Tue JOURNAL oF BioLocicaL CHEMISTRY
Vol. 253, No. 1, Issue of January 10, pp. 261-269, 1978
Printed in U.S.A.

dnaB Protein of Escherichia coli

PURIFICATION AND ROLE IN THE REPLICATION OF $X174 DNA*

(Received for publication, July 20, 1977)

Kuniuiro Uspa,t Roger McMacken,§ anp ArTtHUR KORNBERG

From the Department of Biochemistry, Stanford University School of Medicine, Stanford, California

94305

The dnaB gene product of Escherichia coli has been
purified about 15,000-fold to homogeneity, in 4 to 8% yield,
from wild type cells and from cells which overproduce dnaB
protein 5-fold; the latter cells harbor a ColE1 plasmid
carrying the dnaB gene. The protein is an oligomer of
55,000-dalton subunits; a native molecular weight of 250,000
was estimated from sedimentation in a glycerol gradient.
About 20 such molecules are calculated to be present per E.
coli cell.

Assay for dnaB protein is based on an absolute require-
ment for it, along with 12 other proteins, to reconstitute in
vitro replication of phage @X174 single-stranded DNA to a
duplex replicative form. The inference that dnaB protein is
a constituent of the nucleoprotein intermediate which pre-
cedes dnaG protein (primase) participation in @X174 DNA
replication (Weiner, J. H., McMacken, R., and Kornberg,
A. (1976) Proc. Natl. Acad. Sci. U. S. A. 78, 752-756) was
strengthened by the observation that labeled dnaB protein
is incorporated in the complex and that anti-dnaB antibody
destroys the replicative activity of this intermediate. This
antibody inhibits primer RNA synthesis by primase by
preventing formation of the replication intermediate and
by interfering with its action once formed. It does not,
however, affect the subsequent elongation by DNA polym-
erase III holoenzyme. Anti-dnaB antibody inhibits semicon-
servative E. coli DNA replication in cell lysates; the inhibi-
tion is reversed by dnaB protein.

 

The dnaB gene product of Escherichia coli is essential for
replication of the bacterial chromosome (1-5). It participates
in chromosome growth as shown by immediate cessation of
DNA synthesis which occurs when temperature-sensitive mu-
tants are shifted to a restrictive temperature (5).

* This work was supported in part by grants from the National
Institutes of Health (GM 07581-15), the National Science Foundation
(BMS 06835-A02) (A. K.), and an Eleanor Roosevelt International
Cancer Fellowship (K. U.). The costs of publication of this article
were defrayed in part by the payment of page charges. This article
must therefore by hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.

+ Present address, Department of Medical Chemistry, Kyoto Uni-
versity Faculty of Medicine, Yoshida, Sakyo-Ku, Kyoto 606, Japan.

§ Present address, Department of Biochemistry, School of Hy-
giene and Public Health, The Johns Hopkins University, 615 N.
Wolfe St., Baltimore, Md. 21205.

dnaB protein has also been shown to be necessary for
conversion of phage #X single-stranded DNA! to the duplex
replicative form (RF) (6-11) and to be required for the multi-
plication of RF at the stage of (—)-strand synthesis (12). Two
other small coliphages with SS genomes, M13 and G4, how-
ever, do not require this gene function for their conversion to
RF (6, 7, 10, 13). Hurwitz and associates purified dnaB protein
(14) and found the native molecular weight to be about
250,000. Lark and Wechsler (15) and Kogoma (16) have ana-
lyzed DNA replication in various dnaB mutants in vivo, and
pointed out the likelihood of interactions among dnaB protein
subunits and the possibility of multiple forms of dnaB protein
in cells.

dnaB protein has nucleoside triphosphatase activity and
forms a complex with dnaC protein in the presence of ATP
(14, 17). Recently, the #X SS replicative system has been suc-
cessfully reconstituted (10, 11, 18) from partially purified pro-
teins. dnaB protein is involved together with four other
proteins (DBP, dnaC protein, proteins i and n) in the forma-
tion of a replication intermediate which precedes primase
function (18, 19). Kinetic analysis has suggested that dnaB
protein participates in the intermediate formation as a stoichi-
ometric constituent of the complex rather than as a catalyst
in its formation (19), but the role of this intermediate in
events leading to chain elongation was not understood.

To study the role of dnaB protein in DNA replication, we
have undertaken extensive purification of the protein and an
examination of its properties. We report here the large scale
purification of dnaB protein and some of its physical and
functional properties, particularly in phage #X DNA replica-
tion.

