Vol. 129, No. 2 Printed in U.S.A.

OP BAchzoLoGY, Feb. 1977, p. 658-67 Copyright 0 1977 American Society for Microbiology

JOURNAL

Escherichia coli Membrane Proteins with an Affinity for Deoxyribonucleic Acid MASAMICHI KOHIIYAMA,* REGINE KOLLEK,' WERNER GOEBEL,' AND ADAM KEPES Institut de Recherche en Biologie MolUculaire du Centre de la National Recherche Scientifique et de l'Universitg Paris VII, 75221 Paris Cedex 05 Received for publication 13 July 1976

From the membrane fraction of Escherichia coli K-12 strain, four protein fractions (peaks I, Ha, IIb, and Ill) which have affinity for deoxyribonucleic acid (DNA) have been isolated. The molecular weights of these proteins are between 12,000 and 8,000. Only the peak III fraction contains a protein that binds preferentially to single-stranded DNA, whereas the others contain proteins that bind also to double-stranded DNA. The binding activity ofthe peak Ilb protein is inhibited in the presence of polyuridylic acid. Peak I and peak Ha protein fractions behave like hydrophobic proteins. Ag/4

The replicon hypothesis of Jacob et al. (8) had led many authors to attempt the isolation of deoxyribonucleic acid (DNA) membrane fraction from bacteria (5, 26, 28). The first biochemical analysis of a DNA membrane complex was achieved by Fuchs and Hanawalt (4) who showed that the replication fork is not attached directly to the lipid layer. However, several authors have reported a tight association of the origin of replication with a membrane (3, 29). The precise molecular basis of this DNA membrane association has not yet been clarified. The findings of several DNA-binding proteins in Escherichia coli (7, 20, 23, 26) lead us to think that perhaps the chromosome attachment to membranes is mediated by DNA-binding proteins which are the constituents of cytoplasmic membranes. Thus, we have undertaken a systematic isolation of such DNA-binding proteins from membrane fractions of E. coli. This paper describes the isolation of four membrane proteins from E. coli which have an affimity for DNA. MATERIALS AND METHODS Bacterial strains and media. ApoLA mutant ofE. coli K-12, PA3364 (18) was used throughout this work. Bacteria were grown at 30°C in a complete medium containing peptone (Difco) (33 g), yeast extract (20 g), NaCl (5 g), and thymidine (20 mg) per liter of water. Bacteria were harvested at late log phase. For the preparation of (3H]DNA, M63 medium (17) was used with the following supplements: glucose, (0.6%), Casamino Acid, (0.05%), histidine and arginine (20 jsg/ml each), [pH]thymidine (0.5

1On leave from Geelachaft fOr Molekcularbiologische

Forschung, Stockheim West-Germany.

,ACi per ml), and deoxyadenosine (100 l.g/ml).

32P-labeled phage M13 was prepared according to the

method of Kaerner et al. (10). Preparation of crude membrane fraction. All procedures were carried out at 5°C. All buffers contained 10% glycerol, 0.005 M ,-mercaptoethanol, and 0.0005 M ethylene glycol-bis(beta-aminoethyl ether)-N N-tetraacetic acid. Bacteria (22 g, wet weight) were washed twice with 0.02 M tris (hydroxymethyl) aminomethane - hydrochloride (pH 8.3) and 0.01 M magnesium-acetate, resuspended in 22 ml of the same buffer, and broken by pressure extrusion at 15,000 lb/in2 in a Ribi cell fractionator (Sorvall). The extract was centrifuged at 40,000 x g for 15 min, and the supernatant was recovered carefully without taking the viscous and soft precipitate. The supernatant was centrifuged at 100,000 x g for 90 min, and then the pellets were resuspended with the aid of a Potter homogenizer in 20 ml of 0.01 M Tris-hydrochloride (pH 7.5) with 0.001 M MgCl2. After recentrifugation ofthe suspension at 100,000 x g as above, the crude membrane fraction was resuspended in 10 ml of the same buffer. This procedure of membrane preparation is a modification of the method published previously from this laboratory (9). Preparation of DNA. E. coli [3H]thymidine-labeled DNA was prepared according to the method of Marmur (15) with the following modification. After ribonuclease treatment, DNA was deproteinized at 60C for 5 min by phenol saturated with 0.1 M Tris base. M13 phage DNA was prepared according to the method of Schaller (24). RSC11 plasmid DNA was prepared by the method of Goebel and Bonewald (6). ASay of DNA-binding activity. DNA-binding activity was measured by the method of Tsai and Green (30) which cond of filtering a radioactive DNA-protein complex through nitrocellulose membrane filters. A 0.2-ml amount of reaction mixture contained 0.01 M potassium phosphate (pH 7.2), 0.001 M MgCl2, 100 Ag of bovine serum albumin (heated at 37C for 30 min before use), and 12 pmol

