Proc. Natl. Acad. Sci. USA Vol. 75, No. 2, pp. 799-803, February 1978 Biochemistry
Escherichia coli dnaB mutant defective in DNA initiation: Isolation and properties of the dnaB protein (OX174 DNA replication/ATPase/agarose-ATP/Plbac suppression/PI ban protein)
ERICH LANKA, BRIGITTE GESCHKE, AND HEINZ SCHUSTER Max-Planck-Institut fur Molekulare Genetik, Berlin-Dahlem, Germany
Communicated by Robert L. Sinsheimer, December 5, 1977
ABSTRACT Extracts of the DNA initiation-defective mutant Escherichia coli dnaB252 are inactive in a dnaB complementation assay but yield a ribonucleoside triphosphatase activity of native molecular weight of about 270,000 (60,000dalton polypeptide as subunit) that can be inactivated by antibody to dnaB. On the other hand, extracts of a dnaB252(PI bac) lysogen, in which the dnaB mutation is suppressed in vivo by the constitutive expression of the P1 dnaB analog (ban protein), are active in dnaB complementation and the activity is also sensitive to dnaB antibody. Upon further purification two proteins (with polypeptide molecular weights of 60,000 and 56,000, respectively) are found associated with each other (native molecular weight about 270,000). The larger and the smaller protein are tentatively identified as the dnaB and P1 ban protein. It is suggested that suppression of the dnaB mutation by prophage PI bac is accomplished by a stabilization of dnaB252 by P1 ban subunit molecules in a heteromultimer.
Escherichia coli dna ts mutants are usually divided into two classes, DNA initation- and DNA elongation-defective mutants. The majority of the existing E. coli dna ts mutants are dnaB mutants, which belong to the second class. In turn, most of the dnaB mutants show virtually immediate cessation of DNA synthesis at 420; some, however, exhibit considerable residual synthesis (1). The dna252 mutation was initially classified, phenotypically, as a DNA initation mutant (2); however, recent evidence indicates that this is a dnaB mutation that prevents the initiation and not the elongation of chromosomal replication
(3).
In the present communication it is shown that the dnaB252 mutation is suppressible by prophage Plbac in which the viral dnaB analog (ban) is expressed constitutively (4, 5). By use of a dnaB complementation assay (6, 7), an activity is purified from the E. coli dnaB252(Plbac) lysogen that contains two proteins associated with each other. These proteins are tentatively identified as dnaB and Plban proteins. An extract of E. coli dnaB252 is inactive in a dnaB complementation assay (S. Wickner, cited in ref 3). However, a ribonucleoside triphosphatase activity, sensitive to antibody against dnaB, can be purified from this mutant. Such an activity has been found associated with purified dnaB protein from E. coli wild-type cells (8, 9). These findings are discussed with regard to the functional roles of Plban and dnaB protein in vivo.
Bacteria and Phage. Bacterial strains were E. coli dnaB252 (2, 3), a temperature-resistant revertant selected on tryptonesupplemented agar plates at 420 (this laboratory), BT1000, and BT1071 dnaB (12). Phages were PlCml, PlCmlbac-l, and PlCml Plbac-1 ban-i (4) (kindly provided by A. Jaffe-Brachet and D. Touati-Schwartz) and are abbreviated P1, Plbac, and Plbac ban for convenience. P1 lysogens of E. coli dnaB252 were prepared in this laboratory. Buffers. Buffer A: 20 mM Tris-HCl (pH 7.6)/50 mM NaCl/10 mM MgCl2/0. 1 mM EDTA/0. 1 mM dithiothreitol/ 0.1 mM ATP/12% (wt/vol) glycerol. Buffers B-E: buffer A with the following substitutions (in parentheses): B (1 mM MgCI2), C (MgCI2 and ATP omitted), D (5 mM MgCl2 and 5 mM ATP), and E (1 mM ATP, glycerol omitted). Assays. DNA synthesis with ammonium sulfate fractions was measured as described (10). For the dnaB complementation assay, the ammonium sulfate fraction of the E. coli BT1071 dnaB receptor was prepared as described for strain BC1304 (11). Receptor fractions (about 10 mg of protein per ml) were heated for 2.5 min at 370 in order to inactivate any residual DNA-synthesizing activity; 80-140 ,Ag of receptor protein were used per assay. DNA synthesis was followed for 30 min at 250 as described (10, 11), with 320 pmol (nucleotides) of pX174 DNA. One unit of dnaB complementing activity incorporates 1 nmol of dTMP under the above conditions. For the assay for DNA-dependent ATPase conditions were as described (8) except that 1.5 nmol of qX174 DNA was used and the reaction was terminated after 30 min at 250. One unit of ATPase catalyzed the production of 1 ,umol of 32P, under the above condi-
tions. Protein Fractions. dnaB complementing activity was purified from E. coli dnaB252(Plbac) as described in the legend to Table 2. A DEAE-cellulose fraction from E. coli BT1000 dnaB + (12) and an agarose-ATP fraction from E. coli supF dnaB266(Plbac) (4, 5) were prepared in the same manner. A detailed description will be published elsewhere. Other Methods. Growth and lysis of cells were as described
(11, 13). Sodium dodecyl sulfate (NaDodSO4) gel electrophoresis on 15% (wt/vol) polyacrylamide gel was performed as described by Laemmli (14). Protein was determined by the method of Miller (15) and on NaDodSO4 gels by scanning with an Ortec 4310 densitometer, with bovine serum albumin as standard.
MATERIALS AND METHODS Materials. Agarose-ATP, Type 4 was from PL-biochemicals; [y-32P]ATP, 2 Ci/mmol was from The Radiochemical Centre, Amersham. Other chemicals were as described (10, 11). Antibody to E. coli dnaB (13.5 mg of gamma globulin protein per ml) was a gift of A. Kornberg and R. McMacken.
RESULTS of coli E. Suppression dnaB252 by Prophage P1 bac. Strain dnaB252 was lysogenized with P1, PMbac, or PMbac ban and the colony-forming ability was determined at 300 and 420. Table 1 shows that the dnaB mutation was suppressed only in the PMbac lysogen, but not in the P1 wild-type lysogen [in which
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Abbreviation: NaDodSO4, sodium dodecyl sulfate. 799
800
Proc. Natl. Acad. Sci. USA 75 (1978)
Biochemistry: Lanka et al.
