the bacillus subtilis dinr binding site: redefinition of the

JOURNAL OF BACTERIOLOGY,0021-9193/98/$04.0010

Apr. 1998, p. 2201–2211 Vol. 180, No. 8

Copyright © 1998, American Society for Microbiology

The Bacillus subtilis DinR Binding Site: Redefinition of theConsensus Sequence

KEVIN W. WINTERLING,1,2 DAVID CHAFIN,3 JEFFERY J. HAYES,3 JI SUN,4,5 ARTHUR S. LEVINE,1

RONALD E. YASBIN,5 AND ROGER WOODGATE1*

Section on DNA Replication, Repair, and Mutagenesis, National Institute of Child Health and Human Development,Bethesda, Maryland 20892-27251; Department of Biological Sciences2 and Program in Molecular and CellularBiology,4 University of Maryland, Baltimore County, Baltimore, Maryland 21228; Department of Biochemistry

and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, New York 146423;and Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, Texas 750835

Received 15 August 1997/Accepted 11 February 1998

Recently, the DinR protein was established as the cellular repressor of the SOS response in the bacteriumBacillus subtilis. It is believed that DinR functions as the repressor by binding to a consensus sequence locatedin the promoter region of each SOS gene. The binding site for DinR is believed to be synonymous with theformerly identified Cheo box, a region of 12 bp displaying dyad symmetry (GAAC-N4-GTTC). Electrophoreticmobility 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 indicatesthat 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 affectDinR 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 analysisof the B. subtilis dinR and recA DinR boxes revealed that the DinR box is centrally located within a DNA regionof 31 bp that is protected from hydroxyl radical cleavage in the presence of DinR. Furthermore, while DinR ispredominantly monomeric in solution, it apparently binds to the DinR box in a dimeric state.

Based upon sequence comparisons, it has been hypothesizedthat the Bacillus subtilis protein DinR is the functional ho-molog of the Escherichia coli SOS transcriptional repressor,LexA (20, 21). Indeed, recently published data has firmly es-tablished DinR as the transcriptional repressor of the SOSsystem in B. subtilis (5, 16, 27). Although it is only 34% iden-tical to LexA, DinR demonstrates many biochemical and phys-ical properties that are reminiscent of LexA. For example, likethat of LexA, the deduced amino acid sequence of DinR pre-dicts two distinct domains within the protein. DinR has mosthomology to LexA and other LexA-like proteins in its carbox-yl-terminal domain (10, 27). This C-terminal domain is thoughtto be primarily responsible for the cooperative dimerization ofthe normally monomeric LexA protein at its target site in DNA(8, 22, 23, 25). The C-terminal domain also contains a con-served Ala-Gly cleavage site as well as the appropriatelyspaced serine and lysine residues that have been identified ascritical for autodigestion (14). Indeed, like LexA, DinR under-goes RecA-independent autocatalysis at alkaline pH andRecA-mediated autocatalysis under more physiological condi-tions (16, 27). Such cleavage inactivates repressor function,thereby allowing DinR-regulated genes to be expressed.

Despite the notion that DinR displays transcriptional re-pressor activity that is comparable to that of LexA (16), thereis in fact little homology between the amino-terminal DNAbinding domains of the two proteins (10, 27). In addition to theobvious lack of primary sequence homology, the typical repres-sor-like, helix-turn-helix motif present in LexA is not immedi-ately obvious in DinR. This disparity coincides with the ap-

pearance of completely distinct DNA binding sequences in thetwo repressors. In E. coli and many other gram-negative or-ganisms, the SOS box is a region of 16 bp that displays dyadsymmetry [59-CTGT-(AT)4-ACAG-39] (13, 26). In severalgram-positive bacteria (e.g., B. subtilis and Mycobacteria sp.) (2,4, 17, 19), the binding site for DinR is thought to be thepreviously described Cheo box, a region of 12 bp with dyadsymmetry (59-GAAC-N4-GTTC-39) but no homology to thegram-negative SOS box.

It has recently been suggested that the B. subtilis DinRprotein should be renamed LexA (16). Given the huge differ-ences in the recognition sites between the E. coli LexA proteinand the gram-positive DinR-like proteins, however (4, 17, 19,27; see below), we propose retaining the descriptive nameDinR (damage inducible repressor) rather than renaming theprotein LexA (originally defined as locus for X-ray sensitivityA [7]) and using the term DinR box to describe the binding sitefor DinR to avoid confusion between it and the commonlyreferred to SOS box of E. coli.

We have previously purified the B. subtilis DinR protein tohomogeneity (27) and shown that it does bind to the proposedDinR binding site in the B. subtilis recA promoter region butdoes not bind to certain mutant sequences located within thepreviously identified Cheo box. The availability of the highlypurified B. subtilis DinR protein has enabled us to extend thesestudies and perform a detailed molecular analysis of the B.subtilis DinR box. Indeed, by using a combination of gel elec-trophoretic mobility shift assays, hydroxyl radical footprintprotection assays, and recA-lacZ transcriptional fusions, wehave determined that certain bases within the previously de-fined Cheo box are more critical for binding than others. Thisdata, together with computational analyses of potential bindingsites in other gram-positive organisms, allows us to propose anew consensus DinR box, 59-CGAACRNRYGTTYC-39.

* Corresponding author. Mailing address: Building 6, Room 1A13,NICHD, NIH, 9000 Rockville Pike, Bethesda, MD 20892-2725. Phone:(301) 496-6175. Fax: (301) 594-1135. E-mail: [email protected].

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(This research was conducted by K. Winterling and J. Sun inpartial fulfillment of the requirements for a Ph.D.)

MATERIALS AND METHODS

Bacterial strains and plasmids. The B. subtilis strain used in this study,YB886A, serves as a wild-type strain and is cured of all known prophages. E. coliDH5a (GIBCO-Life Technologies, Gaithersburg, Md.) and GBE180 (DH5apcnB1) were used for routine cloning and maintenance of plasmids (27).

The recA fragment used for mutational analysis of the Cheo box is essentiallythe previously described 202-bp HindIII-Sau3AI fragment encompassing thepromoter of the B. subtilis gene recA (2, 3, 27). Various mutations in the recACheo box were made via site-directed mutagenesis by following the specificationsof the ExSite kit from Stratagene (La Jolla, Calif.) (24).

