DNA-binding Properties of a Lac Repressor Mutant Incapable of ...

THEJOURNAL

OF

BIOLOGICAL CHEMISTRY

Vol. 266, No. 2, Issue of January 15, pp. 1281-1288.1991 Printed in U S A .

DNA-binding Properties of a Lac Repressor Mutant Incapable of Forming Tetramers* (Received for publication, July 2, 1990)

Michael Brenowitz$$, NitaiMandaln 11, Amy Pickar$**, Elizabeth Jamison$, and SankarAdhyall From the $Department of Biochemistry, The Albert Einstein College of Medicine, Bronx, New York 10461 and the ’11Laboratory of Molecular Biology, National Cancer Institute, National Institutesof Health, Bethesda, Maryland 20892

The interaction of proteins bound to sites widely operons of Escherichia coli (7-10). In these systems, the looped separated on the genome is a recurrent motif in both complexes can be formed by the binding of a hi-dentate prokaryotic and eukaryotic regulatory systems. Lac tetramer or the association of DNA-bound protein dimers. repressor mediates the formation of “DNA loops’’ by Thus, the dimer-tetramer association constant and linkage the simultaneous interaction of a single protein tetra- between self-association and DNA binding are critical determer with two DNA-binding sites. The DNA-binding minants of the ability of a DNA-binding protein to mediate properties of a Lac repressor mutant (Ladadi) deficient the formation of a looped complex. in the association of protein dimers to tetramers was The dimer-tetramer association free energy for the Lac investigated. The resultsof quantitative footprint and repressor was recently shown to be -10.6 kcal/mol at 20 “C, gel mobility-shift titrations suggest that the wild-typemuch weaker than previously believed (11).The association Lac repressor (Lad’) binds cooperatively to two op- equilibria of Gal repressor subunits have not yet been charerator sites separated by 11 helical turns on a linear acterized. As one test of the hypothesis that the formation of DNA restriction fragment by the formationof a “looped complex.” LacIndibinds to this two-site operatornon- looped complexes is mediated by the association of protein cooperatively and without formationof a looped com- dimers into bi-dentate tetramers, an analysis of the DNAplex. These results demonstrate that the dimer-tetra- binding properties of a mutant Lac repressor that is deficient mer association of LacI’ is directly responsible for its in its association to tetramerswas conducted. L a c P is unable to cause full repression of transcriptioninitiation and is cooperative binding and its ability to mediate formadefective in mediating DNA loopformation (6,9). In contrast, tion of a looped complex. The Iadimutation disrupts the monomer-dimer as well as eliminating the dimer-tetra-wild-type Lac repressor (LacI+) negatively regulates gene mer association equilibria while theDNA binding af- transcription in vivo by binding to two spatially separated finity of LacIad’to a single siteis unchanged relative to sites on the DNA ( 5 ) and hasbeen shown to form DNA loops the wild-type protein. These results suggestthat DNA i n vitro by gel electrophoresis (8)and electron microscopy (6). binding and dimer-tetramer associationare function- To directly compare the DNA-binding properties of the Gal ally unlinked. The similarity of the DNA-binding prop- repressor, LacI’, and LacPd’, a gal DNA in which OE and/or erties of LacIadiand Gal repressor, a protein believed O1 were changed to a symmetric lac recognition sequence ( 5 ) to function by mediating the formation of a looped was used. complex, are discussed. Despite the high degree of homology between the Gal and Lac repressors, there is a significant difference in the role of DNA looping in regulating the activity of the gal and lac operons. In thelac operon, the rate of transcription initiation The formation of DNA loops by the interactionof proteins is dependent upon the occupancy of the primary operator site bound to sites widely separated on the DNA is known to play by the Lac repressor. The occupancy of the primary operator an important role in the regulation of many biological proc- is increased by the simultaneous interactionof a Lac repressor esses including gene transcription initiation (l), replication tetramer with the primary and pseudo-operators (7). The (2), and recombination ( 3 ) .DNA looping permits formation formation of Lac repressor-mediated DNA loops has been of a higher order DNA multiprotein complex with important demonstrated both i n vivo and in vitro(6-10, 12). In contrast, regulatory consequences. Although the presence of “looped neither operator of the gal operon overlaps the gal promoter. complexes” is well documented in some systems, the mecha- In vivo transcription studies indicate that the simple binding nism(s) by which they regulate cellular activity remains un- of the Gal repressor to both operators (OE and 01) is not known. Two systems in which “looping” has been shown to sufficient for repression (4,5 ) . Whereas both OE and 0, are be a key regulatory component are the gal (4,5 , 6) and lac required for i n vivo negative regulation of the gal operon (1, 5 ) ,it is the formation of a looped complex, mediated by protein * This work was supported in part by National Institutes of Health bound to these two sites, that is believed to prevent transcripGrant GM39929 (to M. B.) and Biomedical Research Support Group funds. The costs of publication of this article were defrayed in part tion initiation. However, i n vitro analysis of Gal repressorby the payment of page charges. This article must therefore be hereby DNA complexes by electron microscopy (6) andDNA-binding marked “aduertisement” in accordance with 18 U.S.C. Section 1734 titration assays (13) did not demonstrate the presence of solely to indicate this fact. looped complexes. § To whom correspondence should be addressed 1300 Morris Park To further study the linkage of repressor self-association Ave., Bronx, NY 10461. and DNA binding in the regulation of gene transcription, a 11 On leave from the Bose Institute, Calcutta, India. ** Participant in the Summer Undergraduate Research Program quantitative analysis of the DNA binding of LacPd’was conconducted by the Sue Golding Graduate Division of the College of ducted. The results of this study demonstrate that Lacyd’ 1) Medicine. has unaltered intrinsic DNA binding affinity compared with

1281

DNA-binding Properties of Laclad'

1282 TTGTGAGCGCTCACAA

TTGTGAGCGCTCACAA

\

/

\

I

11 turns

/

I

FIG. 1. Schematic representation of the gal operon showing

the nucleotide substitutions used to convert OE and 0,from gal to lac recognition sequences (Ok,Of).Diagram is not toscale. The "reduced valence" mutant used intheDNA-bindingstudies contained Ok and OF. The restriction fragments containing Oi/Ok or were otherwise identical.

the wild-type protein, 2) does not bind DNA cooperatively, 3) does not mediate the formation of a looped complex, and 4) has a diminished monomer-dimer association equilibria. A preliminary account of some of this work has been presented ( 14). EXPERIMENTAL PROCEDURES