Table 1. Suppression of E. coli dnaB252 by prophage Plbac
Plating efficiency at 420 relative to 300
dnaB252 strain
Nonlysogen
4.5 X 10-5
Lysogens P1 Plbac Plbac ban
1.4 X l0-5 1.1 1.0 X 10-4
Revertant
0.8
Fraction Extract Ammonium sulfate DEAE-cellulose Agarose-ATPt Glycerol gradientt
Bacteria were grown in TY medium (10) at 30°. During logarithmic growth phase, appropriate dilutions were plated on TY plates (10 g of Bacto Tryptone, 5 g of yeast extract, 10 g of Bacto Agar, and 5 g of NaCl per liter). For the growth of P1 lysogens, medium and plates contained 25 Aig of chloramphenicol per ml.
synthesis of Plban protein is suppressed (4, 5)] or in the Plbac ban lysogen [in which ban protein is defective (4)]. This indicates that the PIban protein is able to assist or substitute for the dnaB protein during E. col DNA initiation, as it does during DNA elongation (4, 5). An attempt was made to imitate, in vitro, the in vivo suppressing action of Plban protein as described earlier (10). Ammonium sulfate fractions were prepared from different strains as described in the legend to Table 2 and tested for their ability to catalyze the conversion of OX174 DNA to its duplex form. DNA synthesis was observed only with fractions from dnaB252(Plbac) and from the revertant strain (Fig. 1). Extracts of dnaB252 were inactive at all temperatures tested (Fig. 1), 60
40
0.
0
2Cu 0
0. C
:'. ~~~Revertant
20
dnaB 252
25
35 30 Temperature, 0C
40
Total Total units protein, mg
Specific activity, units/mg
Yield, %
13,200 1,124 20 20 0.21 0.096
0.73 14 4.9 830 1,180
100 34 12 21 14
-
820 280 98* 175 113
Frozen cells (240 g suspended in 240 ml) were lysed to yield 470 ml of extract (28 mg of protein per ml). All the following operations were done at 4°. Streptomycin sulfate (20%, 118 ml) was added (4% final concentration); the mixture was stirred for 45 min and centrifuged for 45 min at 30,000 rpm. Ammonium sulfate (0.226 g/ml) was added to the supernatant, which was stirred for 30 min and centrifuged for 20 min at 10,000 rpm. The pellet was washed with 50 ml of 50% saturated ammonium sulfate and dissolved in buffer A. After dialysis against buffer A, this fraction (56 ml, 20 mg of protein per ml) was applied to a DEAE-cellulose column (3 X 43 cm). The column was developed with a 3000-ml linear gradient, 0.05-0.6 M NaCl. The dnaB complementing activity eluted at 0.38 M NaCl. After ammonium sulfate precipitation [0.368 mg of (NH4)2SO4/ml, the pellet was dissolved in buffer B and dialyzed against the same buffer. An 0.85-ml aliquot of this fraction (4.3 ml, 4.6mg of protein per ml) was diluted to 5 ml with buffer C and applied to an agarose-ATP column (0.5 X 5 cm). The dnaB complementing activity was eluted with buffer D. An aliquot of the peak fraction was used for glycerol gradient centrifugation as described in the legend to Fig. 3. * Activity remaining after freezing and thawing once. t These steps were performed with only a part of the fraction from the preceding purification step. The values reported assume that the yield and purification would be the same if the entire DEAE-cellulose and agarose-ATP fractions were subjected to the following procedure(s).
as was reported previously (S. Wickner, cited in ref. 3). Like-
dnaB 252(Plbac)
E
Table 2. Purification of dnaB complementing activity from E. coli dnaB252(Plbac)
42
FIG. 1. DNA synthesis with ammonium sulfate fractions. Ammonium sulfate fractions were prepared as described in the legend of Table 2. DNA was synthesized for 30 min at the indicated temperatures (as described in ref. 10) with 90, 120, or 120 'sg of dnaB252(Plbac), dnaB252, or revertant strain protein, respectively. Supplemented DNA synthesis: 120 Atg of protein of dnaB252 (E) or 120 ,ug of protein of the revertant (-), each supplemented with 2.4 jig of protein of BT1000 (DEAE-cellulose fraction).
wise, extracts of dnaB(Plbac ban) were also inactive (data not shown). Fractions from dnaB252 regained activity when supplemented with dnaB protein from BT1000 wild-type cells (Fig. 1), indicating that the failure to synthesize DNA was due to a deficiency in functionally active dnaB protein. Since the latter protein participates in the initiation of 4X174 DNA complementary (-) strand synthesis (16), the Plban protein can also assist in this reaction. These findings prompted us to purify a corresponding activity from the dnaB252(Plbac) lysogen by using a dnaB complementation assay (6, 7). Purification of a dnaB Complementing Activity from E. coli dnaB252(Plbac). As described above, only fractions from dnaB252(Plbac) but not those from the nonlysogen are active in DNA synthesis. This implies that any dnaB complementing activity purified from the lysogen must contain Plban protein (unless a more complicated mode of suppression by prophage Plac is assumed). Cells of dnaB252(Plbac) were grown at 400 to assure that suppression was effective in vivo. The dnaB complementing activity was purified as described in the legend to Table 2. The use of affinity chromatography on an agarose-ATP column greatly improves the dnaB protein isolation as will be described elsewhere. The dnaB complementing activity was eluted from agarose-ATP and subjected to NaDodSO4 gel electrophoresis (Fig. 2). The active fractions contained mainly four proteins with molecular weights of about 70,000, 60,000, 56,000, and 38,000 (Fig. 2). Upon glycerol gradient centrifugation of the agarose-ATP peak fraction, the activity sedimented with a velocity corresponding to a molecular weight of about 270,000, with catalase (molecular weight 250,000) as
Biochemistry: Lanka et al.