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of DNA as nucleotide per 0.2 IACi of E. coli heatdenatured [3H]DNA. It was incubated at 300C for 5 min with the fraction to be assayed. Then the mixture was filtered on nitrocellulose membrane filter (Schleicher and Schull, pretreated as according to Tsai and Green [30]) and washed with 5 ml of washing buffer (0.02 M Tris [pH 7.4], 0.001 M ethylenediaminetetraacetate, 0.05 M NaCl, 0.002 M ,Bmercaptoethanol, 5% glycerol, and 10% dimethyl sulfoxide), followed by two washes with 3 ml of the buffer. The velocity of filtration was regulated to 1 to 2 ml/min. Usually the assay was repeated twice for a given sample. One unit of DNA-binding activity was defined as that retaining 1 nmol of nucleotide on the filter. SDS-polyacrylamide gel electrophoresis. The technique of Weber and Osborn (31) was used. The concentration of acrylamides in gel was 12.5%. The R, values of the marker proteins (purchased from Boehringer) obtained under our conditions were the following: ovalbumin (0.25), D-glyceraldehyde-3phosphate dehydrogenase (0.29), trypsin (0.47), cytochrome c (0.68), and insulin (0.93). Before sodium dodecyl sulfate (SDS) treatment (1% at 1000C for 5 min) samples were usually concentrated by precipitation with 10% trichloroacetic acid, followed by washing with 95% EtOH. When samples did not contain Triton X-100, 25% of trichloroacetic acid was used. This method often failed to precipitate the peak m protein (see text); it was concentrated by lyophilization, followed by dialysis (4 h). DNA polymerases. E. coli DNA polymerases II and III were prepared from PA3364 according to the method of Kornberg and Gefter (12). The phosphocellulose step 2 fractions were used throughout the work. DNA polymerase I was purified from the crude preparation purchased from Boehringer (50 U/ mg of protein) by phosphocellulose column (1.5 by 12 cm) chromatography carried out in the same way as the phosphocellulose step 2 for the above polymerases. All DNA polymerase activities were assayed at 30°C for 15 min in 0.15 ml of reaction mixture containing: 0.066 M Tris-acetate (pH 7.5), 0.01 M MgCl2, 0.001 M dithiothreitol, 3.3 x 10-5 M each of deoxyribonucleoside triphosphate (dNTP), 1 ACi of [3H]thymidine 5'-triphosphate (Amersham), and 1 nmol of calf thymus-activated DNA. Radioactivity in the acid-insoluble fraction was measured. Glycerol gradient centrifugation. A linear gradient solution of 10 to 30% (wt/wt) of glycerol (4.6 ml) was made in the buffer containing 0.01 M potassium phosphate (pH 7.2), 0.05 M KCl, 0.001 M MgCl2, and 0.001 M dithiothreitol. A 0.2-ml sample was layered onto the top of the gradient and centrifuged at 48,000 rpm in the Spinco SW50 rotor, for 2 h and 10 min, for detection of the M13 DNA-protein complex, or for 12 h in the case of determination of sedimentation coefficient. DNA-cellulose chromatography (1). Calf thymus heat-denatured DNA cellulose (gift of Gefter) was used. The protein fraction to be adsorbed was first dialyzed against 50 volumes of 0.01 M Tris (pH 7.5) and 0.001 M MgCl2. The elution of the DNA-binding activity was carried out with dialysis buffer containing various amount of KCl.

659

Protein determination. Protein concentration was determined by the method of Lowry et al. (13) with bovine serum albumin as a standard.

