, Methods in cell biology.

[34]  H. Hansma.  Surface biology of DNA by atomic force microscopy. , 2001, Annual review of physical chemistry.

[35]  M. Rief,et al.  Sequence-dependent mechanics of single DNA molecules , 1999, Nature Structural Biology.

[36]  H. Grubmüller,et al.  Dynamic force spectroscopy of molecular adhesion bonds. , 2000, Physical review letters.

[37]  P. V. von Hippel,et al.  Selection of DNA binding sites by regulatory proteins. , 1988, Trends in biochemical sciences.

[38]  Benjamin C. Jantzen,et al.  Probing protein-DNA interactions by unzipping a single DNA double helix. , 2002, Biophysical journal.

[39]  T M Jovin,et al.  Scanning force microscopy of circular and linear plasmid DNA spread on mica with a quaternary ammonium salt. , 1993, Nucleic acids research.

[40]  H Schindler,et al.  Detection and localization of individual antibody-antigen recognition events by atomic force microscopy. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[41]  D. Mount,et al.  Nucleotide sequence binding specificity of the LexA repressor of Escherichia coli K-12 , 1985, Journal of bacteriology.

[42]  P. Dumoulin,et al.  Orientation of the LexA DNA-binding motif on operator DNA as inferred from cysteine-mediated phenyl azide crosslinking. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[43]  E. Evans,et al.  Strength of a weak bond connecting flexible polymer chains. , 1999, Biophysical journal.

[44]  H. Güntherodt,et al.  Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[45]  M. Rief,et al.  The mechanical stability of immunoglobulin and fibronectin III domains in the muscle protein titin measured by atomic force microscopy. , 1998, Biophysical journal.

[46]  P. Hänggi,et al.  Reaction-rate theory: fifty years after Kramers , 1990 .

[47]  C Sander,et al.  LexA repressor and iron uptake regulator from Escherichia coli: new members of the CAP-like DNA binding domain superfamily. , 1994, Protein engineering.

[48]  D. Mount,et al.  Identification of high affinity binding sites for LexA which define new DNA damage-inducible genes in Escherichia coli. , 1994, Journal of molecular biology.

[49]  G. I. Bell.  Models for the specific adhesion of cells to cells. , 1978, Science.

[50]  Matthias Rief,et al.  Single Molecule Force Spectroscopy on Polysaccharides by Atomic Force Microscopy , 1997, Science.

[51]  H. Kramers.  Brownian motion in a field of force and the diffusion model of chemical reactions , 1940 .

[52]  M. Granger-Schnarr,et al.  Contacts between the LexA repressor–or its DNA‐binding domain–and the backbone of the recA operator DNA. , 1988, The EMBO journal.

[53]  E. Evans,et al.  Dynamic strength of molecular adhesion bonds. , 1997, Biophysical journal.

[54]  O. Berg,et al.  Selection of DNA binding sites by regulatory proteins: the LexA protein and the arginine repressor use different strategies for functional specificity. , 1988, Nucleic acids research.