Materials Media Superbroth contains 12 g of Bactotryptone, 14 g of Bacto yeast extract, 5 ml of glycerol, 3.8 g of KH,PO, and 12.5 g of K,HP04 in 1 liter of distilled water. Bacterial and Plasmid Strains Plasmid pDM1.1 is a derivative of pACYC carrying the ladqgene and a Kn' marker (15) and wasa gift from H. Bujard. The ladq encodes LacI+ under the control of a superactive mutant promoter. The ladq gene in pDM1.l was cloned into the Sal1 site of pBR322. The resulting plasmid pBRF containstwo tandem ladqgenes. Plasmid pBWlOO contains the lad"" mutant gene in the pBR322 background (16). E. coli strain NM75 is F-, leu, proAZ, Eacl::TnlO, gal-, sup" hsdS20 (rB-mB-), rpsLZ0, xy15, mtll, and Aga1R::Cm'. Strain NM18 was transformedseparatelywithpBRIand pBW100. The transformants were used for extraction of I+ (wild-type) and Ied' (mutant) Lac repressors, respectively.

DNA Operators The synthetic constructionof operators in the gal operon in which the recognition sequences (@, @) for Gal repressor were altered to for Lac repressor has been described recognition sequences (@, (5). The resultant symmetric lac recognition sequence is shown in Fig. 1. Linear DNA restriction fragments of 635 bp' which are labeled with 32Pa t only oneend were generated usinga "cut-label-cut" procedure. The plasmids pH107 and pH104 (5)were linearized with EcoRI, labeled with 3zP, and thencleaved with HindIII. The restriction fragments were purified by agarose gel-electrophoresis followed by electroelution (see Methods). The centers of symmetry of @ and Ob are separated by 114 bp, corresponding to 11 helical turns of 10.4 bp of B-DNA. These restriction fragments were used in all of the DNA-binding assays.

e)

Methods Assay of LacP Repressor during Purification LacI+ repressor was assayed during purification based on its binding of "C-labeled IPTG and retentionof the repressor-IPTGcomplex on Millipore filters(17).IPTG-binding buffer contains 10 mM Mg(CH3C02),,200 mM KCI, 0.1 mM EDTA, 6 mM 2-mercaptoethanol, and 10 mM Tris-HCI titrated to pH 7.4. The concentration of the ["CIIPTG stock solution was 1 pmol/ml (2.6 X 10' cpm/pmol). An aliquot of ["CIIPTG stock solution was added to N X 0.25 ml of the binding buffer and mixed well where N is the total numberof assays. 0.25-ml aliquots of this mixture were measured into assay tubes, and the tubes were preincubated a t 4 "C for 5 min. To these The abbreviations used are: bp, base pair(s); IPTG, isopropyl-lthio-0-0-galactopyranoside; DTT, dithiothreitol; FPLC, fast protein liquid chromatography.

tubes were added suitable aliquots of LacI+, the contents mixed well, then incubated a t 4 "C for 30 min. Appropriate control experiments were done without any protein. After 30 min, the entire reaction mixture was filtered on Millipore filters presoaked in binding buffer a t 0 "C and filtered a t low vacuum. The filters were washed twice with 0.2 ml of ice-cold binding buffer,dried, and the radioactive counts of the filterswere measured. During filtration, the filters were never allowed to dry. Purification of Wild-type Lac Repressor (I+) (i) Growth of Bacteria-Bacterial strain NM18/pBRIq was grown to late log-phase in 20 liters of superbroth in a fermenter a t 37 "C. The cells were harvested andwashed with 500ml of 0.01 M Tris-HC1, pH 8.0. The packedcells were stored frozen a t -70 "C. Unless otherwise stated, all the operations during the purification of the repressor were done a t 0-4 "C. (ii) Lysis of Frozen Cells-Sixty-five g of packed frozen cells were thawed on ice (for about 1 h) and then resuspended evenly in 70 ml of lysis buffer (25 mM Tris-HCI, pH 7.4, 5 mM EDTA, 1 mM DTT, 0.1 mM o-nitrophenyl-D-fucoside, and 50 pg/ml phenylmethylsulfonyl fluoride containing 1 mg/ml of lysozyme. The suspension was then kept on ice with frequent stirring. After 30 min, 60 ml of the lysis buffer (without any lysozyme) was added to it, mixed well, and the mixture was incubated a t 15 "Cfor 30 min with frequent stirring. At this stage, the cell suspension became very thick and viscous. Next, 15 ml of 4 M KCI, 3 ml of 0.5 M Mg(CH3C02)2, 7.5 ml of 100% glycerol, and 1.5 mg of DNase I were added to the cell lysate and mixed well. The mixture was incubated further a t 15 "C for 30 min. The lysate was centrifuged at 15,000 rpm for 2 h. The supernatant (185 ml) was used in the next step. (iii) Ammonium Sulfate Fractionation-0.5 volumes of saturated ammonium sulfate solution in the lysis buffer (adjusted to pH 7.4 with 1.0 M KOH) was added to the supernatant,mixed well and kept on ice for 45 min. The precipitates containing repressor were collected by centrifugation a t 12,000 rpm for 30 min and dissolved in 15 ml of KPG buffer (75 mM potassiumphosphate buffer at pH 7.4,5% glucose, 1 mM EDTA, 1 mM DTT, 50 Fg/ml of phenylmethylsulfonyl fluoride, and the solution was ultracentrifuged a t 35,000 rpm. The supernatant wasdialyzed against 1 liter of KPG buffer with two more changes over a period of 30 h. Total protein recovered at this stage was about 230 mg in a volume of 17 ml after dialysis. This solution was further diluted into KPGbuffer to a final protein concentration of 5 mg/ml. (io) FPLC Chromatography on Mono-S Column-A Mono-S prepacked column (5-ml bed volume) was equilibrated with KPGbuffer following the procedure described by the manufacturer (Pharmacia LKB Biotechnology Inc.). Five ml of the dialyzed protein solution a t a concentration of 5 mg/ml was loadedonto the Mono-Scolumn, and the column was washed with KPG buffer and the proteinwas eluted by a 0-0.7 M KC1 gradient in KPGbuffer. The steps for one Mono-S column chromatography run were completed in 25 min by preprogramming. (Tenruns were needed tofractionatethe 230mg of dialyzed protein through the Mono-Scolumn.) More than 90% of the total Azsomaterial came out in the flow-through. T h e L a d + in the fractions of the salt gradient was monitored by ["CJIPTG-binding activity. The other peak was discarded. There were only two A280 peaks eluted after the salt gradient was started themajor peak eluted were at 0.12 M KC1 contained therepressor. Fractions from this peak pooled (about 30 ml), and the protein was precipitated by adding 30 ml of saturated ammonium sulfate in KPG buffer adjusted to pH7.4. After 45 min, the precipitates were collected by centrifugation at 10,000 rpm for 30 min and finally dissolved in KPGbuffer containing 0.4 M KC1 to get a final concentration of the protein at around 30 mg/ml. Total proteinrecovered at this stage (measured before precipitation with ammonium sulfate) was 32 mg. ( u ) FPLC Gel Filtration Chromatography-A prepacked Superose12 column (25-ml bed volume) was washed with water and equilibratedwiththeKPG buffer containing 0.4 M KC1 by passing 2 volumes each a t a flow rate of 0.2 ml/min. About 6 mg of protein in 200 pl of solution from the previous step was loaded/run and eluted with the equilibrating buffer a t a flow rate of 0.4 ml/min. Only one A,, peak was obtained a t 1.57 void volume and coincided with ["C] IPTG-binding activity. The peak fractions were pooled and dialyzed against 500 ml of KPG buffer containing 0.4 M KC1 and 15% glycerol with two more changesover a period of 20 h. Total proteinrecovered at this stage was 20 mg. Sodium dodecyl sulfate-polyacrylamide gel