RESULTS

Isolation of DNA-binding proteins. All procedures were carried out at 5°C. To eliminate DNA, the membrane fractions obtained by the procedure described in Materials and Methods were first treated with diethylaminoethyl (DEAE)-cellulose (0.5 meq/g) in the following way: 10 ml of membrane suspension at protein concentration of 17 mg/ml were passed through the column of DEAE-cellulose (2.5 by 12 cm) equilibrated with 500 ml of 0.01 M Tris-hydrochloride (pH 7.5) and 0.001 M MgCl2. Under these conditions, most of the membrane fractions were not adsorbed to DEAE-cellulose, whereas DNA was retained (M. Roisin and A. Kepes, manuscript in preparation). Usually the turbid flow-through fraction contained onequarter to one-half of input proteins. This step eliminated contaminating soluble proteins as well as proteins loosely bound to membranes. To further purify the membranes, the flowthrough fraction was centrifuged at 100,000 x g for 60 min and the precipitates were resuspended in 10 ml of 0.04 M potassium phosphate (pH 6.5). The membrane suspension was then passed through a column of phosphocellulose (Whatman P11) (2.5 by 3.5) equilibrated with the resuspending buffer (150 ml). The nonadsorbed fraction was kept on ice overnight in the presence of 0.1% Triton X-100. The last two treatments activated the DNA-binding activity 10- to 15-fold. This phosphocellulose treatment was originally set up for elimination of soluble proteins not adsorbed to DEAE-cellulose. However, it turned out that the DNA-binding activity was increased about 10-fold at this step. The treatment with Triton X-100 further enhanced activity but less markedly. The extent of activation was not influenced by variation of the protein-phosphocellulose ratio in the range of 2.4 mg of protein to 4.0 mg for 1 ml of phosphocellulose. The activated membrane fraction was chromatographed on a phosphocellulose column (1.5 by 12) equilibrated with 150 ml of 0.04 M potassium phosphate, pH 6.5, containing 0.1% Triton X-100. About 75% of input protein and 90% of DNA-binding activity were retained by this column. Apparently, the DNA binding-proteins were extracted from the membranes by the Triton X-100 and the phosphocellulose column. The DNA-binding proteins were eluted from the column by linear gradient of 0.15 to 1.5 M KCl in equilibration buffer (total volume 700

J. BACTERIOL.

KOHIYAMA ET AL.

660

ml). Three peaks of DNA-binding activity were detected; the first peak (I), representing onetenth of recovered activity was eluted at 0.4 M KCl, and the second peak (II), which contained three quarters of activity, came down to around 0.8 M KCl, and the third peak (III) was eluted at 1.2 M KCl (Fig. 1). The recovery of the binding activity was 150%. The first peak fractions were pooled and dialyzed for 5 h against 100 times the volume of 0.02 M potassium phosphate (pH 7.5) containing 0.1% of Triton X-100. The dialyzed fraction was put on a DEAE-cellulose column (Whatman DE-32) (1.5 by 6 cm) equilibrated with 100 ml of dialyzing buffer. Elution of the column was carried out with a linear gradient of 0.1 to 1.0 M KCl in the dialysis buffer (total volume, 400 ml). The binding activity eluted as a single peak at 0.2 M KCl. At this stage of purification, the SDS-polyacrylamide gel electrophoresis (31) showed that the material was usually 70% pure (see "Determination of molecular weight"). Because the DEAE-cellulose chromatography of the first peak resulted in a poor recovery of activity (10%), we tried DNA-cellulose (calf thymus single-stranded DNA) as well as rechromatography with phosphocellulose. The binding activity was eluted from DNA-cellulose at 0.8 M KCl with a recovery of 30%. Rechromatography with phosphocellulose gave a similar recovery. The poor recovery of binding protein was probably due to the instability of the first peak fraction; the binding activity decayed at 0°C with a half-life of 3 to 5 days, and when placed at 30°C in the reaction mixture without DNA, the activity was reduced to one-third in 5 min.

The peak II fractions were further purified with a DEAE-cellulose (DE-32) column (1.5 by

,0

0504 MOO ,

+r,

X1

,.

-

_ E..