MATERIALS AND METHODS

Chemicals and Enzymes

DEAE-cellulose (DE-52) and phosphocellulose (P-11) were pur-
chased from Whatman; hydroxyapatite from Clarkson Chemical Co.
(Williamsport, Pa.); Bio-Gel A-1.5m and A-5m (both 200 to 400
mesh) from Bio-Rad; [methylH]dTTP, [5,6-“HJUTP, [a-*P]dGTP,
(**P]orthophosphoric acid, NaB*H, from New England Nuclear;
Freund’s complete adjuvant from Difco Laboratories. [y-*P]ATP

 

1The abbreviations used are: #X, ¢X174; SS, single-stranded;
RF, replicative form; SDS, sodium dodecy! sulfate; albumin, bovine
serum albumin; DBP, DNA-binding protein; holoenzyme, DNA
polymerase III holoenzyme; primase, dnaG protein; NTP, nucleoside
triphosphate.

261
262

was prepared from [**Plorthophosphate. Polypeptide standards (and
their molecular weights) (20) were: egg white lysozyme (14,300)
from Calbiochem; chymotrypsinogen A (25,700) and ovalbumin
(43,000) from Sigma; human hemoglobin (63,000), prepared as de-
scribed previously (21); albumin (68,000) from Pentex; beef liver
catalase (244,000) and Escherichia coli B-galactosidase (540,000)
from Worthington. [(**P]¢X174 am3 phage was a gift of Dr. B. Tye of
this department. Other reagents were from sources previously
described (10, 11, 21).

Buffers

Buffer A is 50 mm Tris-Cl (pH 7.5), 20% (v/v) glycerol, 1 mm
EDTA. NaCl was added as specified. Buffer B is 20 mm potassium
phosphate (pH 6.5), 20% (v/v) glycerol, 1 mm EDTA. KCl was added
as specified. Assay buffer contained 50 mm Tris-Cl (pH 7.5), 10%
sucrose, 20 mm dithiothreitol, and 0.2 mg/ml of albumin.

Bacterial Strains and Growth

E. coli HMS83 (polAl1, polB1, thy, lys), a strain of K12 constructed
by Campbell et al. (22), was used for the main preparation of dnaB
protein. H560 (F+, polAl, endA), and BT1029 (dnaB,,, polAl, endA,
thy) (originally isolated by F. Bonhoeffer and co-workers) were
provided by Dr. Y. Hirota (National Institute of Genetics, Japan).
RLM365 (pLCll-9) is a spontaneous polA* derivative of HMS83
which contains a ColE1 hybrid plasmid (pLCII-9) originally obtained
from clone JA200 (pLCll-9) of the Clarke and Carbon colony bank
(23); the plasmid contains the chromosome of colicinogenic factor E1
and a fragment of the EZ. coli chromosome including the dnaB gene
region and was identified (23) by screening the Clarke and Carbon
colony bank for those clones which could complement thermosensi-
tive dnaB mutants following conjugation.

E. coli HMS83 and RLM365 (pLCll-9) were grown in AZ broth
(which contains per liter: 10 g of K,HPO,, 1.85 g of KH,PQ,, 10 g of
Ardamine Z yeast extract (Yeast Products Inc., Clifton, N.J.), 10 g
of glucose, 50 mg of thymine, 10 mg of vitamin B,) to Asoo = 8 (3/s
log phase) in a Fermacell Fermentor (New Brunswick Scientific
Co.) at 37° with strong aeration. The pH was maintained between
7.0 and 7.2 by addition of NaOH. Cells were harvested in a Sharples
continuous flow centrifuge at 25°, suspended at Asgy = 200 (8 x 10°
cells/ml) in 10% sucrose, 50 mm Tris-Cl (pH 7.5), and frozen in
liquid N,. Cells stored at —20° retained full dnaB activity for at
least 6 months. H560 and BT1029 were grown similarly except that
Hershey broth (7) supplemented with thymine was used and the
harvest was at Aggy = 0.5.

Preparation of Replication Proteins and DNA

Proteins necessary for ¢X SS to RF replication, assayed as
previously described (11), and partially purified from E. coli HMS83
were: DBP (Fraction 3b, 1.63 mg/ml, 28,000 units/mg) (24); protein
n (Fraction IV, 32,000 units/ml, 3,800 units/mg) (11); protein i
(Fraction V, 80,000 units/ml, 210,000 units/mg); protein u (25)
(Fraction V, 18,000 units/ml, 75,000 units/mg); dnaC protein (Frac-
tion V, 10,000 units/ml, 67,000 units/mg); dnaG protein (Fraction V,
50,000 units/ml, 200,000 units/mg); holoenzyme (26) (Fraction IV,
50,000 units/ml, 20,000 units/mg). Procedures for purification of
these proteins will be published elaewhere. DNA-cellulose-binding
fraction was prepared as previously described (11).