x
yj w
Proc. Natl. Acad. Sci. USA 75 (1978)
801
12 13 14 1516 17 x Y2 w 12 1314 15 16 17 Fraction number
FIG. 2. Purification of dnaB252 and Plban protein by agarose-ATP affinity chromatography. DEAE-cellulose fractions of (Left) dnaB252 and (Right) dnaB252(Plbac) (3.9 mg of protein each) were chromatographed on agarose-ATP as described in the legend to Table 2. ATPase (0) or dnaB complementing activity (N) was eluted with buffer D in 0.5-ml fractions. Aliquots (0.1 ml) were used for NaDodSO4 gel electrophoresis (4 V/cm). x, Loading material; w, wash with buffer C. Markers: fl-Galactosidase (d-GAL), bovine serum albumin (BSA), ovalbumin (OVA), and chymotrypsinogen A (CHYA). Agarose-ATP fractions of dnaB252(Plbac) (yr) and of supF dnaB266(Plbac) (Y2) were used as additional markers.
marker (Fig. 3). Only the proteins with molecular weights 60,000 and 56,000 cosedimented with the activity, as revealed by NaDodSO4 gel electrophoresis. The proteins with molecular weights 70,000 and 38,000 to a great extent sedimented faster (Fig. 3). The dnaB complementing activity was purified about 1600-fold (ammonium sulfate fraction - 1), with a 14% overall yield (Table 2). The specific activity is comparable to that of other dnaB ts protein containing activities. It is, however, significantly lower than the activity of wild-type dnaB protein (ref. 8; K. Ueda, R. McMacken, and A. Kornberg, personal communication; E. Lanka and H. Schuster, unpublished data). Purification of dnaB Protein from E. coli dnaB252. Cells of dnaB252 were grown at 25°. Preparation of the ammonium sulfate fraction and DEAE-cellulose chromatography were as described in the legend to Table 2. DEAE-cellulose fractions were tested for N-ethylmaleimide-resistant ATPase (8). A small peak of activity eluted beyond the bulk of other cellular ATPases at approximately the same position at which the dnaB complementing activity of dnaB252(Plbac) emerged in separate experiments. This ATPase activity was further purified by the procedures for dnaB252(Plbac) (see legends to Figs. 2 and 3). The active fractions eluting from agarose-ATP now contained only three proteins (Fig. 2). Their molecular weights were identical to those of three proteins of the corresponding fractions from dnaB252(Plbac); the protein with a molecular weight of 56,000 was missing. The ATPase activity was inac-
tivated by antibody to dnaB as shown below. Upon glycerol gradient centrifugation, the ATPase showed the same sedimentation velocity as the dnaB complementing activity of dnaB252(Plbac) (Fig. 3). Only the protein with molecular weight of 60,000 cosedimented with the ATPase activity, as revealed by NaDodSO4 gel electrophoresis (Fig. 3). Inactivation of ATPase and dnaB Complementing Activity by Antibody to dnaB Protein. Proteins of dnaB252, and of dnaB252(Plbac) were treated with antibody to dnaB (Table 3). dnaB complementing activity from the lysogen is inactivated by the antibody (Table 3). Likewise, the kX174 DNAdependent but not the DNA-independent ATPase activity of both the fractions from dnaB252 and from dnaB252(Plbac) is inactivated by the antibody. The DNA-independent constituent of the total ATPase activity always was relatively higher in the lysogen fractions than in the corresponding fractions from the nonlysogen (Table 3 and Fig. 3). DISCUSSION The dnaB complementing activity of dnaB252(Plbac), when purified to near homogeneity, has a native molecular weight of about 270,000 (Fig. 3) and contains two proteins of molecular weight 60,000 and 56,000 in a molar ratio of about 3:2, respectively (Figs. 2 and 3). Associated with this enzyme complex is an ATPase activity. This enzymatic function as well as the dnaB complementing activity are both inactivated by antibody
Biochemistry: Lanka et al.
802
Proc. Natl. Acad. Sci. USA 75 (1978)
1.5
Catalase
N
co
N C',
/3-Galactosidasei
0.2
r-
(U
C
1.0 >~
1-~~~~~~*-
a5
C.)
cU
cocu
C2 X' -'
0.
Marker
E) ALGAL BSA
OVA j
CHYA
Yi
6
10
14
18
6
10
14
18
Y2
Fraction number FIG. 3. Glycerol gradient centrifugation of dnaB252 and of dnaB252.Plban protein. Agarose-ATP peak fractions (0.2 ml each, Fig. 2) of (Left) dnaB252 (15 Ag of protein) and (Right) dnaB252(Plbac) (8 ,ug of protein) were applied to 4.8-ml 18-43% (wt/vol) glycerol gradients in buffer E. Centrifugation was at 45,000 rpm for 15 hr at 2° (SW65 Spinco rotor). Fractions were tested for dnaB complementation (-) and for 4X174 DNA-dependent (3) and -independent (a) ATPase activity. The number of fractions collected were 24 and 26 for dnaB252 and dnaB252(Plbac), respectively. Two subsequent fractions (0.1 ml each) were pooled and used for NaDodSO4 gel electrophoresis. For explanation of Y1, Y2, and other markers, see the legend to Fig. 2. In separate tubes fl-galactosidase and catalase were added as markers to the agarose-ATP peak fractions and centrifuged in parallel.
to dnaB (Table 3). On the other hand, an ATPase activity purified from dnaB252 has the same native molecular weight as that from dnaB252(Plbac) (Fig. 3) and is related to only one protein of molecular weight 60,000 (Figs. 2 and 3). Its specific activity is about 80% of that of the corresponding ATPase activity of the dnaB-Plban complex from dnaB252(Plbac) if agarose-ATP fractions are compared (Fig. 2). However, upon further purification, the dnaB252 protein loses its activity more
Table 3. Inactivation of dnaB complementation and ATPase activities by antibody directed against dnaB protein
dnaB252
+
dnaB ATPase, % complementation, % 100 (17.4) None 16.4 (16.9) None
dnaB252(Plbac)
+
100 (27.4) 19.9 (27.1)
dnaB
Protein
antibody
100 6.2
Agarose-ATP peak fractions (Fig. 3) of dnaB252 (0.23 zg of protein) and of dnaB252(Plbac) (0.20 ,g of protein) were incubated in a total volume of 30 p1 for 10 min at 00 in the presence or absence of dnaB antibody (about 50 ,Ag of gamma globulin protein). Aliquots were then tested for dnaB complementation and ATPase activity. Numbers in parentheses are ATPase activity without 4X174 DNA.