DNA-binding Properties of Laddi electrophoresis showed more than95% purity. The dialyzed repressor was then distributed in0.5-ml aliquots and storedfrozen a t -70 "C. Purification of Mutant Lac Repressor (LacI"'7

1283

Analysis of Footprint Titrations Individual site-binding equations for LacPd' binding to DNA sites

@ and & can be constructed by considering the relative probability

of each configuration (21, 22) described by a statistical thermodynamic model for the interaction of a monovalent protein to a twoBacterial strain M75/pBW100 was grown as described above for wild-type repressor. Packed frozen cells (120 g) were lysed, and the site operator (13). The equations are lysate was treated successively through ammonium sulfate precipikI[PZl + klkEkI€[P2l2 PI = tation and ultracentrifugation of the dissolved ammonium sulfate (1) 1 + (kl + kE) [PZI + k,k€kI€[P*l2 precipitate asdescribed above forsimilar steps during the purification of the wild-type LacI'. The supernatant after ultracentrifugation was and then treated as follows. (i) Low Salt Precipitation-The supernatant solutionwas dialyzed against 1liter of pH 6.6 10 mM potassium phosphatebuffer containing 1 mM EDTA, 1 mM DTT, and 5% glucose with one change over a period of 20 h. The mutant repressor protein was recovered in the where and YE represent the fractional saturation of sites @ and pellet after centrifugation of the dialyzed material a t 10,000 rpm for Oi, respectively. The microscopic equilibrium constants ( k , ) have 30 min. The protein was dissolved in KPG buffer containing 0.4 M been substituted for the Gibbs free energies using the relation AGi = KCl. -RT In(ki). The AG values are of the following types: 1) binding of (ii) FPLC Gel Filtration Chromatography-The solution was sub- ligand to one site in the absence of binding to others ("intrinsic" gel filtrationchromatography as de- binding free energies,AG, and AGE),and 2) the excess (or cooperative) jected to FPLC Superose-12 scribed for the wild-type repressor. The mutant repressor, as moni- energy for binding to two sites simultaneously (AG,,). This cooperatored by the [I4C]IPTG-binding assay, eluted as amajor peak at tive energy is defined as AGIE= AG0,,l - (AGE + AG,) where AGT,,,] around 1.81 void volume. (A second minor peak eluting much later is the energy of binding both sitessimultaneously. [Pz]represents the did nothave IPTG binding activity and was discarded.) The repressor free concentration of LacIad' dimers. For operators in which either containing fractions were pooled and dialyzed against 1 liter of KPG & or Of- did notbindrepressor ("reduced-valence mutants") the buffer containing 0.4 M KCI, 1 mM EDTA, 1 mM DTT, and 15% individual site-binding equations reduce to glycerol as described for the wild-type repressor. Total protein recovered after dialysis of the Superose fractionswas about 10mg. The mutant repressor thus purified was found to be more than 95% pure as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final preparation was distributed into 0.4-ml aliquots and where; refers to site OE or 01 (21). stored a t -70 'C. Since L a d + binding to only a single DNA site was quantitatively analyzed, dimer-tetramer association would appreciably affect these studies only if DNA binding to the L a d + tetramer was cooperative. Glycerol Gradient Centrifugation An analysis of Lad' tetramer binding cooperativity is beyond the The purified wild-type and mutant Lac repressor proteins were scope of this article. Thus,for these preliminary studies, the analysis subjected to glycerol gradient centrifugationusing molecular markers of Lad' was conducted subject to the assumption that Lad' dimers for size determination. 17.5-35% glycerol gradients in 0.4 M KCI, 1 and tetramers bind DNA equivalently. An analysis of the dimermM EDTA, and 1 mM DTT were layered with 225 pg of LacI', 450 tetramer association of LacI' (11)and its DNA binding to a two-site pg of LacPd', or a mixture of standard protein samplesof known sizes. operator will be presented elsewhere? Centrifugation was done for 44 h at 50,000 rpm. Fractions of eight drops each were collected and checkedfor absorbance a t after Analysis of Gel Mobility-Shift Titrations dilution with 0.3 ml of KPG buffer. The mobility-shift assay measures the fractionof DNA molecules with "i" ligands hound, where ivaries from 0 to the numberof binding DNA-binding Assays sites. A measure of the fractionsof molecules, Oi, that areunliganded, Quantitative DNase I footprint titration experiments were con- singly, and doubly liganded can be written, respectively: ducted as hasbeen described (18, 19). The protocols used to conduct the quantitative gel mobility-shift assays have been described elsewhere (13). Digital representations of titration autoradiograms were obtained using a microcomputer-based video densitometer.' Densitometric analysis was conducted using a microcomputer implementation' of previously developed computer software (18). All experiments were conducted a t 20 t 0.1 "C using linear DNA restriction fragments as described under Materials. The assay buffer in which protein and DNA were equilibrated contained 25 mM Bis-tris, 5 mM MgC12, 1 mM CaC12,2 mM DTT, 50 pg/ml bovine serum albumin, 2 pg/ml calf thymus DNA, and 100 mM KC1 titrated to pH7 with HCI where [Pl] is the concentration of free LacIad' dimer (23). at 20 "C. The fraction of molecules in each configuration was calculated at Theconcentration of DNA was 510 PM in all footprintand = DJDm,alwhere D,is the density each protein concentration as@I,,, mobility-shift titration experiments. Since this concentration is low of band i and D,,,., is the sum of the densities of all the bands in a relative to the equilibrium-binding constants, the concentration of lane (13). These equationsreduce to protein-DNA complexes is negligible relative to the total concentration of protein. Thus, the assumption has been made in all of the 1 =experimentsthat [protein],,,, = [proteinlr,,. The assumption was 1 + k,[P'l also made that the DNA binding activity of the protein preparations is 100%.This assumption is made because of the doubt cast on the accuracy of "DNA binding activity" determinations of CAMP receptor protein (20).Severalfold variability in the activityvalues determined from binding studies conducted under stoichiometric conditions has for analysis of single site-reducedvalence operators where and 8, been observed for both and Lad' andL ~ c I ' ~ It ~should . ~ be noted that represent the fractions of unliganded and singly liganded molecules, a 2-fold error in the DNA binding activity would result in only a 0.4 respectively, and; refers to either site of- or Oh. The procedures used kcal/mol change in the DNA-bindingfree energy a t 20 "C. to fit the footprint and mobility-shift titration data to the appropriate equations are described elsewhere (13). ''P. Reiner and M. Brenowitz, submitted for publication. :'M. Brenowitz, P. Reiner, and B. Turner, unpublished results. 'M. Brenowitz, A. Pickar, and E. Jamison, submitted for publicaM. Brenowitz and E. Jamison, unpublished results. tion.