10 cm) after dialysis against 10 volumes of equilibration buffer (the same as for peak I) for 5 h with four changes of the buffer. The proteins were applied to the column and eluted with a linear gradient of 0.12 to 1.2 M KCl in equilibration buffer (total volume of 600 ml) and then with three times the bed volume of 2 M KCl. Two peaks of DNA-binding activity were obtained: the first peak (Ila) at 0.3 M KCl, the second peak (Ilb) at 1.2 M KCl (Fig. 2). The recovery of the total activity of this step was about 60%. When peak Ha was concentrated by adsorption and stepwise elution from DEAEcellulose, most of the activity was recovered by elution of the column with 0.5 M KCl. However, further elution of the column with 2 M KCl often gave a minor peak of activity (10 to 15% of the major peak) corresponding, by molecular weight and other properties, to peak Ilb. The peak fIb fraction was 80% pure without rechromatography (estimation made by SDSpolyacrylamide gel electrophoresis), whereas the peak Ha fraction even after rechromatography gave several protein bands on SDS-polyacrylamide gel electrophoresis. The peak III was further purified in the following way. The pooled fractions were dialyzed against 50 volumes of 0.04 M potassium phosphate (pH 6.5) buffer for 5 h in the absence of Triton X-100. It was then passed through a phosphocellulose column (1 by 3.5 cm) equilibrated with the dialysis buffer without Triton X-100. The binding activity was eluted with 2 M KCl in dialysis buffer without Triton X-100. The recovery of this step was about 50%. One preparation out of five gave about 90% pure material but often at this stage the preparation gave several bands of protein on SDS-polyacrylamide gel electrophoresis. Fractionation of these proteins is summarized in Fig. 3. Determination of molecular weights. Three methods have been used to determine the mo-

1.5

52M

'i I

In

Im

~i LI e &A/

0)

5000..

2

I

500o > 1 xn n vn/

N

0.5

E5

--L x

ln__ Fractions

FIG. 1. Phosphocellulose chromatography of DNA-binding proteins. Procedures and assay method are described in text. The concentration of KCI was measured by a conductimeter Philips PR.

Fractions

FIG. 2. DEAE-cellulose chromatography of the peak II fraction.

VOL. 129, 1977

DNA BINDING PROTEINS FROM E. COLI MEMBRANES

PA3364

661

22g Prot.: 180 mg act.: 12,000 U Prot.: 112

mg

act.: 18,000 U

Prot.: 60

mg

act.: 10,500 U

Prot.: 31 mg act.: 152,000 U

Prot.: 1.9 mg act.: 22,000 U

Prot.: 9.0 mg act.: 151,000 U

Prot.: 0.6 mg act.: 15,000 U

Prot.: 0.12 mg 2,700 U

Prot.: 0.13 mg act.: 6,000 U

act.:

DEAE-cellulose

Peak

Ia

Prot.: 3.7 mg act.: 75,300 U

Peak Ilb

Prot.: 0.3 mg act.: 5,680 U

FIG. 3. Diagram of protein fractionation. See text for details. Abbreviations: Prot., protein; act., activity; nonads., nonadsorbed; sup, supernatant.

lecular weight of each peak protein; centrifugation on linear gradients of 10 to 30% glycerol, gel exclusion chromatography, and SDS-polyacrylamide gel electrophoresis (31). We tried to determine the molecular weight of peak I protein using a column of Sephadex G75 equilibrated with 0.02 M potassium phosphate (pH 7.2) containing 0.1% Triton X-100 and bovine serum albumin (250 ,g/ml). Forty percent of the input activity was recovered at the exclusion volume. We then tried a column of agarose 1.5 m equilibrated in the same manner as with G-75 and found only 10% of the input activity at the exclusion volume. Expecting to find a high molecular weight, we centrifuged the protein on a linear gradient of 10 to 30% glycerol containing 0.1% Triton X-100 and

albumin (Spinco SW50L, 9 h, 47,000 rpm). The binding activity was found in the upper thirds of the gradient with no clear peak. These results suggested that the peak I protein forms aggregates with micelles of detergent, which interfere with the measurement of its molecular weight. Therefore, we tried gel filtration and centrifugation in the absence of both detergent and albumin. Filtration on a column of Sephadex G-75 gave no clear result due to a poor recovery, whereas centrifugation of the protein on a glycerol gradient enabled us to get 80% of input activity in a single peak corresponding to molecular weight of about 20,000 (Fig. 4). SDS-polyacrylamide gel electrophoresis of peak I preparation demonstrated the existence

662

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KOHIYAMA ET AL.

MARKERS 45

23 12.5

6

x10

lb~~~~~~~~~~~~~~~

-2} iooo t_ =

llb

0.