rapidly than the corresponding protein(s) from the Plbac ly(Fig. 3). The ATPase activity of dnaB252 can also be inactivated by antibody to dnaB (Table 3), indicating that it is indeed the dnaB protein. The protein is inactive in dnaB complementation (Fig. 2). For the dnaB protein from E. coli wild-type strains, molecular weights of about 250,000 for the native molecule and 55,000 for the polypeptide have been reported (refs. 9 and 17; K. Ueda, R. McMacken, and A. Kornberg, personal communication). A ribonucleoside triphosphatase activity was found to be associated with the dnaB protein (8). Our results are in agreement with these earlier findings. The dnaB complementing activity from another strain, E. coli supF dnaB266(Plbac) (4, 5), when purified to homogeneity, also contains two proteins. the molecular weight of one of these is also 56,000 (see y2 in Figs. 2 and 3). This protein is, however, absent in purified preparations of a P1 wild-type and of a Plbac ban lysogen (E. Lanka and H. Schuster, unpublished data). Therefore, the 56,000-dalton polypeptide is tentatively identified as Plban protein. It is assumed that the dnaB and Plban proteins are associated with each other in a heteromul-
sogen
timer.
Inactivation by antibody to dnaB of both the dnaB complementing and the 4X174 DNA-dependent ATPase activity may indicate that dnaB and Plban protein molecules, although
Biochemistry:
Lanka et al.
different in their molecular weights, are rather similar in their antigenic properties. However, binding of antibody molecules to only dnaB monomers in a heteromultimer may also strongly affect the functions of ban monomers in that complex. The ATPase activity of dnaB252(Plbac) differs from that of dnaB252 in its stimulation by kX174 DNA (Table 3 and Fig. 3). Similar differences were found between the ATPase activities from Plbac lysogens and their corresponding nonlysogens of two other E. coli strains (E. Lanka and H. Schuster, unpublished data), indicating an alteration of the dnaB ATPase by the ban protein or an association of an ATPase activity with PMban protein which is different from that of dnaB protein. Our studies in vitro suggest that suppression of the dnaB252 mutation by prophage PMbac is accomplished by an intimate association of dnaB and Plban molecules in a heteromultimer. However, since we do not know whether ban protein free of dnaB is able to initiate kX174 DNA complementary (-) strand synthesis, the assay used in our purification procedure may select against multimers predominantly or solely composed of monomeric ban molecules. The latter may be, however, the most effective in suppression in vivo. The dnaB252 protein, although inactive in the in vitro 4X174 DNA complementary (-) strand synthesis, retains its ATPase activity. Likewise, strain dnaB252, in contrast to elongation-defective dnaB mutants, retains the ability to complete at 420 all chromosomal DNA replication cycles already initiated at 300 (2, 3) and to replicate phage X DNA at 420 after host DNA synthesis has come to a halt (18, 19). These findings suggest that the dnaB ATPase is essential for E. coli DNA elongation and X DNA replication. It then implies that elongation-defective dnaB ts mutants should have a temperature-sensitive ATPase, whereas those exhibiting a considerable residual DNA synthesis at 42° (1) should retain ATPase activity but may be DNA initiation-defective. So far, only one elongation-defective dnaB mutant was tested, and the dnaB protein was found to be temperature sensitive in its ATPase and DNA initiation activity (unpublished results). The agarose-ATP affinity chromatography described above greatly improves the dnaB isolation procedure and facilitates the analysis of more dnaB mutants.
Proc. Natl. Acad. Sci. USA 75 (1978)
803
We thank D. Touati-Schwartz and A. Jaffe-Brachet for supplying uswith the P1 mutants. We thank A. Kornberg and R. McMacken for their generous gift of a dnaB antibody preparation, and D. W. Smith for making available for us his results prior to publication. We are also grateful to M. Mikolajczyk and M. Schlicht for expert technical assistance. We thank J. Womack for helpful advice in preparing the manuscript. 1. Wechsler, J. A. & Gross, J. D. (1971) Mol. Gen. Genet. 113, 273-284. 2. Beyersmann, D., Schlicht, M. & Schuster, H. (1971) Mol. Gen. Genet. 111, 145-158. 3. Zyskind, J. W. & Smith, D. W. (1977) J. Bacteriol. 129, 14761486. 4. D'Ari, R., Jaff-Brachet, A., Touati-Schwartz, D. & Yarmolinsky, M. B. (1975) J. Mol. Biol. 94, 341-366. 5. Ogawa, T. (1975) J. Mol. Biol. 94,327-340. 6. Schekman, R., Wickner, W., Westergaard, O., Brutlag, D., Geider, K., Bertsch, L. L. & Kornberg, A. (1972) Proc. Nati. Acad. Sci. USA 69,2691-2695. 7. Wickner, R. B., Wright, M., Wickner, S. & Hurwitz, J. (1972) Proc. Natl. Acad. Sci. USA 69,3233-3237. 8. Wickner, S., Wright, M. & Hurwitz, J. (1974) Proc. Natl. Acad. Sci. USA 71,783-787. 9. Wright, M., Wickner, S. & Hurwitz, J. (1973) Proc. Natl. Acad. Sci. USA 70,3120-3124. 10. Schuster, H., Mikolajczyk, M., Rohrschneider, J. & Geschke, B. (1975) Proc. Natl. Acad. Sci. USA 72,3907-3911. 11. Schuster, H., Schlicht, M., Lanka, E., Mikolajczyk, M. & Edelbluth, C. (1977) Mol. Gen. Genet. 151, 11-16. 12. Wechsler, J. A., Nuisslein, V., Otto, B., Klein, A., Bonhoeffer, F., Herrmann, R., Gloger, L. & Schaller, H. (1973) J. Bacteriol. 113, 1381-1388. 13. Bouche, J.-P., Zechel, K. & Kornberg, A. (1975) J. Biol. Chem. 250,5995-6001. 14. Laemmli, U. K. (1970) Nature 227,680-685. 15. Miller, G. L. (1959) Anal. Chem. 31, 964. 16. Weiner, J. H., McMacken, R. & Kornberg, A. (1976) Proc. Natl. Acad. Sci. USA 73,752-756. 17. Schekman, R., Weiner, J. H., Weiner, A. & Kornberg, A. (1975) J. Biol. Chem. 250,5859-5865. 18. Lanka, E. & Schuster, H. (1970) Mol. Gen. Genet. 106, 274285. 19. Gross, J. D. (1972) Current Topics in Microbiology and Immunology (Springer-Verlag, Berlin), Vol. 57, pp. 39-73.