Media and growth conditions. B. subtilis strains were maintained on tryptoseblood agar base medium, and liquid cultures were grown in antibiotic medium 3(Difco Laboratories, Detroit, Mich.) or nutrient broth (Oxoid Ltd., Basingstoke,United Kingdom) with aeration at 37°C. E. coli strains were grown on Luria-Bertani agar or in Luria-Bertani broth. Ampicillin (50 mg/ml), chloramphenicol(20 mg/ml), kanamycin (30 mg/ml), and isopropyl-b-D-thiogalactopyranoside(IPTG) (1 mM) (Gold Biotechnology, Inc., St. Louis, Mo.) were added asrequired.

b-Galactosidase assays. DNA damage-inducible promoter activity of the recA-lacZ and din-lacZ fusions was examined by measuring b-galactosidase activity aspreviously described (27). Briefly, B. subtilis cultures were grown with aeration at37°C in nutrient broth supplemented with 0.1% yeast extract. Cultures weregrown to early exponential phase, when an aliquot was removed and the culturewas divided in half. Mitomycin (0.5 mg/ml) was added to one-half of the culture,and 1-ml aliquots were taken from the induced and uninduced cultures after 90min of additional incubation. After the absorbance (optical density at 595 nm) ofeach sample was spectrophotometrically measured, the cells were harvested andwashed in 0.5 ml of 25 mM Tris-HCl (pH 7.4). The supernatant was decanted,and the pellet was placed in a dry ice-ethanol bath. The pellet was subsequentlyresuspended in 0.64 ml of Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM

KCl, 1 mM MgSO4, and 50 mM b-mercaptoethanol [pH 7.0]) (15), 0.16 ml of alysozyme solution (2.5 mg/ml in Z buffer) was added, and the sample wasincubated at 37°C for 5 min. Eight microliters of 10% Triton X-100 was added,and the samples were warmed to 30°C. b-Galactosidase activity was calculated bythe method of Miller (15).

Electrophoretic mobility shift assays. The exact location of each DinR boxrelative to the 235 and 210 promoter elements as well as the translational startsite of each gene is shown in Fig. 1. The wild-type and mutant recA DinR boxesused in this assay were gel-purified synthetic oligonucleotides that varied inlength from 60 to 67 bp. Complementary single-stranded oligonucleotides wereannealed together by mixing equimolar amounts of each oligonucleotide, thusproducing short regions of double-stranded DNA (27). In addition, oligonucle-otides were similarly synthesized, purified, and annealed so that they corre-sponded to the wild-type promoter sequences of dinA and dinB. All of theseprobes were designed so that the DinR binding site was centered, with approx-imately 26 bp of wild-type sequence flanking each side.

dinC contains two putative DinR binding sites, which are located close to itspromoter elements (Fig. 1). One putative DinR site is located between the 235and 210 promoter elements (at 256 to 268 bp relative to the initiator codon)and was designated the 224 site for convenience (Fig. 1). The other site islocated about 30 bp upstream of the first site (at 286 to 298 bp relative to theinitiator codon) and is called the 253 site (Fig. 1). To assess the ability of DinRto bind to each of these sites, three probes were synthesized, one that containedthe 224 site, one that contained the 253 site, and one that contained both the224 and the 253 sites.

The dinR gene has three potential binding sites, two of which are located closeto the promoter region (Fig. 1) and are denoted the 239 and 267 DinR bindingsites. The third site is further upstream and is called the 2104 DinR box. Thislatter box has previously been shown to play no apparent role in regulating dinR(5) and was therefore not studied further in the gel mobility shift assay. As aconsequence, we synthesized only three probes, one that contained only the 239or the 267 site and one that contained both of these sites.

The oligonucleotides were designed so that when they were annealed together,both ends had 59 T and/or A extensions that could be labeled with [32P]dATP

FIG. 1. Location of DinR boxes in the promoter regions of the B. subtilis recA, dinC, and dinR genes. The respective 235 and 210 promoter elements for each geneare in slightly larger, bold-faced letters. The DinR boxes are double underlined. The recA-DinR box is centered at 251 relative to the 235 promoter. The twodinC-DinR boxes are located at 224 and 253 relative to the 235 and 210 elements. The three dinR-DinR boxes are located at 239, 267, and 2104 relative to the235 promoter.

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and/or [32P]dTTP by a Klenow fill-in reaction. Reaction mixtures (20 ml each)containing approximately 3.0 ng of labeled probe and various amounts of DinRwere incubated at room temperature for 25 min in binding buffer (150 mM NaCl,20 mM Tris-HCl [pH 7.5], 0.2 mM EDTA, 1.0 mM MgCl2, 5% glycerol [vol/vol],50 mg of bovine serum albumin [BSA] per ml). Protein-DNA complexes wereseparated in native polyacrylamide gels (5 or 6% acrylamide). Gels were driedand subsequently exposed to Kodak XAR X-ray film for appropriate periods oftime. Dried gels were also exposed to Molecular Dynamics phosphor screens andscanned into the Molecular Dynamics PhosphoImager. Subsequent quantitationwas performed with Image Quant version 1.1 software.

Determination of the oligomeric state of DinR in solution and DinR bound totarget DNA. To determine the oligomeric state of DinR in solution, we comparedits relative sedimentation in a glycerol gradient to that of the E. coli LexAprotein. Highly purified DinR and LexA proteins (5 mg each) were loaded ontoseparate 5 to 30% linear glycerol gradients in buffer D [10 mM piperazine-N,N9-bis(2-ethanesulfonic acid) (PIPES)–NaOH (pH 7.0), 0.1 mM EDTA, 10% (vol/vol) glycerol, 200 mM NaCl]. Each gradient also contained 5 mg of BSA, 5 mg ofovalbumin, and 2 mg of cytochrome c as internal molecular weight standards.Ultracentrifugation was carried out for 26 h at 49,000 rpm with a SW60Ti rotor.Fractions (100 ml each) were collected, and proteins were separated in sodiumdodecyl sulfate-polyacrylamide gel electrophoresis gradient gels containing 9 to19% polyacrylamide. Proteins were subsequently visualized by staining the gelwith silver.