L /

I~~~~~~~~~~~~~~~

01

1000

500

1

0

X

1

2

3

(~

4

gradient volume (ml) FIG. 4. Glycerol grad ient centrifugation ofpeaks I and IIa. The conditions for centrifugation are described in the text. A 0.2-ml amount of the peak I fraction obtained from the phosphocellulose chromatography was dialyzed for 1 h against 100 ml of 0.02 M potassium phosphate (pH 7.2) without Triton X100 and then put onto the glycerol gradient. After centrifugation, about 25 fractions were collected and assayed for DNA-binding activity. The volume of each fraction was measured by a pipette and summed for the presentation of the coordinate. A 0.2-ml portion of the peak IIa fraction obtained by DEAE-cellulose chromatography was treated in the same way.

of a major protein band (70%o pure) with a molecular weight of 12,000 in three independent preparations. The molecular weights of the minor peaks were variable. Therefore, we concluded that the molecular weight of peak I protein is 12,000 (Fig. 5). aThe same sort of diffrculty was encountered in measuring the molecular weight of the peak IIa protein: in the presence of bothlTiton X-100 and albumin, gel filtration (Sephadex G-75 or agarose [1.5 mp) indicated a high molecular weight (1,500,000) (Fig. 6). Under the same

conditions, glycerol gradient centrifugation gave no clear peak. As in the case of the peak I treation on an agarose protein, we first

FIG. 5. SDS-polyacrylamide electrophoresis of DNA-binding proteins. A Kipp and Zoner densitometer traced the gels of each protein preparation after electrophoresis and staining (31) as described in the text.

1. 5-m column equilibrated with phosphate buffer containing albumin but not Triton X100. No clear peak in activity was detected (less than 1% recovery). Thinking that the agarose gel adsorbed the peak IIa protein, we used other types of gel (Sephadex and Bio-Gel P) without success. Thus, the presence of Triton X-100, but not bovine serum albumin, apparently stabilizes the peak IIa activity, probably because of aggregation of protein with deter-

gent.

The centrifugation of the peak IIa fraction on the glycerol gradient prepared without detergent enabled us to obtain a single peak activity slightly lighter than the peak I protein (Fig. 4). The instability of this peak activity was striking; at O°C, the half-life was about 45 min. Because the final DEAE-cellulose fraction was not pure enough to allow the determination of the molecular weight, further purification was carried out by DNA-cellulose chromatography. The binding activity was eluted 0.r at 4 M of KCI. This fraction showed one major protein band (about 80%o pure) at a molecular weight of 10,000 (Fig. 5). The peak IIb protein was easily chromatographed on a column of Sephadex G-75 in the

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DNA BINDING PROTEINS FROM E. COLI MEMBRANES

663

examined whether the DNA-binding activity measured by nitrocellulose filtration repre1500 4, 4 sented the formation of a DNA-protein complex by using the peak I obtained by phosphocellulose chromatography and 3H-labeled M13 phage M13 phage DNA, incubated with the DNA. 1b 1000 c 1000lb peak I, sediments faster (36.8S) than the phage DNA alone (23S). Furthermore, more than 90% s 500 l l of the faster sedimenting DNA was trapped by 500 the filter (Fig. 8). This result demonstrated the formation of a stable complex between DNA and the peak I protein and also shows that the 0 X | |0 efficiency of nitrocellulose filtration for detection of the complex is high. Specificity for DNA. Since molecules such as i1500 ribonuclease can bind tightly to DNA (2), the proteins isolated thus far were not necessarily la specific DNA-binding proteins. Therefore, we tested whether the four isolated fractions 1000could discriminate DNA from ribonucleic acid (RNA). If a protein has more affinity for DNA than RNA, the amount of radioactive DNA l 500 trapped on a filter should not be affected by the presence of cold RNA. After 5 min of incubation of each fraction in the presence of a constant amount of [3H]DNA and various amounts of 10 20 30 40 50 polyuridylic acid, the mixture was immediately Fractions filtered and the radioactivity retained on the FIG. 6. Sephadex G-75 chromatography of peaks filter was counted. The results of this experiIIa and IIb. A column of Sephadex G-7c (1.5 by 23 ment demonstrated that only the peak llb procm) was equilibrated with 0.02 M potassium phos- tein was unable to discriminate DNA from phate (pH 7.2), 0.05 MKCI, 0.1% Triton X-100, and RNA (Fig. 9). 200 g of bovine serum albumin per ml. Peak IIa or Using the small circular plasmid RSCII DNA IIb (0.7 ml) obtained by DEAE-cellulose chromatog- (4), we then examined whether these proteins raphy was put on the column and 0.7 ml of the could distinguish single-stranded DNA (heat fraction was collected in each tube. The exclusion denatured) from double-stranded DNA. Similar