eo

eo

1284

Properties DNA-binding

of Ladadi

arations were used for the quantitative analysis of the site specific DNA-binding characteristics of the proteins. Purification and Molecular Weight Characterization of L a d ' DNA Binding of Lad+-An analysis of any mutantprotein and LacPdi.The Iadimutation inthe Lac repressor gene results must be made in comparison to the wild-type protein from in the replacement of 31 native COOH-terminal aminoacids which it is derived. The LacI' tetramer hasbeen shown to be with 16 other amino acids (6, 16) in the translated protein. capable of binding two operators simultaneously (cf. 6-10, The calculated molecular masses of the constituentpolypep- 12). A mobility-shift titration of LacI' to a DNA fragment tides of LacI' and LacIRdiare 38.5 and 37.7 kDa, respectively, containing both the Ok and Of. binding sites is shown in Fig. based on their sequence. The LacI' tetramer and Lacydi dimer 4B. The band at the bottom is free DNA, whereas no band is are 154 and 74 kDa, respectively. While the calculated PI discernible where the DNA with only one site occupied by values ofLacI' and LacIadiare 6.42 and 8.02, isoelectric LacI' ought to appear, as deduced by comparison with mobilfocusing of the two proteins showed their actual PI values to ity-shift titrations of operators containingonly a single bindbe approximately equal (data notshown). ing site (data not shown). The bands at the top represent LacIadihas been shown previously to be defective in repress- complexes in which both DNA-binding sites are occupied. It ing operons that require bipartite lac operators and in its is clear that complexes in which both sites are occupied by ability to form DNA loops (6, 9, 16). These phenotypes were LacI' appear quickly, with the obvious absence of a detectable traced to the inability of LacIadito form tetramers. We have amount of singly liganded complexes as binding intermedipurified this mutantprotein, as well as thewild-type protein, ates. The absence of appreciable populations of binding interas described under "Experimental Procedures." The native mediates is characteristic of highly concerted transitions. In molecular weights of LacI' and LacIadiwere found to be 154 other words, the binding of LacI' to thebipartite operator, as and 65 kDa by gelfiltration chromatography (6). These results assayed by the mobility-shift method, is cooperative. are in good agreement with the calculated molecular weights. Careful examination of the complexes which appear in the The size difference of the two proteins was further confirmed titration shown in Fig. 4B reveals that theinitial appearance by glycerol gradient centrifugation of the two proteins using of the slow mobility band is followed bya transition,at higher molecular weight standards (Figs. 2 and 3). The molecular protein concentrations, to a band of slightly increased mobilmasses of the two proteinsdetermined from the gradient ity. That the slowest mobility band is the looped complex centrifugations are 158 and 64 kDa, respectively. These prep- formed by the simultaneous binding of a single LacI' tetramer to Ok and OF and the faster band is the "tandem complex" formed by the binding of two LacI' tetramers is suggested by two lines of evidence. 1)Kramer et al. (9) demonstrated that 0.20 the relative mobilities of the looped and tandem complexes vary as a functionof the separation of the DNA-binding sites. When the binding sites are separated by 11 helical turns of 0.15 10.4 bp, as is true in the studies presentedherein, the mobility of the two complexes was almost identical. 2) The densities 8 of the two "shifted" bands were integrated and analyzed separately. Their populations are consistent with the tran2 0.10 RESULTS

0.05

-

10 15 20 FRACTION NUMBER

-[ L a c P ~Dimerl-b

FIG. 2. Glycerol gradient sedimentation of LacI+and LacIndi of 0.2-ml fractions repressors. Details are as given in text. The after dilution with 0.3 ml of KPG buffer is plotted against fraction number. Open symbols (dashedline) representsedimentation of LacI"". Closed symbols (solid line) represent Lad'.

-[LacI' Tetramer] -b

x

4

6

8

10

12

14

16

18

20

22

FRACTION NllMRFR

FIG. 3. Glycerol gradient sedimentation of LacI+and LacIadi (filleddiamonds) along with standard molecular weight markers (open circles).The standard markers are myoglobin (17 kDa),ovalbumin(44 kDa),and globulin (158 kDa). The fraction numbers containing the known markers are plotted against their molecular weights.