Soluble extract of BT1029 was prepared as described for Fraction
I from HMS83 (see “Results”) except that heating at 37° was for 1.5
min instead of 4 min and centrifugation was for 30 min at 53,000 x
g rather than at 32,000 x g.

@X SS was prepared by the method of Ray (21, 27).

Preparation of Antibodies

Antibody against protein i was prepared as previously described
(19). Antibody against dnaB protein was prepared essentially in the
same way by an initial injection of 155 wg of purified dnaB protein
(Fraction IV, 900,000 units/mg) followed by a booster (75 ug) 3
weeks later. Blood was collected 8 days after the booster, and the y-
globulin was purified to homogeneity (28). Control y-globulin was
prepared from serum of unimmunized rabbits.

Assays of dnaB Protein

dnaB protein activity was assayed by measuring conversion of
@X SS to RF in cooperation with other E. coli proteins. Three
different systems were used.

dnaB Gene Product of E. coli

Assay A-—The partial reconstitution assay identical with the
“Stage III’ system of Schekman ef al. (11) consisted of 10 to 15 pl of
assay buffer, DNA-cellulose-binding fraction (15g of protein), pro-
tein i (0.06 yg), dnaC protein (0.3 ug), a dnaB sample, and a
mixture containing 100 nmol of spermidine-Cl, 20 nmol of ATP, 2.5
nmol each of GTP, CTP, and UTP, 0.3 nmol (as nucleotide) of X
DNA (SS), 0.1 yg of rifampicin, 120 nmol of MgCl, 0.45 nmol of
(8H]dTTP (350 Ci/mol), and 1.2 nmol each of dATP, dGTP, and
dCTP (total volume, 25 yl). The components, mixed at 0° in this
order, were incubated for 10 min at 30°. Incubation was terminated
by chilling the mixture on ice and by adding about 40 pl of 0.2 m
sodium pyrophosphate and 0.5 ml of 10% trichloroacetic acid. The
resulting precipitate was collected on glass fiber filters (Whatman
GF/C), washed three times with 3 ml of 1 nN HCl, 0.1 m sodium
pyrophosphate and once with 3 ml of ethanol, dried, and counted in
a toluene-based scintillation fluid in a Nuclear Chicago scintillation
spectrometer.

Assay B—The total reconstitution assay was identical except that
(a) DNA-cellulose-binding fraction was replaced by five purified
proteins (DBP (4.9 yg), protein n (4.2 xg), dnaG protein (0.5 ug),
holoenzyme (1.3 wg), and protein u (2.0 ug)), (6) ¢X SS was first
mixed with DBP, and (c) ATP concentration was lowered (6.7 nmol/
25 wd.

Assay C— The complementation assay was performed by incubat-
ing a soluble extract (Fraction I, preheated at 37° for 10 min) of
BT1029 with the dnaB sample, #X SS and the low molecular weight
components of Assay A at 30°.

In all assays, one unit is defined as incorporation of 1 pmol of
total deoxynucleotide into an acid-insoluble form in 1 min; the
value for dTTP incorporation was multiplied by 4.

Two-stage DNA Replication

This was carried out as before (19). In the first stage (replication
intermediate formation), the reaction mixture (incubated 20 min at
30°) contained dnaB protein, dnaC protein, DBP, protein i, protein
n, protein u, @X SS, spermidine, ATP, MgCl, and assay buffer in
the same amounts as in Assay B, but in a total volume of 15 wl. The
second stage (primer synthesis and DNA elongation) was carried
out at 30° for 3 min after addition of anti-protein i y-globulin (8 yg),
and the amounts of primase, GTP, CTP, UTP, rifampicin, {SH]dTTP,
dATP, dGTP, dCTP, and holoenzyme used in Assay B.

Three-stage DNA Replication

This consisted of a first stage as above (incubation time 10 min)
and a second stage (primer synthesis) performed by adding primase,
the three rNTPs and rifampicin, as above, and incubating for 10
min at 30°. The third stage (DNA elongation) (3 min at 30°) was
performed by adding the remaining components of the two-stage
replication (see above).