VO

Cyt C

Vt

volume was determined by dextran blue and the total volume was determined by phenol red.

p0

Yt

presence of Triton X-100 (Fig. 6). From the position of the peak, we estimate its molecular weight to be 7,000. SDS-polyacrylamide gel C,oo electrophoresis of the fraction obtained by 4 DEAE-cellulose chromatography demonstrated i one protein band (always diffused in form) whose peak corresponded to a molecular weight < 5dI of 11,000. With the peak III protein, filtration on both a Bio-Gel P30 column and on a Sephadex G-75 gave an estimated molecular weight of 6,000 o0._ ._. (Fig. 7). SDS-polyacrylamide electrophoresis of 20 40 so the fraction further purified by a DNA-cellulose Fractions column (elution by 1.4 M KCI) revealed a wide FIG. 7. Bio-Gel P30 chromatography of peak III. protein band corresponding to a molecular A column of Bio-Gel P30 (200 to 400 mesh, Bio-Rad) weight of 8,000 -9,000. (1.5 23 cm) was equilibrated with 0.02 M potassiumby phosphate (pH 7.2) and 0.25 MNaCl. A 0.7-ml Evidence for a DNA-protein complex. Thus amount ofpeak III concentrated by the rechromatogfar we have only shown that there are several raphy with phosphocellulose was put on the column. activities which caused the retention of Seven-tenths milliliter of the fraction was collected in [3H]DNA on nitrocellulose filters. We therefore each tube.

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KOHIYAMA ET AL.

itself, when present at more than 2.10-3 M, increased the amount of DNA retained on the filter. Thus, concentrations of spermidine up to 1.5 x 10-3 M were used in the experiment. Only the peak fla activity was not affected by the presence of spermidine, whereas the activities of peaks I, Ill, and especially IIb were sensitive to its presence (Fig. 11). Effect of MgCI2 and KCI. The efficiency of the lactose operon-lac repressor binding as

40

E~v1000-

500-

o~~~ 1

10

20

Fractions FIG. 8. Cosedimentation of the peak I protein and M13 DNA. [3H]DNA (0.5 nmol) (20,000 cpm) was added into 0.2 ml of the assay mixture with (b) or without (a) the peak I fraction from the phosphocellulose chromatography (5.8 U). After 5 min of incubation at 30°C, the mixture was layered onto the glycerol gradient and centrifuged. Fractions of a 0.2-ml volume were collected in each tube. One-tenth milliliter was precipitated by 5% trichloroacetic acid and the radioactivity of the precipitates was counted (filled circles). One-tenth milliliter of each peak fraction was filtered directly onto a nitrocellulose membrane as described for the DNA-binding-activity assay (triangles).

to its inability to discriminate DNA from RNA, the peak IIb protein bound equally well to both single- and double-stranded DNA. Apart from some slight differences in efficiency, the peak I and peak IIa proteins could attach to both types of DNA. Only the peak III protein bound preferentially to single-stranded DNA (Fig. 10). Effect of spermidine. According to Schekman et al. (25), the essential role of the E. coli DNA-binding protein in in vitro DNA synthesis can be replaced by spermidine. We examined the effect of spermidine on the DNA binding activity of four protein fractions. Spermidine

2

3

4

1

3

2

Col N Cle Acid (A260 nW X-r3) FIG. 9. Effect of polyuridylic acid on DNA binding. The DNA-binding activity of each of the four fractions was assayed as described in the text in the presence of varying amounts of E. coli B heat-denatured DNA (Choay) (circles) or of polyuridylic acid

(Miles) (triangles). I

Ii

Ia

0

a

[N

[3H]I-DNA (nmol) FIG. 10. Affinity for single- or double-stranded DNA. The DNA-binding activity of the peak fractions (each presented at 1 U) was assayed using as substrate either RSC11 native (open circles) or heatdenatured (closed circles) [3H]DNA.