FIG. 4. Autoradiograms of a mobility-shift titrations of the Oi/Ot operator with LacIadi(A) and LacI' (B).Protein concentrations for the LacIEdi titration are shown in Fig. 8. Protein concentrations for the Lad+ titration arelane 1, 0; lane 2, 0.6pM; h e 3, 1.5 pM; lane 4, 3.0 pM; lane 5, 6.0 pM; lane 6,14.8 pM; lane 7, 59.2 pM; lane 8, 148 p ~ lane ; 9, 296 p ~ lane ; 10, 592 p ~ lane ; 11, 1.5 nM; lane 12, 14.8 nM; lane 23, 18.5 nM. Concentrations of Ladadi and Lad' are expressed in moles of dimer and tetramer, respectively. The assignl u t i l r UT ~ codr

bald tu a partioular oonfiyration w a n mado an slooorib9d

in thetext. The density preceding the band representing thecomplex containing two LacIadimolecules ( A ) was included in the densitometric analysis (35). Techniques of image analysis were used to alter the contrast and the aspect ratios of the autoradiogram images in this figure. However, densitometric analysis was conducted on unprocessed digital images.

DNA-binding Propertiesof Laclad' sient appearance of looped complexes followed bythe appearance of tandem complexes at higher protein concentrations." In the DNase I footprint titration autoradiogram, bands between Ob and Of. become hypersensitive to DNase I upon LacI' binding (Fig. 5). The relative hypersensitivity of the band indicated in Fig. 5 is quantitated inFig. 6C. The hypersensitivity reaches a maximum at thepoint approximately at which the fractional saturation of Oh and 0: is unity.5 The fact that the integrated optical density of standard blocks located outside the region between Ob and 0: did not change with increasing LacI' shows that theDNase I hypersensitivity A B C ll W H

.

H

If

.. I

PI

FIG.5. Several lanes from an autoradiogram of a DNase I footprint titration experiment of LacI+ binding to the Ok/Ok operator. The arrom indicates the representative hypersensitive band quantitated in Fig. 6. The LacI' concentrations are 1.5 pM ( A ) , 37 nM ( B ) ,and 74 nM (c).

1285

is limited to the region between the two sites (Fig. 6, A and

B). Periodic DNase I hypersensitivity of base pairs between two separated protein-binding sites that are "in phase" has been shown to correlate with the presence of a DNA loop (cf. 24). This hypersensitivity has a periodicity of one helical turn and is believed to result from a deformation of the DNA helix on the outside of the loop. Although the fact that the resolution of our titration autoradiograms does not permit a determination of the periodicity of the Lac1'-induced hypersensitivity, our results are consistent with the interpretation that the observed DNase I hypersensitivity is an indicator of the formation of a DNA loop. The decrease in hypersensitivity at high LacI' concentrations (Fig. 6C) is consistent with the mass action-driven transition from looped to tandem complexes. Taken together these results suggest that thecooperative binding of LacI' to the restriction fragment containing and Of. is the result of the formation of a DNA loop; a conclusion consistent with the observation of looped complexes in electron micrographs of LacI' bound to the same operator (6). Although a quantitative analysis of the cooperative binding of LacI' to the Ok/Ok two-site operator is beyond the scope of this article, the analysis of LacI' to a single site-reduced valence operator (OblO?) was conducted. This analysis yields the intrinsic binding free energy AGEand represents the DNA binding affinity of LacI' under these experimentalconditions in the absence of any cooperative or bi-dentate interactions. Evidence that LacI' does not dissociate to monomers is found in the dissociation studies of Royer et al. (11)who observed no species smaller than dimers. DNase I footprint and gel mobility-shift titrations of Ok/OF with LacI' were analyzed by least squares minimization of Equation 3 and Equations 7 and 8, respectively, where the protein concentration, [P2] is LacI' dimer, since each dimer in the tetramer binds DNA. The values of AGE resolved from these titrations are shown in Table I. DNA Binding of LacZ""-The DNA binding of LacIadito the Ob/OF single site operator was analyzed in order to measure the protein-DNA interaction in the absence of potential site-site interactions. Binding curves were obtained from moTABLE I Gibbs free energies of binding Lacl' and Ladadito Ok/O$ singk site operator Experiment

ACE

acnim,.

s'

kcaljmol & 65% confidence limits

LacI' Footprint Gel shift

-1.8

.. ..

1

-2.2 I 1E-13

1E-12

1E-11

1E-10

1E-9

LacPd' Footprint I 1E-8

1E-7 1E-6

[LOCI+ tetramer] (e) [Laclad'dimer] (A)

FIG. 6. A and €3, integrated OD values of standard blocks located outsidethe region between Ok and 0: (19). C, relativeDNase I hypersensitivity of a band located between Ok and 0: as a function of protein concentration as determined from footprint titration autoradiograms. Protein concentrations areexpressed as LacI' tetramer (using the assumption stated Fig. in 2) and LacP"' as dimer assuming the protein existssolely in that form. Diamonds represent data from titration of Ok/O; with LacI'; triangles represent data from titration of Ok/O(. with LacI""'.

Gel shift

-13.7 ? 0.2 -12.7 2 0.1

-b

-b

0.070 0.069

-11.5 f 0.2 -14.1 ? 0.1 -11.1 ? 0.2 -13.4 f 0.2

(-20)'

0.084

(-8) (-20)' (-8)

0.050 0.138 0.107

Square rootof the variance.

'The assumption was made

in the analysis of LacI' that theprotein does not dissociate to monomers (11). The shapeof the titration curves is sensitive toA&,, only over a very narrow rangeof values. AGDim,,and AGEare infinitely correlated parameters outside of this range, a fact that precludes the unique determination of their values. The value of AGoimer of-8 represents the lower limit of the curve where the numericalcorrelation between value of A G i r n e r AGoimcrandAGE reaches 0.998 (data not shown). The of -20 exceeds the upper limit and corresponds to a non-dissociable dimer. Thus, its exactvalue will have no effect on the determination of AGE. See Ref. 13 for a detailed discussion of this issue.