E. coli Chromosome Replication on Cellophane Discs

The procedure (29, 30) consisted of prelabeling H560 cells with
(“C]thymidine (1.8 Ci/mol) for 1 h at 37°, washing, and resuspending
the cells in nonradioactive medium, and spreading and lysing the
cells with lysozyme and Brij 58 on cellophane discs placed on agar.
The lysate was incubated for 30 min at 30° in a DNA synthesis
mixture which included ("H]thymidine (315 Ci/mol). The amount of
DNA synthesis was corrected for variation in the number of cells on
a disc by the '*C value.

Assay of ATPase

The reaction mixture (25 yl) contained 15 yl of assay buffer, 120
nmol of MgCl,, 20 nmol of [y-"P]ATP (2 to 6 Ci/mol), 0.3 nmol of ¢X
SS DNA (as nucleotide), and 0.1 yg of rifampicin; DNA was omitted
in assays of the replication intermediate. Incubation was for 10 min
at 30°, and the reaction was terminated by addition of 100 yl each of
cold 7% perchloric acid and Norit (20% (w/v) in H,O). The mixture
was stirred on a Vortex mixer, and centrifuged for 10 min at 5,000
x g. *P was measured in a 25-1 aliquot of the supernatant. One
unit is the release of 1 pmol of inorganic phosphate (measured as
82P unadsorbed to Norit) in 1 min.

Immunodiffusion

Double diffusion (31) was performed on a microscope slide coated
with 0.8% agarose containing 10 mM Tris-Cl (pH 8.0), 0.14 m NaCl,
and 1% Triton X-100. Wells were punched out with a template
dnaB Gene Product of E. coli

(Gelman Instrument Co., Ann Arbor, Mich.), and samples of 1 to 5
wl were added. Precipitates developed within 3 days at room temper-
ature. The slide was treated with 0.5% phosphomolybdie acid for 10
min (to brighten the precipitin pattern (32)) and photographed
under darkfield illumination.

SDS-Polyacrylamide Gel Electrophoresis

Analytical electrophoresis was carried out in a slab gel of 10%
polyacrylamide and 0.1% SDS. The buffer system of Marco e¢ al.
(33) and the Studier apparatus (34) were used. Samples were
precipitated with 10% trichloroacetic acid, dissolved in 50 yl of
sample buffer (2 m Tris, 30% glycerol, 10% SDS, 1 m 2-mercaptoeth-
anol, 0.1% bromphenol blue), neutralized with 5 yl of 1 N HCl, and
heated for 2 min at 100° before application to the slab. A constant
current of 12 mA per slab (15 x 11 x 0.1 cm) was applied until the
dye marker entered the gel; 36 mA was used thereafter.

Glycerol Gradient Sedimentation

A 0.1-ml mixture of dnaB protein and standard proteins was
applied to a 5-ml glycerol gradient (15 to 35%) containing 20 mm
potassium phosphate (pH 6.8), 100 mm KCl, 1 mm EDTA, 1 mu
dithiothreitol, and 10 mm MgCl,. Gradients were centrifuged for 15
h at 0° at 50,000 rpm in a Beckman SW 50.1 rotor. Two-drop
fractions were collected from the tube bottom.

Gel Filtration

Gel filtration of dnaB protein was performed by applying a 0.14-
ml sample to a column of Bio-Gel A-1.5m (200 to 400 mesh, 0.7 x 21
cm) and eluting with Buffer B containing 0.1 m KC] and 50 ug/ml of
albumin at a flow rate of 2 ml/h. Three-drop fractions were collected.

Gel filtration of the replication intermediate containing *H-la-
beled dnaB protein (see below) was performed by filtering 78 x1 of a
first stage reaction mixture (from a two-stage DNA replication
assay; see above) through Bio-Gel A-5m (200 to 400 mesh, 0.7 x 8
cm) (19). Four-drop fractions were collected, chilled, and assayed for
both replication activity (by incubating with the second stage
mixture) and radioactivity.

3H-labeling of dnaB Protein

Radioactive labeling of dnaB protein was performed at 0-2° by a
modification of the reductive alkylation method of Rice and Means
(35). The dnaB protein (Fraction V; 12 ug in 100 ul of 0.1 M
potassium phosphate (pH 6.5), containing glycerol, KCl, and EDTA)
was brought to pH 8 with 0.2 m NaQH, and treated with 160 nmol of
formaldehyde. After 30 s, NaB#H, (160 nmol, 6 Ci/mmol, freshly
dissolved in 0.01 m NaOH to 20 mm) was added. After 2 min, 6 yl of
2 ™ Tris-Cl (pH 6.5) was added and the total mixture was dialyzed
against 300 ml of Buffer A. The outer liquid was changed after 2 h
and again after 4 h. Specific radioactivity of the preparation was 6
x 10* cpm/pg (0.15 pCi/yg); only about 10% of the original dnaB
activity was recovered. For preparation of replication intermediate,
3H-labeled dnaB protein was further purified on a Bio-Gel A-5m
column; only the fractions containing active dnaB protein were
used.