Escherichia coli dnaB mutant defective in DNA initiation: Isolation and properties of the dnaB protein (OX174 DNA replication/ATPase/agarose-ATP/Plbac suppression/PI ban protein)
ERICH LANKA, BRIGITTE GESCHKE, AND HEINZ SCHUSTER Max-Planck-Institut fur Molekulare Genetik, Berlin-Dahlem, Germany
Communicated by Robert L. Sinsheimer, December 5, 1977
ABSTRACT Extracts of the DNA initiation-defective mutant Escherichia coli dnaB252 are inactive in a dnaB complementation assay but yield a ribonucleoside triphosphatase activity of native molecular weight of about 270,000 (60,000dalton polypeptide as subunit) that can be inactivated by antibody to dnaB. On the other hand, extracts of a dnaB252(PI bac) lysogen, in which the dnaB mutation is suppressed in vivo by the constitutive expression of the P1 dnaB analog (ban protein), are active in dnaB complementation and the activity is also sensitive to dnaB antibody. Upon further purification two proteins (with polypeptide molecular weights of 60,000 and 56,000, respectively) are found associated with each other (native molecular weight about 270,000). The larger and the smaller protein are tentatively identified as the dnaB and P1 ban protein. It is suggested that suppression of the dnaB mutation by prophage PI bac is accomplished by a stabilization of dnaB252 by P1 ban subunit molecules in a heteromultimer.
Escherichia coli dna ts mutants are usually divided into two classes, DNA initation- and DNA elongation-defective mutants. The majority of the existing E. coli dna ts mutants are dnaB mutants, which belong to the second class. In turn, most of the dnaB mutants show virtually immediate cessation of DNA synthesis at 420; some, however, exhibit considerable residual synthesis (1). The dna252 mutation was initially classified, phenotypically, as a DNA initation mutant (2); however, recent evidence indicates that this is a dnaB mutation that prevents the initiation and not the elongation of chromosomal replication
(3).
In the present communication it is shown that the dnaB252 mutation is suppressible by prophage Plbac in which the viral dnaB analog (ban) is expressed constitutively (4, 5). By use of a dnaB complementation assay (6, 7), an activity is purified from the E. coli dnaB252(Plbac) lysogen that contains two proteins associated with each other. These proteins are tentatively identified as dnaB and Plban proteins. An extract of E. coli dnaB252 is inactive in a dnaB complementation assay (S. Wickner, cited in ref 3). However, a ribonucleoside triphosphatase activity, sensitive to antibody against dnaB, can be purified from this mutant. Such an activity has been found associated with purified dnaB protein from E. coli wild-type cells (8, 9). These findings are discussed with regard to the functional roles of Plban and dnaB protein in vivo.
Bacteria and Phage. Bacterial strains were E. coli dnaB252 (2, 3), a temperature-resistant revertant selected on tryptonesupplemented agar plates at 420 (this laboratory), BT1000, and BT1071 dnaB (12). Phages were PlCml, PlCmlbac-l, and PlCml Plbac-1 ban-i (4) (kindly provided by A. Jaffe-Brachet and D. Touati-Schwartz) and are abbreviated P1, Plbac, and Plbac ban for convenience. P1 lysogens of E. coli dnaB252 were prepared in this laboratory. Buffers. Buffer A: 20 mM Tris-HCl (pH 7.6)/50 mM NaCl/10 mM MgCl2/0. 1 mM EDTA/0. 1 mM dithiothreitol/ 0.1 mM ATP/12% (wt/vol) glycerol. Buffers B-E: buffer A with the following substitutions (in parentheses): B (1 mM MgCI2), C (MgCI2 and ATP omitted), D (5 mM MgCl2 and 5 mM ATP), and E (1 mM ATP, glycerol omitted). Assays. DNA synthesis with ammonium sulfate fractions was measured as described (10). For the dnaB complementation assay, the ammonium sulfate fraction of the E. coli BT1071 dnaB receptor was prepared as described for strain BC1304 (11). Receptor fractions (about 10 mg of protein per ml) were heated for 2.5 min at 370 in order to inactivate any residual DNA-synthesizing activity; 80-140 ,Ag of receptor protein were used per assay. DNA synthesis was followed for 30 min at 250 as described (10, 11), with 320 pmol (nucleotides) of pX174 DNA. One unit of dnaB complementing activity incorporates 1 nmol of dTMP under the above conditions. For the assay for DNA-dependent ATPase conditions were as described (8) except that 1.5 nmol of qX174 DNA was used and the reaction was terminated after 30 min at 250. One unit of ATPase catalyzed the production of 1 ,umol of 32P, under the above condi-
tions. Protein Fractions. dnaB complementing activity was purified from E. coli dnaB252(Plbac) as described in the legend to Table 2. A DEAE-cellulose fraction from E. coli BT1000 dnaB + (12) and an agarose-ATP fraction from E. coli supF dnaB266(Plbac) (4, 5) were prepared in the same manner. A detailed description will be published elsewhere. Other Methods. Growth and lysis of cells were as described
(11, 13). Sodium dodecyl sulfate (NaDodSO4) gel electrophoresis on 15% (wt/vol) polyacrylamide gel was performed as described by Laemmli (14). Protein was determined by the method of Miller (15) and on NaDodSO4 gels by scanning with an Ortec 4310 densitometer, with bovine serum albumin as standard.
MATERIALS AND METHODS Materials. Agarose-ATP, Type 4 was from PL-biochemicals; [y-32P]ATP, 2 Ci/mmol was from The Radiochemical Centre, Amersham. Other chemicals were as described (10, 11). Antibody to E. coli dnaB (13.5 mg of gamma globulin protein per ml) was a gift of A. Kornberg and R. McMacken.
RESULTS of coli E. Suppression dnaB252 by Prophage P1 bac. Strain dnaB252 was lysogenized with P1, PMbac, or PMbac ban and the colony-forming ability was determined at 300 and 420. Table 1 shows that the dnaB mutation was suppressed only in the PMbac lysogen, but not in the P1 wild-type lysogen [in which
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Abbreviation: NaDodSO4, sodium dodecyl sulfate. 799
800
Proc. Natl. Acad. Sci. USA 75 (1978)
Biochemistry: Lanka et al.