DNA-binding Properties of Laddi

1286

the two constants canbe resolved from a single titration curve only over a very narrowrange of values. When the selfassociation constant is either much greater or much smaller than the DNA-binding constant the shapes of the binding 2 0.6 } curves reach a “convergencelimit” at which pairs of values of E0 0.4 A G D and ~ ~AGE ~ ~will describe the data equally well (cf. 13, 23). The binding of LacPdi to @/OF is best described by values of A G D and ~ ~AGE ~ ~at the convergence limit where A G D ~>> ~ ~AGE. , The value of AGDime,of -8 kcal/mol represents this limit. Thus, the value of AGE shown in Table I is a n upper limit of its true value. It must be emphasized that although these data indicate the linkage of protein self-associationandDNAbinding forLacIadi, directprotein selfassociation studies are required to determine the true value of AGD~,,,~~. value The of AGDimer of -8 was used to calculate the concentrationof LacPdi dimers in the analysis of binding to the two-site operatordescribed below. I I -8 -6 -1 2 -10 An autoradiogram of a mobility-shift titration of LacPdi log [ ~ a c l a d monomer i toto11 with Ok/Ok is shown in Fig. 4A. In contrast to LacI’ (Fig. FIG. 7. A , binding curves determined from mobility-shift experi- 4B),appreciable concentrations of singly liganded intermements of LacPd’ binding to the Oh/Of single site (reduced valence) diates are present (Fig. 4A). The binding curves determined operator. Triangles indicate data for the band containing unliganded from the mobility-shift titrations of Lacydi binding the @/ DNA; diamonds indicate the band containing DNA with oneLacIadi two-site operator are shown in Fig. 8A. These data were molecule bound. Open and closed symbols represent replicate experi- fit to Equations 4-6 with the constraint that LacPdi binds a footprint titration ments. B, individual site binding curve from experiment of LacIad’binding to the@/@ single site operator. Solid equivalently to 0; and OF (see “Experimental Procedures”). of the lines indicate the binding curves calculated from the best-fit values This constraint is justified based upon the identity sequences of o“, and OF and the fact that footprint titrations shown in Table I where AG,,,,, = -8. Ladad’ concentrations are expressed in terms of total LacPd’ monomer. of Ok/O:’ (Table I) and Ok/Oy (data not shown) areidentical. By constraining AGI = AGE, it is possible to resolve unique bility-shifttitrations (Fig. 7A). These curvesdescribe the values of AGE and thecooperative free energy,AGIE (13).The fraction of DNA molecules that arefree and the fraction with fact that AGIE is zero, within experimental error, indicates one LacPdimolecule bound (see “Experimental Procedures”). that binding of LacPdi is non-cooperative (Table11). In contrast, the individual site binding curves that are obA similar conclusion is reached from the footprint titration Ok/Ok two-site operator (Fig. tained from footprint titrations (Fig. 7 B ) describe the frac- studies of LacPdi binding to the tional saturation of a DNA site. The two experiments yield 8B). These data were fit to Equations 1 and 2 without concomplementary information about the DNA-binding charac- straints (see “Experimental Procedures”). Once again, AGIE teristics of a protein. The mobility-shift titrations of LacIndiwith the Ok/Of single site operatorwere fitted using techniques of non-linear least squares analysis to Equations 7 and 8 as described under “Experimental Procedures.”Correspondingly, the footprint titrations were fitted t o Equation 3. Initially, the assumption was made that LacIad’exists in solution as a non-dissociable dimer (Figs. 2 and 3). The values of the intrinsic DNA binding affinity, AGE, determined subject to this assumption are listed as the uppervalue ineachpairinTable I. However, an inspection of plots of thetitrationdataandthebest-fit binding curves revealed systematic deviations of the curves from the data. Both the mobility-shift and footprint titration data exhibited steepertransitionsthanpredicted by this model (data not shown). o.6 0.4 An alternative model assumesthatLacPdi undergoes a monomer-dimerequilibrium and that the dimer has much greater DNA binding affinity than the monomer. This linkage of protein self-association and DNA binding was first demonstrated for cI-repressor (25, 26) and may be a more general log [ ~ a c ~ a monomer di total] phenomenonthan previously thought (cf. 13, 27). An imFIG. 8. A , bindingcurves frommobility-shift experiments of proved fit of the data to both the mobility-shift and footprintLacIad’binding to theOh/OF two site operator.Triangles indicate data titration datawas obtained when the associationof monomers for band containing unligandedDNA; squares indicate band containt o dimers was assumed to be weak. Note the decrease in the ing DNA with one LacPd’ molecule bound diamonds indicate band containing DNA with two LacIad’molecules bound. B, individual site square root of the variance (Table I) and the fact that the of LacIad’binding binding curves from a footprint titration experiment predicted curves fit the dataover the entire range(Fig. 7). to the Ok/Ok operator. Diamonds indicate binding to Ok triangles A fundamental problem with attempting to determine both indicate binding to Oh. Solid and dashed lines indicates the binding self-association (AGDim,,)and DNA-binding (AGE) constants curves calculated from the best-fit values to Oh and O,“, respectively, from titration curves is the inherent correlation between the shown in Table 11. Ladad’ concentrations are expressed in terms of two parameters in the binding equations. Unique values of total monomer. r

I

\P

@/e

I

DNA-binding Properties of L d " "