The oligomeric state of DinR when it is bound to its target sequence wasdetermined by employing an electrophoretic mobility shift assay-based protocoldeveloped by Orchard and May (18). The DNA fragments used in this experi-ment were identical to those described in the preceding section. The protein-DNA complexes were formed as described above and separated in native poly-acrylamide gels whose acrylamide concentrations ranged from 4.5 to 10% (4.5, 5,6, 7, 8, 9, and 10%). In addition to the DinR-DNA complexes, a sample ofpurified DinR and a set of nondenatured protein standards (Sigma ChemicalCo., St. Louis, Mo.) were determined empirically. The lanes containing theDinR-DNA complexes were excised from each of the gels, dried, and subse-quently exposed to Kodak XAR X-ray film for appropriate periods of time. Theremainder of the gel, containing the purified DinR and the nondenatured proteinstandards, was stained with Coomassie brilliant blue R-250. The distances mi-grated by each of the DinR-DNA complexes, the DinR protein alone, and eachof the standards were measured and then divided by the distance travelled by thedye (bromophenol blue) in each lane. This calculation yields the relative mobility(Rf) of each protein or protein-DNA complex. The logarithm of the Rf was thenplotted for each protein standard as a function of gel concentration. The slope ofthe line for each protein standard, called the retardation coefficient (Kr), wassubsequently plotted as a function of the molecular weight of each standard. TheKr was determined for each DinR-DNA complex, for the free (unbound) DNA,and for the DinR alone. The derived standard curve was subsequently used tocalculate the molecular weight of each protein-DNA complex, the DNA, and theDinR. Subtracting the molecular weight of the DNA from that of the DinR-DNAcomplex yields the apparent molecular weight of the protein associated with eachprotein-DNA complex.

Labeled DNA for hydroxyl radical footprint analysis. Approximately 15 mg ofPCR primer, DINR01 (59-GCGAAGCTTCTCATGATCATAACCTC-CAAC-39),RECA01 (59-GCGAAGCTTACATGATTTTCTGATACATTA-39), or RECA02(59-CGCGAATTCCTTTTATGTTACACTACATA-39), was 59 radiolabeled inseparate reactions with T4 polynucleotide kinase. Briefly, 10 U of T4 polynucle-otide kinase (New England Biolabs) was used to singly radioactively label eachPCR primer in a 20-ml reaction mixture containing 15 mg of PCR primer(DINR01, RECA01, or RECA02), 13 T4 PNK reaction buffer (70 mMTris z HCl [pH 7.6], 10 mM MgCl2, 5 mM dithiothreitol), and 50 mCi of[g-32P]dATP at 6,000 Ci/mmol for 30 to 60 min at 37°C. Radioactive primerswere used in standard PCRs to obtain the dinR and recA promoter sequences in100-ml reaction mixtures containing 10 ml of labeled primer (from the labelingreaction), 13 Vent polymerase buffer, 6 mg of complementary unlabeled PCRprimer, 270 ng of B. subtilis YB886A chromosomal DNA, 10 mM each de-oxynucleoside triphosphate, and 2 U of Vent DNA polymerase (New EnglandBiolabs). PCR DNA was precipitated and separated on a native 8% polyacryl-amide gel made with 13 Tris-borate-EDTA. After separation, the wet gel wasexposed to autoradiography film to identify the radioactive PCR product. Theradioactive dinR and recA DNAs were gel purified by soaking a crushed gel slicein 700 ml of TE (10 mM Tris-Cl [pH 8.0], 1 mM EDTA) buffer overnight. Theradioactive DNAs were filtered through series 8000 microcentrifuge filtrationdevices (Lida Manufacturing Corporation), precipitated, dissolved in TE buffer,and stored at 3,000 to 5,000 cpm/ml.

Hydroxyl radical footprint analysis. Equal volumes of labeled DNA (approx-imately 60,000 cpm) and DinR (diluted into glycerol-free 23 binding buffer [27])were incubated at room temperature for 25 min. Labeled DNA from dinR-DinRor recA-DinR complexes was gel purified in the same manner as the labeled PCRproducts described above. In a typical 40-ml reaction mixture, 2 ml of 1 mMFe-EDTA (50 mM final) and 2 ml of 20 mM sodium ascorbate (1 mM final) werepipetted onto the side of the reaction tube. Hydroxyl radical cleavage wasinitiated by adding 2 ml of a 1:250 dilution of 30% hydrogen peroxide solutioninto the existing drop (0.0075% final) and the DNA solution for 2.5 min. Cleav-age was stopped by adding 1/10 volume of stop solution containing 50% glycerol

and 10 mM EDTA. Protein-DNA complexes were separated by loading thecleavage reaction mixtures immediately onto a native 5% polyacrylamide–0.53Tris-borate-EDTA gel.

G-specific reaction. In a typical 200-ml reaction mixture, approximately 20,000cpm of singly labeled DNA was added to 20 ml of 103 G-specific reaction buffer(0.5 M sodium cacodylate-10 mM EDTA) and 160 ml of water. One microliter ofstraight dimethyl sulfate was added to the tube. The reaction mixture was mixedimmediately and incubated for 1 min before being briefly spun in a microcen-trifuge. Fifty microliters of stop solution was added (1.5 M sodium acetate, 1 Mb-mercaptoethanol, 0.004 mg of sonicated calf thymus DNA per ml), and theDNA was precipitated and dissolved in 90 ml of TE buffer. Ten microliters ofpiperidine was added and incubated at 90°C for 30 min. The DNA was dried tocompletion in a speed-vac concentrator. The dried DNA was dissolved twice in20 ml of water and redried. The DNAs were finally dissolved in 40 to 50 ml of TEbuffer and stored at 4°C.

Sequencing gel analysis of hydroxyl radical footprint. Approximately 5,000cpm from each sample was placed into separate Eppendorf tubes and driedcompletely in a speed-vac concentrator. Dried DNAs were dissolved in 4 ml offormamide loading buffer (100% formamide, xylene cylanol, bromophenol blue)and heated to 95°C for 2 min. Samples were immediately placed on ice andloaded onto a 6% polyacrylamide-8 M urea sequencing gel. After analysis, gelswere dried and exposed to autoradiography film or to a Molecular DynamicsPhosphorImager screen.