Table 1. Suppression of E. coli dnaB252 by prophage Plbac
Plating efficiency at 420 relative to 300
dnaB252 strain
Nonlysogen
4.5 X 10-5
Lysogens P1 Plbac Plbac ban
1.4 X l0-5 1.1 1.0 X 10-4
Revertant
0.8
Fraction Extract Ammonium sulfate DEAE-cellulose Agarose-ATPt Glycerol gradientt
Bacteria were grown in TY medium (10) at 30°. During logarithmic growth phase, appropriate dilutions were plated on TY plates (10 g of Bacto Tryptone, 5 g of yeast extract, 10 g of Bacto Agar, and 5 g of NaCl per liter). For the growth of P1 lysogens, medium and plates contained 25 Aig of chloramphenicol per ml.
synthesis of Plban protein is suppressed (4, 5)] or in the Plbac ban lysogen [in which ban protein is defective (4)]. This indicates that the PIban protein is able to assist or substitute for the dnaB protein during E. col DNA initiation, as it does during DNA elongation (4, 5). An attempt was made to imitate, in vitro, the in vivo suppressing action of Plban protein as described earlier (10). Ammonium sulfate fractions were prepared from different strains as described in the legend to Table 2 and tested for their ability to catalyze the conversion of OX174 DNA to its duplex form. DNA synthesis was observed only with fractions from dnaB252(Plbac) and from the revertant strain (Fig. 1). Extracts of dnaB252 were inactive at all temperatures tested (Fig. 1), 60
40
0.
0
2Cu 0
0. C
:'. ~~~Revertant
20
dnaB 252
25
35 30 Temperature, 0C
40
Total Total units protein, mg
Specific activity, units/mg
Yield, %
13,200 1,124 20 20 0.21 0.096
0.73 14 4.9 830 1,180
100 34 12 21 14
-
820 280 98* 175 113
Frozen cells (240 g suspended in 240 ml) were lysed to yield 470 ml of extract (28 mg of protein per ml). All the following operations were done at 4°. Streptomycin sulfate (20%, 118 ml) was added (4% final concentration); the mixture was stirred for 45 min and centrifuged for 45 min at 30,000 rpm. Ammonium sulfate (0.226 g/ml) was added to the supernatant, which was stirred for 30 min and centrifuged for 20 min at 10,000 rpm. The pellet was washed with 50 ml of 50% saturated ammonium sulfate and dissolved in buffer A. After dialysis against buffer A, this fraction (56 ml, 20 mg of protein per ml) was applied to a DEAE-cellulose column (3 X 43 cm). The column was developed with a 3000-ml linear gradient, 0.05-0.6 M NaCl. The dnaB complementing activity eluted at 0.38 M NaCl. After ammonium sulfate precipitation [0.368 mg of (NH4)2SO4/ml, the pellet was dissolved in buffer B and dialyzed against the same buffer. An 0.85-ml aliquot of this fraction (4.3 ml, 4.6mg of protein per ml) was diluted to 5 ml with buffer C and applied to an agarose-ATP column (0.5 X 5 cm). The dnaB complementing activity was eluted with buffer D. An aliquot of the peak fraction was used for glycerol gradient centrifugation as described in the legend to Fig. 3. * Activity remaining after freezing and thawing once. t These steps were performed with only a part of the fraction from the preceding purification step. The values reported assume that the yield and purification would be the same if the entire DEAE-cellulose and agarose-ATP fractions were subjected to the following procedure(s).
as was reported previously (S. Wickner, cited in ref. 3). Like-
dnaB 252(Plbac)
E
Table 2. Purification of dnaB complementing activity from E. coli dnaB252(Plbac)
42
FIG. 1. DNA synthesis with ammonium sulfate fractions. Ammonium sulfate fractions were prepared as described in the legend of Table 2. DNA was synthesized for 30 min at the indicated temperatures (as described in ref. 10) with 90, 120, or 120 'sg of dnaB252(Plbac), dnaB252, or revertant strain protein, respectively. Supplemented DNA synthesis: 120 Atg of protein of dnaB252 (E) or 120 ,ug of protein of the revertant (-), each supplemented with 2.4 jig of protein of BT1000 (DEAE-cellulose fraction).
wise, extracts of dnaB(Plbac ban) were also inactive (data not shown). Fractions from dnaB252 regained activity when supplemented with dnaB protein from BT1000 wild-type cells (Fig. 1), indicating that the failure to synthesize DNA was due to a deficiency in functionally active dnaB protein. Since the latter protein participates in the initiation of 4X174 DNA complementary (-) strand synthesis (16), the Plban protein can also assist in this reaction. These findings prompted us to purify a corresponding activity from the dnaB252(Plbac) lysogen by using a dnaB complementation assay (6, 7). Purification of a dnaB Complementing Activity from E. coli dnaB252(Plbac). As described above, only fractions from dnaB252(Plbac) but not those from the nonlysogen are active in DNA synthesis. This implies that any dnaB complementing activity purified from the lysogen must contain Plban protein (unless a more complicated mode of suppression by prophage Plac is assumed). Cells of dnaB252(Plbac) were grown at 400 to assure that suppression was effective in vivo. The dnaB complementing activity was purified as described in the legend to Table 2. The use of affinity chromatography on an agarose-ATP column greatly improves the dnaB protein isolation as will be described elsewhere. The dnaB complementing activity was eluted from agarose-ATP and subjected to NaDodSO4 gel electrophoresis (Fig. 2). The active fractions contained mainly four proteins with molecular weights of about 70,000, 60,000, 56,000, and 38,000 (Fig. 2). Upon glycerol gradient centrifugation of the agarose-ATP peak fraction, the activity sedimented with a velocity corresponding to a molecular weight of about 270,000, with catalase (molecular weight 250,000) as
Biochemistry: Lanka et al.
x
yj w
Proc. Natl. Acad. Sci. USA 75 (1978)
801
12 13 14 1516 17 x Y2 w 12 1314 15 16 17 Fraction number
FIG. 2. Purification of dnaB252 and Plban protein by agarose-ATP affinity chromatography. DEAE-cellulose fractions of (Left) dnaB252 and (Right) dnaB252(Plbac) (3.9 mg of protein each) were chromatographed on agarose-ATP as described in the legend to Table 2. ATPase (0) or dnaB complementing activity (N) was eluted with buffer D in 0.5-ml fractions. Aliquots (0.1 ml) were used for NaDodSO4 gel electrophoresis (4 V/cm). x, Loading material; w, wash with buffer C. Markers: fl-Galactosidase (d-GAL), bovine serum albumin (BSA), ovalbumin (OVA), and chymotrypsinogen A (CHYA). Agarose-ATP fractions of dnaB252(Plbac) (yr) and of supF dnaB266(Plbac) (Y2) were used as additional markers.