1287

tional studies? The linkage of protein dimerization andDNA binding is consistent with the mechanismby which proteins utilizing the "helix-turn-helix" motif recognize specific DNA sequences (cf. 28, 29). The fact that bothGal and Lac represExperiment AGE AGI AGEI Sa sors bend DNA upon site-specific binding (30) also argues kcal/mol& 65% confidence limits that thetwo monomers must actcooperatively, i.e. that proper b 0.2 k 0.2 0.075 Gel shift -13.4 & 0.2 positioning of the recognition helix in the major groove can Footprint -13.6 & 0.2 -13.7 k 0.2 -0.3 & 0.2 0.064 only be achieved by distorting the structure of one of the Square root of the variance. macromolecules. The cocrystal complexes of other proteins Analysis was conducted subject t o the numerical constraint that as being the more that have been solved all point to the DNA AGE = AGI (see text). malleable of the two macromolecules (cf. 28, 29). Evidence is approximately equal tozero within experimental error. As from circular dichroism (31) and ethylation interference(32) was the case with the titrations of the Ok/Op operator, the studies of Gal repressor-operatorcomplexes suggeststhat the site-specific binding of Gal represbinding curves predicted from the best-fit values shown in DNA is distorted upon the sor. That similar DNA structural changes occur upon Lac Table 11 represent the dataover the entire range. is by the increased The non-cooperative binding of LacPdi toa two-site oper- repressor binding to its operatorsuggested affinity of Lac repressor for a n operator (such as the symator suggests that this mutant protein is unable to associate metric sequence used in these studies) lacking the central into a bi-dentate tetramer and mediate formation of a looped complex. However, formation of a looped complex need not base pair of the wild-type lac operator sequence (33). The fact that the Pdimutation affects both the monomerrequire a net negativecooperativefreeenergy.Additional evidence that Lacydi does not mediate looped complex for- dimer and dimer-tetramerequilibria suggests that theregions mation is found in that the bandsbetween Ok and @ do not of Lac repressor which mediate thesetwo levels of interaction exhibit DNase I hypersensitivity upon titrating the Ok/O? are contiguous. However,consideration shouldbe given to the fact that single amino acid substitutions, let alone a deletion operator with LacPdi, as was observed for LacI' (Fig. 6C). of 31 amino acids, can have effects far removed from the site of the perturbation. Thus, itdifficult is to draw any structural DISCUSSION conclusions from the studies presented herein. In a similar In both prokaryotes and eukaryotes the of transcription rate fashion, the apparentindependence of DNA binding and the initiation can be regulated by widely separated control ele- association of dimers to tetramers does not show that these ments on the DNA. One mechanism of communication be- functionsaremediated by distant regions of the protein. tween such control elements is thought involve to association Hopefully, the ultimate solution of the structure of Lac reof specifically bound proteinmolecules, resulting in theloop- pressor at atomic resolution (34) will providea structural ing of the intervening DNA (cf. 1). Despite the ubiquity of framework within which these functional studies can be inDNA loops, the molecular mechanism by whichthey modulate terpreted. transcription initiation is unknown. The association reaction The DNA binding and DNase I hypersensitivity results can involve self-association of a single type of protein or the obtained for LacI' andLacydi clearly show a correlation association of two (or more) different proteins. The simplest between the loss of binding cooperativity and the loss of the system in which to study this phenomenon is the self-associ- ability to mediate the formation of a DNA loop. This correation of a single protein which binds to two specific sites. In lation strongly supports that the observed cooperativity of this context, Lac repressor is anideal modelprotein. However, binding of LacI' is a direct resultof the bi-dentate interaction a complex series of equilibria exist in even this simplest of of a single LacI' tetramer with two operator sites. That the systems. It was to dissect out individual equilibria that these DNase I hypersensitivity isa measure of DNA loop formation studies of the Laclad' were conducted. is supported by the visualization of DNA loops by electron It was unexpected that theIndimutation weakens the mon- microscopy of Ok/O? and LacI' but not with LacIadi(6). The omer-dimer aswell as eliminating the dimer-tetramer equilib- fact that appreciable concentrations of the tandem complex of LacI' ria. Royer et al. (11) did not detect the presence are observed a t moderate concentrations of LacI+ (Figs. 4B monomers suggesting that the monomer-dimer interface is and 6C) suggests that the stability of the looped complex is stable over the concentration range that the protein binds the not appreciably greater than the tandem complex for linear DNA. It is clear that the monomer-dimer association free DNA. ~ , ismuch lessnegative than the energy, A G D ~of~ ~LacIndi The net stability of the Lac1'-mediated looped complex is intrinsicDNA-binding freeenergy, AGE. However,only a a result of a balance between negative free energy contribulower limit of its value could be estimated from the analysis of the binding curves. When this weak dimerization reaction tions resulting from the increased local concentration of the second DNA site and a positive free energy contribution for is taken into account, the values of AGEdetermined forLacI' and LacPdi are remarkably similar, suggesting that the intrin- the energy required to bendduplex DNA thatis much shorter sic affinityof LacPd' for its specific sites is notgreatly altered than its persistence length(10). Thus, the ability of a looped relative to LacI'. The concurrence of the DNA-binding ener- complex to act asa metabolic regulator is dependent upon a is 5 -8 since set of thermodynamic parameters (10).A determination of gies for LacI' and LacIad' suggeststhat AGDime, it is unlikely that a deletion mutation, which would weaken the mechanism by which a looped complex regulates tranthe oligomerization of a protein, would strengthen its DNA scription initiation must include the determination of these of the thermodynamic binding affinity. These resultsimply that while the monomer- parameters and ultimately the solution dimer and DNA-binding equilibria are linked, DNA binding linkage of LacI' DNA binding andself-association. The footprint and mobility-shift titrations of LacIadiwere and dimer-tetramer associationoccur independently. However, a conclusive answer to this question requires the direct consistent within 0.5 kcal/mol; the mobility-shift titrations characterization of the monomer-dimer association reaction reported weaker binding affinity. Thisdifference is similar in magnitude to that observed in studies of Gal repressor (13). of LacPdi. Can we infer any structural correlations from these func- Adifference of -1.0 kcal/mol was observed in the LacI' TABLEI1 Gibbs free energies of binding Laclad'to the OklOk two site operator A value of AGDimer of -8 kcal/rnol is assumed inthese calculations.

DNA-binding Properties of LacIadi

1288

titrations. The phenomenological theory developed by Cann (35) suggests that measurable levels of dissociation occur during electrophoresis even for tight binding repressors such as LacI' and LacIad'.A second possibility is that dissociation of the complexes occurs upon gel loading prior to electrophoresis. However, studies with Gal repressor suggest that this latter phenomenon cannot account for the differences in the apparent binding affinity (13). Although the results obtained from the two techniques differ by relatively small amounts, the issue of the validity of the equilibrium constants obtained from mobility-shift titrations is an important one and will be considered in detail elsewhere.'j The bipartite operators of the gal operon of E. coli negatively regulate the operon by binding a repressor capable of forming a DNA loop. Evidence that it is the formation of a looped complex,rather than a specific unique property of Gal repressor, that is responsible for regulation was obtained from in viuo transcription studies in which the OB and 0: sites of the gal operon were replaced by the and @ operator sites (5). Conversion of the operator sites resulted in the control by Lac repressor. I n vitro electron micrographs confirmed the presence of Lac repressor-mediated loops (6) in agreement with the results reported here. Thus, either Gal or Lac repressor can repress transcription of the gal operon. It was therefore somewhat of a surprisethat theDNA-binding characteristics of Gal repressor were more similar to LacIadithan to LacI+. Like LacPd', Gal repressor does not bind a two-site operator cooperatively nor is there evidence from DNase I hypersensitivity thatit can form a loopedcomplex (13). Cooperative binding by Gal repressor was only observed at low temperature (13), a characteristic apparently not shared by LacPd' (data not shown). Another similarity between Gal repressor and LacPdi isthat their monomer-dimer self-association equilibrium constants appear to be less than their DNA-binding constants (13). Thus, how does Gal repressor regulate transcription initiation? It is possible that Gal repressor mediates the rate of transcriptioninitiation by a mechanism other than DNA looping, although the ability of the Lac repressor to substitute as thenegative regulatory element of the gal operon ( 5 ) argues against this possibility. The apparentinability of Gal repressor to bind cooperatively or form a DNA loop invitro may be compensated by a requirement for a small effector molecule, DNA supercoiling, bending by a bound protein, and/or binding of an accessory protein to Gal repressor to facilitate selfassociation in uiuo (see Ref. 13 for a more extensive discussion). The similarity of the DNA-binding characteristics of Gal repressor and LacPd' supports the possibility that a weak dimer-tetramer association by Gal repressor is compensated for by additional components or processes i n uivo. CONCLUSIONS