RESULTS

Regulation of damage-inducible genes in B. subtilis. All liv-ing organisms are exposed to a variety of synthetic and naturalDNA-damaging agents. The differential regulation of a re-sponse for coping with such damage would allow cells to re-spond to the extent of DNA damage by inducing only proteinsthat are required to efficiently repair all of the damaged DNA.In E. coli, differential regulation of SOS genes occurs and isachieved, at least in part, by variation of the affinity of thetranscriptional repressor, LexA, for its binding site (reference13 and references therein). Analysis of the levels of b-galacto-sidase produced by various din-lacZ fusions revealed that thebasal level of B. subtilis din expression also varies considerably(Fig. 2). Obviously, such differences in expression could, intheory, be achieved by differential promoter activity or by al-teration of the affinity of the repressor for its binding site.While previous studies have demonstrated that DinR binds tothe Cheo box upstream of recA, dinB, dinC, and dinR (5, 16,27), the relative affinity of DinR for each site is largely un-

FIG. 2. Expression of B. subtilis dinA-lacZ, dinB-lacZ, dinC-lacZ, dinR-lacZ,and recA-lacZ transcriptional fusions. The various constructs were present in asingle copy due to integration at the amyE locus. The SOS regulon was induced,where noted, by the addition of 0.5 mg of mitomycin per ml, cultures wereharvested 90 min later, and the level of b-galactosidase activity was determined.The data for the dinR-lacZ fusion was taken from the study of Haijema et al. (5),and that for the recA-lacZ fusion, taken from the study of Winterling et al. (27),was used for comparison.

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known. Interestingly, gel electrophoretic mobility shift assaysof the dinA and dinB DinR boxes (Fig. 3) revealed that DinRbinds to both boxes with roughly the same affinity and thatthese affinities are qualitatively similar to those of the recA-DinR box (27) and the dinR-DinR boxes (see below). Suchobservations suggest, therefore, that at least for the din genesassayed here, differential expression is achieved via differentialpromoter activities rather than differential operator affinities.This conclusion is also supported by our observation that all ofthe fusions were induced to the same extent (fourfold) undermild inducing conditions. If regulation was primarily at thelevel of operator binding, one might have expected the din-lacZ fusions to exhibit much more variable induction ratios.

Binding of DinR protein to the two DinR boxes upstream ofdinC. Unlike many din genes, dinC contains two DinR boxes,located close to its promoter. The location of each of thesesites makes them potential transcriptional repressor bindingsites. (As noted above, for the sake of simplicity, we denotedthese sites the 253 box and the 224 box (with respect to their

locations near the 235 and 210 promoter elements, respec-tively) (Fig. 1). Indeed, DNase I and hydroxy radical footprint-ing experiments have shown that both sites are apparentlyprotected in the presence of DinR (16). The relative affinity ofDinR for each DinR box is, however, unknown. To determinethe affinity of DinR for each of these sites, DNA mobility gelshift assays were performed with a probe containing only the253 site, a probe containing only the 224 site, and a probethat contained both the 253 and the 224 sites (Fig. 4A).Experiments revealed that under these assay conditions, DinRbinds specifically to each of the three probes (Fig. 4B). Visualinspection of the shifted complexes suggests, however, thatthere is no significant difference in the affinity for one probeover another. With the probe containing both DinR boxes,there was, however, a second, larger shifted complex detect-able at higher concentrations of DinR. We suggest, therefore,that each DinR box serves equally well as a potential bindingsite but that at higher cellular concentrations there is a greaterlikelihood of DinR occupancy at both sites.

Self-regulation of dinR. E. coli LexA not only serves as therepressor of a number of unlinked genes in the SOS regulonbut also acts as a negative regulator of its own synthesis. Wepresume, therefore, that like LexA, DinR regulates its ownexpression. Indeed, gel mobility shift assays with crude cellextracts suggest that this may be the case (5). Although dinRcontains three potential binding sites, Haijema et al. havedemonstrated that the DinR box, which we denote the 2104box, apparently plays no role in regulating DinR expression(5). The two remaining sites are located at 267 and 239,respectively (Fig. 1 and 5A). These sites have identical coreCheo box sequences and differ from each other only in the N4region. More importantly, however, they diverge from the con-sensus sequence, having a 39 T instead of C (GTTC3GTTT).Thus, we were interested in determining if DinR bound pref-

FIG. 3. Binding of DinR to the DinR box located upstream of dinA and dinB.Radiolabeled dinA and dinB promoter fragments (3.0 ng or ;3.4 nM) wereincubated with various concentrations of DinR (nanomolar monomer) as indi-cated at the top of each lane. Reactions were performed at room temperature for25 min. Protein-DNA complexes were separated in native polyacrylamide gels(5% acrylamide) and visualized by exposure to X-ray film.

FIG. 4. Binding of DinR to the two DinR boxes located upstream of dinC. (A) Nucleotide sequence of the region upstream of dinC. The two previously identifiedCheo boxes are indicated in bold-faced type, and the positions of the three probes used in the in vitro binding studies are indicated below the sequence as P1 (224DinR box), P2 (253 DinR box), and P3 (both DinR boxes). (B) The individually radiolabeled fragments (P1, P2, or P3; 3.0 ng or ;3.4 nM) were incubated with variousconcentrations of DinR (nanomolar monomer) as indicated at the top of each lane. Reactions were performed at room temperature for 25 min. Protein-DNAcomplexes were separated in native polyacrylamide gels (5% acrylamide) and visualized by exposure to X-ray film.



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erentially to one site or equally well to both sites (as is the casefor the two dinC DinR boxes). Indeed, the DNA mobility shiftassays revealed that DinR binds specifically to all three probes(Fig. 5B), and analysis of the shifted complexes revealed that

the affinities of DinR for the 239 and the 267 binding sites arequalitatively similar. As in the case of dinC, however, a secondshifted complex is clearly detectable when DinR is incubatedwith the probe containing both putative binding sites, suggest-

FIG. 5. Binding of DinR to the two DinR boxes located upstream of dinR. (A) Nucleotide sequence of the region upstream of dinR. The two previously identifiedCheo boxes are indicated in bold-faced type, and the positions of the three probes used in the in vitro binding studies are indicated below the sequence as P1 (239DinR box), P2 (267 DinR box), and P3 (both DinR boxes). (B) The individually radiolabeled fragments (P1, P2, or P3; 3.0 ng or ;3.4 nM) were incubated with variousconcentrations of DinR (nanomolar monomer) as indicated at the top of each lane. Reactions were performed at room temperature for 25 min. Protein-DNAcomplexes were separated in native polyacrylamide gels (6% acrylamide) and visualized by exposure to X-ray film.

FIG. 6. Expression of B. subtilis recA-lacZ transcriptional fusions. The effects of single base pair changes within the B. subtilis recA DinR box were determined byquantitating b-galactosidase activities generated from recA-lacZ transcriptional fusions expressed in B. subtilis. The histogram represents the level of b-galactosidaseactivity of each individual mutant construct. The effects of substitutions at each nucleotide in the DinR box were analyzed and are depicted as follows: G, u; A, ; T,

; C, f. The consensus sequence that resulted in equal to or less than 17 Miller units of b-galactosidase activity from the recA-lacZ fusion is given below the histogram.For comparison, the wild-type sequence for the recA DinR box is also listed.