marker (Fig. 3). Only the proteins with molecular weights 60,000 and 56,000 cosedimented with the activity, as revealed by NaDodSO4 gel electrophoresis. The proteins with molecular weights 70,000 and 38,000 to a great extent sedimented faster (Fig. 3). The dnaB complementing activity was purified about 1600-fold (ammonium sulfate fraction - 1), with a 14% overall yield (Table 2). The specific activity is comparable to that of other dnaB ts protein containing activities. It is, however, significantly lower than the activity of wild-type dnaB protein (ref. 8; K. Ueda, R. McMacken, and A. Kornberg, personal communication; E. Lanka and H. Schuster, unpublished data). Purification of dnaB Protein from E. coli dnaB252. Cells of dnaB252 were grown at 25°. Preparation of the ammonium sulfate fraction and DEAE-cellulose chromatography were as described in the legend to Table 2. DEAE-cellulose fractions were tested for N-ethylmaleimide-resistant ATPase (8). A small peak of activity eluted beyond the bulk of other cellular ATPases at approximately the same position at which the dnaB complementing activity of dnaB252(Plbac) emerged in separate experiments. This ATPase activity was further purified by the procedures for dnaB252(Plbac) (see legends to Figs. 2 and 3). The active fractions eluting from agarose-ATP now contained only three proteins (Fig. 2). Their molecular weights were identical to those of three proteins of the corresponding fractions from dnaB252(Plbac); the protein with a molecular weight of 56,000 was missing. The ATPase activity was inac-
tivated by antibody to dnaB as shown below. Upon glycerol gradient centrifugation, the ATPase showed the same sedimentation velocity as the dnaB complementing activity of dnaB252(Plbac) (Fig. 3). Only the protein with molecular weight of 60,000 cosedimented with the ATPase activity, as revealed by NaDodSO4 gel electrophoresis (Fig. 3). Inactivation of ATPase and dnaB Complementing Activity by Antibody to dnaB Protein. Proteins of dnaB252, and of dnaB252(Plbac) were treated with antibody to dnaB (Table 3). dnaB complementing activity from the lysogen is inactivated by the antibody (Table 3). Likewise, the kX174 DNAdependent but not the DNA-independent ATPase activity of both the fractions from dnaB252 and from dnaB252(Plbac) is inactivated by the antibody. The DNA-independent constituent of the total ATPase activity always was relatively higher in the lysogen fractions than in the corresponding fractions from the nonlysogen (Table 3 and Fig. 3). DISCUSSION The dnaB complementing activity of dnaB252(Plbac), when purified to near homogeneity, has a native molecular weight of about 270,000 (Fig. 3) and contains two proteins of molecular weight 60,000 and 56,000 in a molar ratio of about 3:2, respectively (Figs. 2 and 3). Associated with this enzyme complex is an ATPase activity. This enzymatic function as well as the dnaB complementing activity are both inactivated by antibody
Biochemistry: Lanka et al.
802
Proc. Natl. Acad. Sci. USA 75 (1978)
1.5
Catalase
N
co
N C',
/3-Galactosidasei
0.2
r-
(U
C
1.0 >~
1-~~~~~~*-
a5
C.)
cU
cocu
C2 X' -'
0.
Marker
E) ALGAL BSA
OVA j
CHYA
Yi
6
10
14
18
6
10
14
18
Y2
Fraction number FIG. 3. Glycerol gradient centrifugation of dnaB252 and of dnaB252.Plban protein. Agarose-ATP peak fractions (0.2 ml each, Fig. 2) of (Left) dnaB252 (15 Ag of protein) and (Right) dnaB252(Plbac) (8 ,ug of protein) were applied to 4.8-ml 18-43% (wt/vol) glycerol gradients in buffer E. Centrifugation was at 45,000 rpm for 15 hr at 2° (SW65 Spinco rotor). Fractions were tested for dnaB complementation (-) and for 4X174 DNA-dependent (3) and -independent (a) ATPase activity. The number of fractions collected were 24 and 26 for dnaB252 and dnaB252(Plbac), respectively. Two subsequent fractions (0.1 ml each) were pooled and used for NaDodSO4 gel electrophoresis. For explanation of Y1, Y2, and other markers, see the legend to Fig. 2. In separate tubes fl-galactosidase and catalase were added as markers to the agarose-ATP peak fractions and centrifuged in parallel.
to dnaB (Table 3). On the other hand, an ATPase activity purified from dnaB252 has the same native molecular weight as that from dnaB252(Plbac) (Fig. 3) and is related to only one protein of molecular weight 60,000 (Figs. 2 and 3). Its specific activity is about 80% of that of the corresponding ATPase activity of the dnaB-Plban complex from dnaB252(Plbac) if agarose-ATP fractions are compared (Fig. 2). However, upon further purification, the dnaB252 protein loses its activity more
Table 3. Inactivation of dnaB complementation and ATPase activities by antibody directed against dnaB protein
dnaB252
+
dnaB ATPase, % complementation, % 100 (17.4) None 16.4 (16.9) None
dnaB252(Plbac)
+
100 (27.4) 19.9 (27.1)
dnaB
Protein
antibody
100 6.2
Agarose-ATP peak fractions (Fig. 3) of dnaB252 (0.23 zg of protein) and of dnaB252(Plbac) (0.20 ,g of protein) were incubated in a total volume of 30 p1 for 10 min at 00 in the presence or absence of dnaB antibody (about 50 ,Ag of gamma globulin protein). Aliquots were then tested for dnaB complementation and ATPase activity. Numbers in parentheses are ATPase activity without 4X174 DNA.
rapidly than the corresponding protein(s) from the Plbac ly(Fig. 3). The ATPase activity of dnaB252 can also be inactivated by antibody to dnaB (Table 3), indicating that it is indeed the dnaB protein. The protein is inactive in dnaB complementation (Fig. 2). For the dnaB protein from E. coli wild-type strains, molecular weights of about 250,000 for the native molecule and 55,000 for the polypeptide have been reported (refs. 9 and 17; K. Ueda, R. McMacken, and A. Kornberg, personal communication). A ribonucleoside triphosphatase activity was found to be associated with the dnaB protein (8). Our results are in agreement with these earlier findings. The dnaB complementing activity from another strain, E. coli supF dnaB266(Plbac) (4, 5), when purified to homogeneity, also contains two proteins. the molecular weight of one of these is also 56,000 (see y2 in Figs. 2 and 3). This protein is, however, absent in purified preparations of a P1 wild-type and of a Plbac ban lysogen (E. Lanka and H. Schuster, unpublished data). Therefore, the 56,000-dalton polypeptide is tentatively identified as Plban protein. It is assumed that the dnaB and Plban proteins are associated with each other in a heteromul-
sogen
timer.