VOL. 129, 1977

DNA BINDING PROTEINS FROM E. COLI MEMBRANES

665

that they are different proteins. First, the binding activity of the peak I fraction is unstable at every stage ofpurification, whereas the activity of peak Ha fraction is stable at 00C until elimination of Triton X-100 by fractionation according to molecular size. The molecular weight of peak I protein is bigger than that of the peak Ha protein. Finally, the presence of spermidine does not influence the binding activity of peak Ha, whereas it reduces binding capacity of the peak I protein. The properties of the peak llb protein are the following: (i) its low molecular weight even in the presence ofTriton X-100 and (ii) its pronounced sensitivity to spermidine and a tight binding to RNA. The peak Ill frac-

'20e°60-< 3000

00

1000

o

0

_tI__ ____o

0.5

1

0

___

0

a___ _

1

Spermnci (103M) FIG. 11. Effect of spermidine on DNA binding. The DNA-binding activity of each peak fraction was tested in the presence of varying amounts of spermidine (open circles). The control contained no peak fractions (closed circles).

KCI O.IM

02M

E

1000

tested by the filter technique is increased if MgCl2 is present (21). The same situation was found with the activities of the four bindingprotein fractions; all of them required 10-3 M of MgCl2 for maximum activity. The binding ac~50Otivity of all four fractions was inhibited by KCI (Fig. 12). Effect of DNA-binding proteins on DNA synthesis. The DNA-binding protein isolated by Sigal et al. (26) can stimulate DNA synthesis catalyzed by the DNA polymerase II or by the in vitro system for single-stranded DNA CE,~ ~ ~ ~ 1 replication (25). To distinguish our proteins O 2.lr3M 1O-M from this DNA-binding protein, we examined Mg Cl2 respectively the effect of four protein fractions on DNA synthesis carried out by each of three FIG. 12. Effect of KCI or MgCl2 on DNA-binding DNA polymerases ofE. coli. Even at a saturat- activity ofpeak I. The activity was measured under ing concentration of the protein, none of the standard conditions in various amounts of KCl (triDNA syntheses was considerably affected by angles) and MgCl2 (circles). these protein fractions (Table 1). TABLz 1. Effect of DNA-binding proteins on DNA DISCUSSION polymerases Summarzing the characteristics of the four [3Hlthymidine 5'-triphosphate incorporated (pmol) fractions of DNA-binding protein (Table 2), we Polymerase want to emphasize their differences which led Peak Peak Peak Peak us to think it probable that these fractions conPa III Ha Ilb tain four distinct proteins. The peak I fraction 8.3 7.7 9.0 I 9.7 10.3 resembles the peak Ha fraction because of their II 5.4 5.4 5.1 5.5 7.6 instability, behavior with Triton X-100, and mII 9.5 8.9 10.4 9.4 9.2 inability to distinguish single from doublea Each was present at 1 U/assay. stranded DNA. However, several facts indicate

666

KOHIYAMA ET AL.

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TABLE 2. Characteristics of DNA-binding-protein fractions Peak

Distinction of DNA from poly(U)a

I Ila Ilb

Im a

+ +

+

Characteristics Double- or singleAggregation with Tristranded DNA ton X-100

Both Both Both Single-stranded

Spermidine inhibition

+ +

+

-

+ +

-

poly(U), Polyuridylic acid.

tion is easily distinguished by its finn binding to phosphocellulose and its specificity for single-stranded DNA. Although these observations favor the idea offour distinct proteins, the conclusive evidence, such as differentiation by immunology or by mutation, is still required. Judging from the ability to form aggregates in the presence of Triton X-100, two proteins, peak I and Ha, among the four studied in the present work can certainly be hydrophobic suggesting a membranous origin. The rapid decrease in binding activity in the absence of Triton X-100 may be explained by their hydrophobic character which tends to induce aggregation of molecules (14). The other two proteins may also be constituents of membranes unless they are trapped (from the soluble fraction) inside vesicles. The chromosomal sites involved in membrane attachment were first reported as the replication fork (27). However, because of the absence of lipids in the purified replication fork (4) and of the success in isolation of a stable membranous complex containing the replication origin (3), the current idea had been that the replication origin is the unique site for chromosome attachment to membranes (29). Recently Parker and Glaser have shown that the replication fork exists as a phospholipase-sensitive and lysozyme-resistant complex (19). For demonstration of such a complex, they have used almost the same filtration technique as we have used here. Since their method should permit detection of our DNA-binding proteins, it raises a question as to why they could show the perferential complexing of the growing point. If the number of DNA-binding-protein molecules is very limited, it will explain why the membrane attachment site is restricted. However, the calculated number of the peak IIb protein molecules, for example, in a cell, is about 600. One tentative explanation is that most of the present binding proteins (if they participate in the attachment of replication fork) are masked, or inhibited, except those which are located around the replication fork. The idea of DNAbinding inhibitors is supported by two observations: (i) the DNA-binding activity of DNA-free