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ing that both DinR binding sites can be occupied if there isenough DinR.

Detailed molecular analysis of the DinR box located up-stream of recA. Based upon previous studies (2, 4, 16, 17, 19,27), DinR or a DinR-like protein unequivocally recognizes andbinds to specific sequences located upstream of a number ofdin genes. In the case of B. subtilis recA, this site is locatedupstream of the 235 promoter element (approximately at251) (Fig. 1). By performing detailed mutational studies onthis DinR binding site, we have been able to determine whichof the bases within the formerly identified Cheo box are im-portant for regulated expression of RecA (Fig. 6). As expected,certain base changes within the core Cheo box resulted inincreased basal expression of recA-lacZ transcription in theabsence of exogenous DNA damage. It seems very unlikelythat these changes affect promoter activity per se, given thelocation of the DinR box upstream of the 235 region, and weinterpret the data as reflecting specific changes in operator

affinities. In general, lowest expression was achieved when thesequence matched the consensus Cheo box (2). In some in-stances, changes in the sequence resulted in constitutive ex-pression (i.e., the most-59 G of the Cheo box to either A, T, orC), while certain other changes were accommodated (i.e., theC3T transition in the 59 GAAC side of the Cheo box) (Fig. 6).Interestingly, substitutions in the two outermost nucleotides inthe N4 region also appear to affect recA-lacZ expression,whereas those in the very middle of this region appear to havelittle effect (Fig. 6).

As part of these studies, we have analyzed the effects ofchanges in the 59 and 39 bases that flank the Cheo box. Inter-estingly, while changes in the 59 cytosine to thymine or adeninehad very little effect on expression of the recA-lacZ fusion, achange to a guanine resulted in constitutively high levels ofexpression (Fig. 6). Likewise, changes from the 39 guanine tothymine had little effect, while those to adenine or cytosineresulted in constitutive expression. Assuming that a b-galacto-sidase level of 17 Miller units or lower represents the baselinefor recA-lacZ expression from the various mutant DinR boxes,the lowest level of expression (and therefore the tightest DinRbinding) appears to be attained when the DinR box is 59-T/CGAAT/CG/ANNCGTG/TCG/T-39 (where the previously de-scribed consensus Cheo box is underlined) (Fig. 6). This se-quence fits remarkably well with the newly derived consensusDinR binding site (see below).

FIG. 7. Footprinting analysis of DinR bound to the recA operator. (A) Thecoding and noncoding strands of a radiolabeled recA promoter fragment wereincubated with increasing concentrations of purified DinR and subjected tohydroxyl radical cleavage (lanes 4 and 5 and 9 and 10, respectively). LabeledDNA was also cleaved in the absence of DinR (lanes 3 and 8). Each experimentwas run alongside naked DNA (Control) and a G sequencing reaction (G-Rxn).The boxed regions denote the location of the DinR box on each strand. The 251site is representative of the most intense region of protection as well as the centerof each DinR box. (B) Nucleotide sequence of the recA operator and regionsprotected from hydroxyl radical cleavage. The sizes of the bars correspond to theintensities of protection. The center of the protected region is represented by ashaded circle that corresponds to the center of the DinR box. (C) Three-dimensional representation of the bases within the recA operator that are pro-tected by DinR during hydroxyl radical cleavage, indicating that the protectedsites lie on one face of the DNA.



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Hydroxyl radical footprinting of the B. subtilis recA and dinRDinR boxes. Given that we and others (4, 27) have demon-strated that nucleotide changes in the core Cheo box abolishbinding of DinR or a DinR-like protein, as measured by gelmobility shift assays, the assumption that the binding occurs atthe Cheo box seems more than reasonable. Unfortunately, theresults of gel mobility shift assays can often be misleading whenbinding is weak (see reference 1 for a detailed discussion). Asa consequence, binding often needs to be analyzed by alterna-tive methods, such as DNA-footprint analysis. Indeed, Milleret al. (16) and Durbach et al. (4) have used such an approachto show that the core Cheo box is protected from DNase I andhydroxyl radical cleavage. Nevertheless, we were encouragedto perform additional footprint analyses to firmly establish thatDinR does bind to the DinR box proposed in this study. To thisend, we performed hydroxyl radical protection assays in thepresence and absence of DinR with the B. subtilis recA anddinR promoter/operator sequences (Fig. 7 and 8). As notedpreviously, DinR binds specifically to the recA promoter/oper-ator region to form a single major protein DNA species asevidenced by nondenaturing gel mobility shift (27). When thisspecies is analyzed by hydroxyl radical footprinting, a patternof protection is observed which is consistent with a singlehigh-affinity binding site. A single region of protection is ob-served on each DNA strand. Within each region, a stretch of 3to 4 bases is most strongly protected by DinR. These stronglyprotected stretches are each flanked by two regions of weakerprotection (Fig. 7A). When the protection pattern is plotted ona linear representation of the recA sequence (Fig. 7B), it isclear that these stretches of protection are separated by about10 bp and staggered on opposite strands by about 2 to 4 bp,with a total of 30 to 31 bp being protected from cleavage.These results indicate a cross-groove pattern of protectionmost likely caused by a protein lying along one side of theDNA helix. In support of this interpretation, a plot of pro-tected positions on a three-dimensional representation of theDNA double helix reveals that all protected sites lie along oneface of the DNA (Fig. 7C), exactly as has been found forfootprints of other bacterial repressor-DNA complexes (6).Furthermore, and perhaps more importantly, the center of thefootprint coincides exactly with the center of the proposedDinR box (CGAATATGCGTTCG) found at this position.

The nondenaturing gel mobility shift assay suggests thatDinR apparently binds to the dinR promoter/operator to formtwo major protein-DNA species (Fig. 5). Presumably, the fast-er-migrating band contains only one DinR-DNA complex,whereas the slower-migrating band contains two. Interestingly,footprints of the larger probe containing all three potentialDinR binding sites (239, 267, and 2104; Fig. 1) revealed thatwhen fully loaded, DinR apparently protects all three regions.Indeed, these regions are most clearly demarcated by the mostintense area of protection, found in the center of the footprintpattern (Fig. 8). When cross-strand offset is corrected for (seeabove), the centers of these three regions of protection can beidentified. In concordance with the recA footprint, the regionsof protection coincide exactly with the centers of the predictedDinR box elements within the dinR promoter. As noted, wefind that with the larger probe used in the footprinting assay,the 2104 DinR box does bind DinR, even though it has pre-viously been reported to play no role in regulating dinR (5).

