The Bacillus subtilis DinR Binding Site: Redefinition of the Consensus Sequence

ABSTRACT Recently, the DinR protein was established as the cellular repressor of the SOS response in the bacterium Bacillus subtilis. It is believed that DinR functions as the repressor by binding to a consensus sequence located in the promoter region of each SOS gene. The binding site for DinR is believed to be synonymous with the formerly identified Cheo box, a region of 12 bp displaying dyad symmetry (GAAC-N4-GTTC). Electrophoretic mobility shift assays revealed that highly purified DinR does bind to such sites located upstream of the dinA, dinB,dinC, and dinR genes. Furthermore, detailed mutational analysis of the B. subtilis recA operator indicates that some nucleotides are more important than others for maintaining efficient DinR binding. For example, nucleotide substitutions immediately 5′ and 3′ of the Cheo box as well as those in the N4 region appear to affect DinR binding. This data, combined with computational analyses of potential binding sites in other gram-positive organisms, yields a new consensus (DinR box) of 5′-CGAACRNRYGTTYC-3′. DNA footprint analysis of the B. subtilis dinR and recA DinR boxes revealed that the DinR box is centrally located within a DNA region of 31 bp that is protected from hydroxyl radical cleavage in the presence of DinR. Furthermore, while DinR is predominantly monomeric in solution, it apparently binds to the DinR box in a dimeric state.

[1]  D. Sinderen,et al.  Regulated expression of the dinR and recA genes during competence development and SOS induction in Bacillus subtilis , 1996, Molecular microbiology.

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

[3]  G. May,et al.  An EMSA-based method for determining the molecular weight of a protein--DNA complex. , 1993, Nucleic acids research.

[4]  J. Carey.  [8] Gel retardation , 1991 .

[5]  N. Guillén,et al.  Identification of dinR, a DNA damage-inducible regulator gene of Bacillus subtilis , 1991, Journal of bacteriology.

[6]  G B Koudelka,et al.  DNA twisting and the affinity of bacteriophage 434 operator for bacteriophage 434 repressor. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[7]  R. Woodgate,et al.  Characterization of DinR, the Bacillus subtilis SOS repressor , 1997, Journal of bacteriology.

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

[9]  J. Hayes,et al.  The missing nucleoside experiment: a new technique to study recognition of DNA by protein. , 1989, Biochemistry.

[10]  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.

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

[12]  S. Harrison,et al.  Effect of non-contacted bases on the affinity of 434 operator for 434 repressor and Cro , 1987, Nature.

[13]  P Howard-Flanders,et al.  DNA repair and genetic recombination: studies on mutants of Escherichia coli defective in these processes. , 1966, Radiation research.

[14]  D. Cheo,et al.  Molecular characterization of regulatory elements controlling expression of the Bacillus subtilis recA+ gene. , 1992, Biochimie.

[15]  F. Movahedzadeh,et al.  Mycobacterial recA is cotranscribed with a potential regulatory gene called recX , 1997, Molecular microbiology.

[16]  D. Cheo,et al.  Cloning and characterization of DNA damage-inducible promoter regions from Bacillus subtilis , 1991, Journal of bacteriology.

[17]  F. Movahedzadeh,et al.  Characterization of Mycobacterium tuberculosis LexA: recognition of a Cheo (Bacillus-type SOS) box. , 1997, Microbiology.

[18]  M. Granger-Schnarr,et al.  The carboxy‐terminal domain of the LexA repressor oligomerises essentially as the entire protein , 1988, FEBS letters.

[19]  V. Mizrahi,et al.  SOS induction in mycobacteria: analysis of the DNA‐binding activity of a LexA‐like repressor and its role in DNA damage induction of the recA gene from Mycobacterium smegmatis , 1997, Molecular microbiology.

[20]  M. Daune,et al.  Large-scale purification, oligomerization equilibria, and specific interaction of the LexA repressor of Escherichia coli. , 1985, Biochemistry.

[21]  Roger Woodgate,et al.  The SOS Response , 1998 .

[22]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .

[23]  J. Carey.  Gel retardation. , 1991, Methods in enzymology.

[24]  J. W. Little,et al.  Autodigestion of lexA and phage lambda repressors. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[25]  J. W. Little.  Autodigestion of lexA and Phage 1 Repressors , 1984 .

[26]  D. Mount,et al.  Repression of the E coli recA gene requires at least two LexA protein monomers. , 1991, Biochimie.

[27]  D. Cheo,et al.  Inducible DNA repair and differentiation in Bacillus subtilis: interactions between global regulons , 1992, Molecular microbiology.

[28]  Bradley T. Smith,et al.  The Bacillus subtilis dinR Gene Codes for the Analogue of Escherichia coli LexA , 1996, The Journal of Biological Chemistry.

[29]  N. Guillén,et al.  Expression of the Bacillus subtilis dinR and recA genes after DNA damage and during competence , 1992, Journal of bacteriology.