membrane preparations is only linear with a limited range of membrane concentration and further addition of the preparation does not increase the amount of DNA retained on the filter; (ii) the DNA-binding activity is always enhanced considerably after treatment of the membrane fraction by phosphocellulose in the absence ofTriton X-100. If some inhibitors exist they could be adsorbed to phosphocellulose. In fact, the materials eluted from phosphocellulose by 1 M KCI reduced the DNA-binding activity of the activated membrane fraction. The characterization of inhibitors will be studied in the near future. If some of our proteins participate in chromosome attachment to membranes at the initiation site, one of the present proteins should display an increased affinity for the origin of replication. The DNA fragments enriched for initiation site were prepared from bacteria labeled with [3Hlthymidine and 5-bromodeoxyuridine at the starting point of replication. Only the peak Ha protein showed threefold increased affinity with DNA of initiation site compared with the bulk of the chromosome. However, subsequent tests revealed this to be due to the increased affinity of the peak Ha protein for DNA containing 5-bromodeoxyuridine moiety. Therefore, the initiation site should be prepared by some other methods. The obvious approach to identify the physiological function of these proteins is to examine mutants of E. coli, especially those altered in DNA replication. Until now, we have tested a dnaA mutant (CRT-46) (11), three dnaH types obtained from D. Glaser (K; 50, 66, and 133), and recA36 obtained from J. George (15). The only striking difference between mutants and the wild type is that crude-membrane preparation of both KI 50 and 66 are completely adsorbed by a DEAE-cellulose column, with which only nucleic acids and soluble proteins should be trapped. The detailed description as well as analysis of all types of dna mutants will be completed in the near future. Finally some remarks should be presented concerning the differences between known DNA-binding proteins in E. coli and our pro-

VOL. 129, 1977

DNA BINDING PROTEINS FROM E. COLI MEMBRANES

teins. The first DNA-binding protein isolated in E. coli (26) has the following characteristics: (i) it has a molecular weight of 23,000; and (ii) it has a stimulatory effect on DNA synthesis carried out by the DNA polymerase II (16) as well as on in vitro synthesis of OX-174 phage DNA (25). The present four proteins can be easily distinguished from this protein by their molecular weight, their membranous origin, and their inability to stimulate DNA polymerase II activity. Very recently, a histone-like protein has been found in E. coli (23). In spite of its dominant presence in soluble fractions and its different behavior with DEAE-cellulose (not adsorbed; Rouviere-Yaniv, personal communication), because of the similarity in molecular weight between that protein and ours, a serological test is required for the definite distinction. The protein X obtained from the membrane fraction of E. coli can also be considered as a DNA-binding protein (7). However, its molecular weight is about four times greater than those of the present proteins. ACKNOWLEDGMENT This work was supported by a grant from the NATO Scientific Affairs and Delegation G6n6rale a la Recherche Scientifique et Technique. LITERATURE CITED 1. Alberts, B., and G. Herrick. 1971. DNA-cellulose chromatography. Methods Enzymol. 21:198-217. 2. Felsenfeld, G., G. Sandeen, and P. H. von Hippel. 1963. The destabilizing effect of ribonuclease on the helical DNA structure. Proc. Natl. Acad. Sci. U.S.A. 50:644651. 3. Fielding, P., and C. F. Fox. 1970. Evidence for stable attachment of DNA to membrane at the replication origine of E. coli. Biochem. Biophys. Res. Commun. 41:157-162. 4. Fuchs, E., and P. C. Hanawalt. 1970. Isolation and characterization ofthe DNA replication complex from E. coli. J. Mol. Biol. 52:301-322. 5. Ganesan, A. T., and J. Lederberg. 1965. A cell membrane bound fraction of bacterial DNA. Biochem.

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