Determination of the oligomeric state of DinR. Sequenceanalysis of the DinR box shows two regions of dyad symmetry.This configuration appears to represent two potential half sitesthat could theoretically each be bound by a DinR monomer.LexA has previously been shown to exist primarily as a mono-

mer in solution (22, 23) and dimerizes upon binding to the SOSbox (8).

Since LexA and DinR are functionally analogous (16, 27),we were interested in determining the oligomeric state of DinRin solution and bound to target DNA. The solution state wasdetermined by comparison of its sedimentation on a glycerolgradient to that of the E. coli LexA protein and to knownprotein standards. Under these assay conditions, LexA andDinR sediment virtually identically (Fig. 9). Unlike sodiumdodecyl sulfate-polyacrylamide gel electrophoresis, which largelyseparates proteins based upon the length of the polypeptide,glycerol gradient sedimentation is a measure of molecular size

FIG. 8. Footprinting analysis of DinR bound to the dinR operator. Thecoding strand of a radiolabeled dinR promoter fragment was incubated withincreasing concentrations of purified DinR and subjected to hydroxyl radicalcleavage (lanes 4 and 5). Labeled DNA was also cleaved in the absence of DinR(lane 3). The experiment was run alongside naked DNA (Control) and a Gsequencing reaction (G-Rxn). The boxed regions denote the locations of theDinR boxes. The 239, 267, and 2104 sites are representative of the mostintense regions of protection as well as the centers of the respective DinR boxes.



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rather than mass and assumes that the unknown proteins (inthese experiments, LexA or DinR) occupy the same shape andspecific volume as the known standards. This does not appearto be a valid assumption in the case of LexA and DinR, as theobserved size of both proteins was larger (;40 kDa) than thatpredicted for a LexA or DinR monomer (;23 kDa). Basedupon its sedimentation, however, which was identical to that ofLexA (22, 23), we conclude that DinR exists predominantly asa monomer in solution.

To determine if DinR binds to target DNA as a monomer,dimer, or higher-order oligomer, we used the previously de-scribed method of Orchard and May (18) (Fig. 10). Althoughit is possible to use the theoretical molecular masses of freeDNA and DinR protein in these calculations, as noted above,under nondenaturing conditions in which proteins are sepa-rated by molecular size, conformation, and charge, the pre-dicted value does not always match that obtained experimen-tally. As a consequence, we determined empirically theapparent molecular masses of the following complexes: freeDNA, DinR monomer, DinR dimer, DinR-dinR, and DinR-recA (Fig. 10). The molecular mass of the DinR-dinR complexwas found to be ;127 kDa, and the molecular mass of theDinR-recA complex was ;136 kDa. The molecular mass of the

DNA, as determined empirically was ;48 kDa. Thus, aftersubtracting the molecular mass of the DNA from the DNA-protein complexes, we found that the molecular masses ofDinR bound to dinR and to recA are ;79 kDa and ;88 kDa,respectively. As noted above, DinR has a predicted molecularmass of ;23 kDa. In our gel electrophoresis assay, however,the monomeric weight of DinR was found to be ;60 kDa, andthat of the trace amounts of dimeric DinR that were detectablewas ;77 kDa. The difference in these values from those thatwere predicted presumably arises through the unique electro-static and structural characteristics of DinR. Thus, the closestfit to the estimated mass of the protein bound to DNA (;79 to88 kDa) is that of the empirically determined dimeric DinRprotein (;77 kDa). Based upon these observations, we there-fore conclude that DinR, like LexA (8), binds to its targetsequence as a dimer.

DISCUSSION

A new consensus binding site for DinR. Based upon thecombined transcriptional fusion studies, gel mobility shift as-says, and hydroxyl radical footprinting, we have derived a newconsensus binding site for DinR. Upon reanalysis of the pre-viously identified B. subtilis din genes and those of (presumablydamage inducible) recA and lexA/dinR from a variety of gram-positive bacterial species, we find that the 59 residue of theDinR box is generally cytosine and the 39 residue is generallyguanine (Table 1). In addition, the N4 region does not appearto accommodate all nucleotides and retain equal DinR bindingefficiency (Fig. 6) (4, 27). Using such an approach, we deriveda consensus DinR binding site of 59-CGAACRNRYGTTCG-39. Footprint analysis of several din genes reveals that thissequence is centrally located within a DNA region of 31 bp thatis protected by DinR from hydroxyl radical cleavage (Fig. 7 and8) (4), definitive evidence that this sequence is the one that isrecognized and bound by DinR. Furthermore, we found thatlike E. coli LexA (8, 22, 23, 25), DinR is predominantly mo-nomeric in solution but binds to its target DNA as a dimer(Fig. 10), presumably because each monomer binds coopera-tively to one half site.

Obviously, there are minor differences in the DinR boxsequence from one gene to another. These slight deviationsfrom the consensus are also seen in the SOS box of E. coli andare deemed essential for the differential regulation of SOSgenes, allowing the cell to provide a graded response to DNAdamage. We expected that deviations from the consensusDinR box would have direct effects on the affinity of DinR foreach site, but as evidenced by the data from the present study,this is not always the case. For example, based upon the tran-scriptional studies with recA, we would expect a cytosine at thevery 39 end of the newly defined DinR box to lead to a com-plete loss of DinR binding. Such changes are found in the 267DinR box of dinR (Fig. 1); yet, paradoxically, this sequence stillbinds DinR. Thus, efficient binding at any site is most likelydetermined by the sequence context of the entire DinR box. Innaturally occurring DinR boxes, compensatory mutations mayhave arisen so that unregulated expression of (a potentiallytoxic) protein does not occur. We suggest, therefore, that theconsensus sequence of the DinR box should be used as anindicator of potential DinR binding but that definitive in vitrostudies (gel mobility shift assay and footprinting) should beperformed on each site before any site is assumed to be boundby DinR.