Inactivation by antibody to dnaB of both the dnaB complementing and the 4X174 DNA-dependent ATPase activity may indicate that dnaB and Plban protein molecules, although
Biochemistry:
Lanka et al.
different in their molecular weights, are rather similar in their antigenic properties. However, binding of antibody molecules to only dnaB monomers in a heteromultimer may also strongly affect the functions of ban monomers in that complex. The ATPase activity of dnaB252(Plbac) differs from that of dnaB252 in its stimulation by kX174 DNA (Table 3 and Fig. 3). Similar differences were found between the ATPase activities from Plbac lysogens and their corresponding nonlysogens of two other E. coli strains (E. Lanka and H. Schuster, unpublished data), indicating an alteration of the dnaB ATPase by the ban protein or an association of an ATPase activity with PMban protein which is different from that of dnaB protein. Our studies in vitro suggest that suppression of the dnaB252 mutation by prophage PMbac is accomplished by an intimate association of dnaB and Plban molecules in a heteromultimer. However, since we do not know whether ban protein free of dnaB is able to initiate kX174 DNA complementary (-) strand synthesis, the assay used in our purification procedure may select against multimers predominantly or solely composed of monomeric ban molecules. The latter may be, however, the most effective in suppression in vivo. The dnaB252 protein, although inactive in the in vitro 4X174 DNA complementary (-) strand synthesis, retains its ATPase activity. Likewise, strain dnaB252, in contrast to elongation-defective dnaB mutants, retains the ability to complete at 420 all chromosomal DNA replication cycles already initiated at 300 (2, 3) and to replicate phage X DNA at 420 after host DNA synthesis has come to a halt (18, 19). These findings suggest that the dnaB ATPase is essential for E. coli DNA elongation and X DNA replication. It then implies that elongation-defective dnaB ts mutants should have a temperature-sensitive ATPase, whereas those exhibiting a considerable residual DNA synthesis at 42° (1) should retain ATPase activity but may be DNA initiation-defective. So far, only one elongation-defective dnaB mutant was tested, and the dnaB protein was found to be temperature sensitive in its ATPase and DNA initiation activity (unpublished results). The agarose-ATP affinity chromatography described above greatly improves the dnaB isolation procedure and facilitates the analysis of more dnaB mutants.
Proc. Natl. Acad. Sci. USA 75 (1978)
803
We thank D. Touati-Schwartz and A. Jaffe-Brachet for supplying uswith the P1 mutants. We thank A. Kornberg and R. McMacken for their generous gift of a dnaB antibody preparation, and D. W. Smith for making available for us his results prior to publication. We are also grateful to M. Mikolajczyk and M. Schlicht for expert technical assistance. We thank J. Womack for helpful advice in preparing the manuscript. 1. Wechsler, J. A. & Gross, J. D. (1971) Mol. Gen. Genet. 113, 273-284. 2. Beyersmann, D., Schlicht, M. & Schuster, H. (1971) Mol. Gen. Genet. 111, 145-158. 3. Zyskind, J. W. & Smith, D. W. (1977) J. Bacteriol. 129, 14761486. 4. D'Ari, R., Jaff-Brachet, A., Touati-Schwartz, D. & Yarmolinsky, M. B. (1975) J. Mol. Biol. 94, 341-366. 5. Ogawa, T. (1975) J. Mol. Biol. 94,327-340. 6. Schekman, R., Wickner, W., Westergaard, O., Brutlag, D., Geider, K., Bertsch, L. L. & Kornberg, A. (1972) Proc. Nati. Acad. Sci. USA 69,2691-2695. 7. Wickner, R. B., Wright, M., Wickner, S. & Hurwitz, J. (1972) Proc. Natl. Acad. Sci. USA 69,3233-3237. 8. Wickner, S., Wright, M. & Hurwitz, J. (1974) Proc. Natl. Acad. Sci. USA 71,783-787. 9. Wright, M., Wickner, S. & Hurwitz, J. (1973) Proc. Natl. Acad. Sci. USA 70,3120-3124. 10. Schuster, H., Mikolajczyk, M., Rohrschneider, J. & Geschke, B. (1975) Proc. Natl. Acad. Sci. USA 72,3907-3911. 11. Schuster, H., Schlicht, M., Lanka, E., Mikolajczyk, M. & Edelbluth, C. (1977) Mol. Gen. Genet. 151, 11-16. 12. Wechsler, J. A., Nuisslein, V., Otto, B., Klein, A., Bonhoeffer, F., Herrmann, R., Gloger, L. & Schaller, H. (1973) J. Bacteriol. 113, 1381-1388. 13. Bouche, J.-P., Zechel, K. & Kornberg, A. (1975) J. Biol. Chem. 250,5995-6001. 14. Laemmli, U. K. (1970) Nature 227,680-685. 15. Miller, G. L. (1959) Anal. Chem. 31, 964. 16. Weiner, J. H., McMacken, R. & Kornberg, A. (1976) Proc. Natl. Acad. Sci. USA 73,752-756. 17. Schekman, R., Weiner, J. H., Weiner, A. & Kornberg, A. (1975) J. Biol. Chem. 250,5859-5865. 18. Lanka, E. & Schuster, H. (1970) Mol. Gen. Genet. 106, 274285. 19. Gross, J. D. (1972) Current Topics in Microbiology and Immunology (Springer-Verlag, Berlin), Vol. 57, pp. 39-73.