The interaction of proteins bound to sites widely separated on the genome is a recurrent motif in both prokaryotic and eukaryotic regulatory systems. One mechanism by which bound proteins can actat a distance is by their self-association to form higher order oligomers. The recent demonstration that LacI' tetramers dissociate to dimers with dissociation constants comparable to site-specific DNA binding (11) suggests that the thermodynamic linkage of protein self-associD. Senear and M. Brenowitz, manuscript in preparation.

ation and DNA binding may provide a subtle modulator of cellular processes. The inability of LacIadi to associate to tetramers coupled with its inability to mediate the formation of looped complexesis direct evidence of the coupling of selfassociation and the formation of looped complexes. In addition, the fact that the titration curves of LacPdi with DNA containing a single binding site show a weakening of the monomer-dimer association reaction relative to thewild-type repressor suggests that the monomer-dimer and dimer-tetramer interfaces may be interrelated. Although an unequivocal determination of the DNA binding affinity of LacPd' cannot be made in the absence of an independent determination of AGDimer, the results suggest that the DNA binding affinity of this mutant is unchanged relative to the wild-type. However, the uncertainty inherent in the analysis procedure precludes a definitive conclusion that dimer-tetramer association and DNA binding are unlinked in the Lac repressor. Acknowledgments-We thank B. Muller-Hillfor the gift of plasmid pBWlOO bearing the LacIadigene and Tomasz Heyduk, James Lee, and Paul Sollitti for critically reviewingthe manuscript. REFERENCES 1. Adhya, S. (1989) Annu. Reu. Genet. 2 3 , 207-230 2. Echols, H. (1986) Science 2 3 3 , 1050-1056 3. Moitoso deVargas, L., Kim, S. & Landy, A. (1989) Science 2 4 4 , 14571461 4. Majumdar, A. & Adhya, S. (1984) Proc. Natl. Acad. Sci. U. S. A. 85,96839687 5. Haber, R. & Adhya, S. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,9683-9687 6. Mandal, N., Su., W., Haber, R., Adhya, S. & Echols, H. (1990) Genes & Deuel. 4,410-418 7. Borowiec, J. A,, Zhang, L., Sasse-Dwight,S. & Gralla, J. D. (1987) J. Mol. Bid. 1 9 6 , 101-111 8. Kramer, H., Niemoller, M., Amouyal, M., Revet, B., von Wilcken-Bergmann, B. & Muller-Hill, B. (1987) EMBO J. 6 , 1481-1491 9. Oehler, S., Eisman, E. R., Kramer, H. & Muller-Hill, B. (1990) EMBO J. 9,973-979 10. Bellomy, G. R., Mossing, M. C. & Record, M. T. (1988) Biochemistry 2 7 , 3900-3906 11. Royer, C., Chakerian, A. E. & Matthews, K. S. (1990) Biochemistry 2 9 , 4959-4966 12. Whitson, P. A., Olson, J. S. & Matthews, K. S. (1986) Biochemistry 2 5 , 3852-3858 13. Brenowitz, M., Jamison, E., Majumdar, A. & Adhya, S. (1990) Biochemistry 29,3374-3383 14. Pickar, A., Jamison, L. & Brenowitz, M. (1990) Biophysical J. 5 7 , 64 (abstr.) 15. Deuschle. U.. Gentz. R. & Buiard. , H. (1986) , . Proc. Natl. Acad. Sci. U. S. A. 83,4134-4137 ' 16. Lehming, N., Sartorius, J., Oehler, S., von Wilcken-Bergman, B. & MullerHill, B. (1988) Proc. Natl. Acad. Sei. U. S. A. 8 5 , 7947-7951 17. Gilbert, W. & Muller-Hill, B. (1966) Proc. Nutl. Acad. Sci. U. S. A. 5 6 , 1 sa1-1 nan 18. Brenowitz, M., Senear, D. F., Shea, M. A. & Ackers, G. K. (1986) Methods Enzymol. 130,132-181 19. Brenowitz, M. & Senear, D. F. (1989) in Current Protocols in Molecular Biology, Su plement 7 (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., S e i h a n , J. G., Smith, J. A,, and Stouhl, K., eds) John Wiley & Sons, New York 20. Heyd"_k, T.& Lee, J. C. (1990) Proc. Natl. Acad. Sci. U. S. A. 87,17441'148

21. Ackers, G. K., Johnson, A. D. & Shea, M. A. (1982) Proc. Natl. Acad. Sci. U. S. A. 7 9 , 1129-1133 22. Ackers, G. K., Shea, M. A. & Smith, F. R. (1983) J. Mol. Biol. 1 7 0 , 223242 23. Senear, D. F., Brenowitz, M., Shea, M. A. & Ackers, G. K. (1986) Biochemistry 2 5 , 7344-7354 24. Hochshild, A. & Ptashne, M. (1986) Cell 44,681-687 25. Pirrotta, V., Chadwick, P. & Ptashne, M. (1970) Nature 227,41-44 26. Johnson, A. D., Pabo, C. 0. & Sauer, R. T.(1980) Methods Enzymol. 6 5 , 839-856 27. Bowie, J. U. & Sauer, R. T. (1989) Biochemistry 2 8 , 7139-7143 28. Ag arwal, A. K., Rodgers, D. W., Drottar, M., Ptashne, M. & Harrison, S. (1988) Science 242,899-907 29. Jordan, S. R. & Paho, C. 0.(1988) Science 242,893-899 30. Zwieb, C., Kim, J. & Adhya, S. (1989) Genes & Deuel. 3,606-611 31. Wartell, R. M. & Adhya, S. (1988) Nucleic Acids Res. 1 6 , 11531-11541 32. Ma'umdar, A & Adhya, S. (1989) J. Mol. Biol. 2 0 8 , 217-223 33. Sadler, J. R.,'Sasmor, H. & Betz, J. L. (1983) Proc. Natl. Acad. Sci. U. S. A. 80,6785-6789 34. Pace, H. C., Lu P. & Lewis, M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1870-1873 35. Cann, J. R. (1989) J . Biol. Chem. 2 6 4 , 17032-17040

6.