The binding site for DinR was originally proposed to be aregion of 12 bp with dyad symmetry (59-GAAC-N4-GTTC-39).The spacing of these two half sites is much closer than that of

FIG. 9. Determination of the oligomeric state of DinR in solution. A mixtureof proteins containing DinR or LexA and standards was sedimented on a 5 to30% glycerol gradient. Fractions (100 ml each) were collected, and proteins wereseparated by electrophoresis on 9 to 19% gradient polyacrylamide gels. Proteinswere visualized by silver staining the gels. Every other fraction is shown sequen-tially, starting with the top fraction (more slowly sedimenting, smaller proteins)at the far left. (A) BSA (5 mg), ovalbumin (5 mg), LexA (5 mg), and cytochromec (2 mg). (B) BSA (5 mg), ovalbumin (5 mg), DinR (5 mg), and cytochrome c (2mg). Since LexA and DinR sediment identically, we assume that DinR, likeLexA, is predominantly a monomer in solution.



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the E. coli SOS box [59-CTGT-(AT)4-ACAG-39]. The datapresented in this paper suggests, however, that the sequencerecognized by DinR is at least 2 bp larger than that previouslypredicted. The new DinR box (59-CGAACRNRYGTTCG-39)still retains dyad symmetry, but we find that mutations all alongthe binding element have some effect on binding affinity.Clearly, unambiguous delineation of the nucleotides withineach half site which make interactions with the DinR mono-mers sequence specific will require classical structural charac-terization. However, the mutation analysis combined with foot-printing data and analogy to other repressor/operatorstructures suggests that the outer 4 to 5 bp make interactionswith DinR sequence specific, while the inner 4 bp act as aspacer element. Mutations in the outer 5 bp of the operatorhave the greatest effect on affinity, while those in the central4-bp spacer element have a qualitatively weaker effect on DinRbinding. The hydroxyl radical footprinting results indicate thatthe physical center of the protein-DNA complex coincidesexactly with the center of the genetically defined DinR box(Fig. 7A and B). Furthermore, the footprinting data revealsthat the outer 5 bp within this element are oriented such thatthe major groove edge directly faces the DinR protein, whilethe minor groove of the 4-bp spacer element is oriented towardthe protein in the center of the complex (Fig. 7C). In addition,X-ray and biochemical analyses of other repressor/operatorcomplexes show that changes in uncontacted spacer elementscan have substantial effects on operator affinity. This effect isdue to subtle sequence-dependent changes in DNA structure

and consequently to the relative orientation and presentationof contacted residues at opposite ends of the element (11, 12).

These studies, together with previously published studies(16, 27), clearly demonstrate that the E. coli LexA and B.subtilis DinR proteins are both structurally and functionallyrelated. The major difference between the proteins is the siteto which they specifically bind to DNA. Nuclear magneticresonance-based studies have shown that the Ser39, Asn41,Ala42, Glu44, and Glu45 residues of LexA interact with theCTGT half site (9). With the exception of Ser39, these residuesare not conserved in the B. subtilis DinR protein or in relatedgram-positive DinR-like proteins (10, 27), so perhaps the dif-ference in the DNA binding site is not too surprising. Thequestion of which DinR residues make contact with its DNAbinding site will undoubtedly be resolved by the eventual X-ray- or nuclear magnetic resonance-derived analysis of DinRstructure.

Affinity of DinR for each DinR box. One interesting featureof this study is our observation that all the DinR operatorsequences appear to bind DinR protein with roughly equalaffinities. A simple explanation is that the gel mobility shiftassay used to quantitate binding is not sensitive enough toidentify possible differences (1). Indeed, more quantitative ex-periments currently in progress will allow us to determine thedissociation constant of DinR for each DinR box. A finding ofan equal affinity of DinR for each DinR box would contrastdramatically with the finding that E. coli uses differential bind-ing of LexA to its SOS box to regulate damage-inducible gene

FIG. 10. Determination of the oligomeric state and molecular weight of DinR bound to the dinR and recA operators. The retardation coefficient (Kr) of each proteinstandard was plotted against the molecular weight of the protein to generate the standard curve. The standard curve and the Krs for DinR (A) and the DinR-DNAcomplexes (B) were used to determine their respective molecular weights. (A) Molecular weight standards: urease trimer, BSA dimer, BSA monomer, ovalbumin,carbonic anhydrase, and a-lactalbumin (h); DinR monomer (F); and DinR dimer (Œ). (B) Molecular weight standards: urease trimer, BSA dimer, BSA monomer,ovalbumin, carbonic anhydrase, and a-lactalbumin (h); DNA probe, (F); DinR-dinR (}); and DinR-recA (Œ). Based upon these observations, we conclude that DinRbinds to its operator sequence in a dimeric state (see Results for detailed description of calculations).



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expression. Unlike E. coli, however, in which SOS regulationappears to be straightforward, B. subtilis has at least four dif-ferent modes of SOS induction (28), and additional factors,such as activator proteins (21), seem likely to provide theancillary functions to induce the regulon under various envi-ronmental and developmental stimuli. The availability of aninducible SOS response is important and has been conservedin both gram-positive and gram-negative organisms, but theprocess of evolution has allowed the development of divergentregulatory mechanisms.

ACKNOWLEDGMENTS

We thank Gerry Barcak for E. coli GBE180, John Little for thehighly purified E. coli LexA protein, and Rick Wolf and PhilFarabaugh for their comments on the manuscript.

This research was partially supported by NSF grant MCB-9219436to R.E.Y., Public Health Service grant RO1GM52426 to J. J. Hayes,

and University of Rochester Cancer Center training grant no.CA09363D-16A1 to D.C.

REFERENCES

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3. Cheo, D. L., K. W. Bayles, and R. E. Yasbin. 1992. Molecular characteriza-tion of regulatory elements controlling expression of the Bacillus subtilisrecA1 gene. Biochimie 74:755–762.

4. Durbach, S. I., S. J. Andersen, and V. Mizrahi. 1997. SOS induction inmycobacteria: analysis of the DNA-binding activity of a LexA-like repressorand its role in DNA damage induction of the recA gene from Mycobacteriumsmegmatis. Mol. Microbiol. 26:643–653.

5. Haijema, B. J., D. van Sinderen, K. Winterling, J. Kooistra, G. Venema, andL. W. Hamoen. 1996. Regulated expression of the dinR and recA genesduring competence development and SOS induction in Bacillus subtilis. Mol.Microbiol. 22:75–85.

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TABLE 1. Comparison of putative DinR boxes from the din genes of B. subtilis and the genes of several gram-positive bacteria

Organism Genea DinR binding siteb Accession no.

Bacillus subtilis recA (251) CGAATATGCGTTCG* U56238

dinA CGAACTTTAGTTCG* M64048

dinB AGAACTCATGTTCG* M64049

dinC (224) CGAACGTATGTTTG* M64050

dinC (253) AGAACAAGTGTTCG*

dinR (239) CGAACCTATGTTTG* M64684

dinR (267) CGAACAAACGTTTC*

dinR (2104) GGAATGTTTGTTCG*

Bacteroides fragilis recA (220) CGAATTAAACTTTG M63029

recA (2107) CGAACGGATCATCG

Clostridium perfringens recA AGAACTTATGTTCG U61497

Corynebacterium glutamicum recA CGTAGGAATTTTCG U14965

Corynebacterium pseudotuberculosis recA AGAATGGTCGTTAG U30387

Deinococcus radiodurans recA CGATCCTGCGTAAG U01876

Mycobacterium leprae recA CGAACAGATGTTCG X73822

Mycobacterium smegmatis recA CGAACAGGTGTTCG* X99208

Mycobacterium tuberculosis recA CGAACAGGTGTTCG* X58485

lexA CGAACACATGTTTG* X91407

Staphylococcus aureus recA CGAACAAATATTCG L25893

Streptococcus mutans recA CGAACATGCCCTTG M81468

Streptomyces lividans recA CGAACATCCATTCT X76076

Thermotoga maritima recA CGAATGTCAGTTTG L23425

a The location of the DinR box with respect to identified promoter elements is noted in parentheses.b The consensus DinR binding site, CGAACRNRYGTTCG, is defined as occurring in at least 70% of the sequences analyzed. With standard nomenclature, R 5

G or A and Y 5 C or T. The most-59 and most-39 nucleotides of the DinR box are in bold-faced type, while those of the previously identified Cheo box are underlined.Sequences with an asterisk denote sites that have been experimentally shown to physically bind DinR (or a DinR-like protein): B. subtilis recA (16, 27), dinA (Fig. 2),dinB (Fig. 2; [16]), dinC (Fig. 3; [16]), and dinR (Fig. 4; [5]); M. smegmatis recA (4, 19); M. tuberculosis recA (19) and lexA (17). The consensus sequence of the derivedDinR box is 59-CGAACRNRYGTTCG-39.



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9. Knegtel, R. M. A., R. H. Fogh, G. Ottleben, H. Ruterjans, P. Dumoulin, M.Schnarr, R. Boelens, and R. Kaptein. 1995. A model for the LexA repressorDNA complex. Proteins 21:226–236.

10. Koch, W. H., and R. Woodgate. 1998. The SOS response, p. 107–134. In J. A.Nickoloff and M. F. Hoekstra (ed.), DNA damage and repair: DNA repairin prokaryotes and lower eukaryotes. Humana Press, Totowa, N.J.

11. Koudelka, G. B., P. Harbury, S. C. Harrison, and M. Ptashne. 1988. DNAtwisting and the affinity of bacteriophage 434 operator for bacteriophage 434repressor. Proc. Natl. Acad. Sci. USA 85:4633–4637.

12. Koudelka, G. B., S. C. Harrison, and M. Ptashne. 1987. Effect of non-contacted bases on the affinity of 434 operator for 434 repressor and Cro.Nature 326:886–888.

13. Lewis, L. K., G. R. Harlow, L. A. Gregg-Jolly, and D. W. Mount. 1994.Identification of high affinity binding sites for LexA which define new DNAdamage-inducible genes in Escherichia coli. J. Mol. Biol. 241:507–523.

14. Little, J. W. 1984. Autodigestion of LexA and phage repressors. Proc. Natl.Acad. Sci. USA 81:1375–1379.

15. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.

16. Miller, M. C., J. B. Resnick, B. T. Smith, and C. M. Lovett, Jr. 1996. TheBacillus subtilis dinR gene codes for the analogue of Escherichia coli LexA.Purification and characterization of the DinR protein. J. Biol. Chem. 271:33502–33508.

17. Movahedzadeh, F., M. J. Colston, and E. O. Davis. 1997. Characterization ofMycobacterium tuberculosis LexA: recognition of a Cheo (Bacillus-type SOS)

box. Microbiology 143:929–936.18. Orchard, K., and G. E. May. 1993. An EMSA-based method for determining

the molecular weight of a protein-DNA complex. Nucleic Acids Res. 21:3335–3336.

19. Papavinasasundaram, K. G., F. Movahedzadeh, J. T. Keer, N. G. Stoker,M. J. Colston, and E. O. Davis. 1997. Mycobacterial recA is cotranscribedwith a potential regulatory gene called recX. Mol. Microbiol. 24:141–153.

20. Raymond-Denise, A., and N. Guillen. 1991. Identification of dinR, a DNAdamage-inducible regulator gene of Bacillus subtilis. J. Bacteriol. 173:7084–7091.

21. Raymond-Denise, A., and N. Guillen. 1992. Expression of the Bacillus subtilisdinR and recA genes after DNA damage and during competence. J. Bacte-riol. 174:3171–3176.

22. Schnarr, M., M. Granger-Schnarr, S. Hurstel, and J. Pouyet. 1988. Thecarboxy-terminal domain of the LexA repressor oligomerises essentially asthe entire protein. FEBS Lett. 234:56–60.

23. Schnarr, M., J. Pouyet, M. Granger-Schnarr, and M. Daune. 1985. Large-scale purification, oligomerization equilibria, and specific interaction of theLexA repressor of Escherichia coli. Biochemistry 24:2812–2818.

24. Sun, J. 1997. Ph.D. thesis. University of Maryland, Baltimore County.25. Thliveris, A. T., J. W. Little, and D. W. Mount. 1991. Repression of the E.

coli recA gene requires at least two LexA protein monomers. Biochimie73:449–456.

26. Wertman, K. F., and D. W. Mount. 1985. Nucleotide sequence bindingspecificity of the LexA repressor of Escherichia coli K-12. J. Bacteriol. 163:376–384.

27. Winterling, K. W., A. S. Levine, R. E. Yasbin, and R. Woodgate. 1997.Characterization of DinR, the Bacillus subtilis SOS repressor. J. Bacteriol.179:1698–1703.

28. Yasbin, R. E., D. L. Cheo, and K. W. Bayles. 1992. Inducible DNA repair anddifferentiation in Bacillus subtilis: interactions between global regulons. Mol.Microbiol. 6:1263–1270.



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the bacillus subtilis dinr binding site: redefinition of the

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