Other Methods

B-Galactosidase activity was determined by the method of Craven
etal. (36), catalase by the method of Beers and Sizer (37), hemoglobin
by absorbance at 405 nm, and protein by the method of Lowry et al.
(38) with albumin as the standard.

RESULTS

Purification

dnaB protein was purified approximately 15,000-fold from a
lysate of HMS83 cells (Table I). (Unless noted, all operations
were performed at 0-4°).

Preparation of Soluble Extract—Frozen Escherichia coli
HMS83 cell paste (2900 g) was thawed at 0-4° in 300- to 500-g
portions, and adjusted to pH 7.5 with solid Tris base. To the
suspension were added lysozyme, NaCl, and spermidine: Cl,
to achieve final concentrations, respectively, of 0.2 mg/ml, 0.1
Mm, and 0.01 m. After gentle mixing, the suspension was

 

 

 

 

 

 

 

263
TABLE I
Purification of dnaB protein
Fraction actaty Total protein opens Yield ae
ie mg unitsimg | % ~fold
I. Lysate 22,300} 343,000 65 |100 1
II. Ammonium sulfate; 7,100 6,440 1,100/ 32 17
Il. DEAE-cellulose 2,150 27 80,000! 9.6] 1,230
IV. Phosphocellulose 830 1.91] 435,000; 3.7 6,690
V. Hydroxyapatite 830 0.83 1,000,000} 3.7 | 15,400

 

distributed in 250-m1 centrifuge bottles and, after 45 min at
0°, the bottles were placed in a 37° bath for 4 min, mixed by
inversion each min, and then centrifuged for 60 min at 27,000
x g in a Sorvall GSA rotor. The supernatant fluid was
decanted (Fraction I; 10 liters).

Ammonium Sulfate Fractionation—Solid ammonium sul-
fate (0.226 g/ml) was added to Fraction I. The mixture was
stirred for 30 min; the precipitate was collected by centrifuga-
tion (17,000 x g for 25 min), resuspended in a solution (/a
volume of Fraction I) of ammonium sulfate in Buffer A (0.2 g
of salt to each mi of Buffer A), and collected again by
centrifugation (17,000 x g, 25 min). The precipitate was
washed with Buffer A containing ammonium sulfate (0.16 g
of salt added to each milliliter of buffer) (1/so volume of
Fraction I), collected by centrifugation (31,000 x g for 15 min)
and dissolved in a minimal volume of Buffer B containing 1
mm dithiothreitol? (Fraction II, 132 ml).

DEAE-cellulose — Fraction II was dialyzed against 2.5 liters
of Buffer B containing 0.08 mM KCl and 1 mm dithiothreitol for
9h, and clarified by centrifugation (27,000 x g for 20 min).
Forty-milliliter portions were diluted with 2 volumes of Buffer
B containing 1 mo dithiothreitol to achieve the same conduc-
tivity as the buffer used to equilibrate the column (see below).
The diluted sample was applied (over a 6-h period) to a
DEAE-cellulose column (7.5 x 12.5 cm) equilibrated with
Buffer B containing 0.08 m KC] and 1 mm dithiothreitol. The
column was washed with 100 ml of the equilibrating buffer
and then with 820 ml of Buffer B containing 0.27 m KCl and 1
mM dithiothreitol. Elution of adsorbed dnaB protein was with
750 ml of Buffer B containing 0.37 mM KCl at 150 ml/h; 12.5-ml
fractions were collected. dnaB protein activity appeared in
the first fractions in which conductivity was increasing. Peak
fractions were combined, diluted 3.5-fold with Buffer B, and
applied (over a 6-h period) to a second DEAE-cellulose column
(10.4 cm’, 1.6 x 5.2 cm) equilibrated with Buffer B containing
0.1 Mm KCl. The column was washed with 75 ml of Buffer B
containing 0.27 m KCl and dnaB protein was eluted with a
60-ml gradient of KCI from 0.27 to 0.40 m in Buffer B at 50
ml/h; 1.7-ml fractions were collected. The dnaB protein peak
fractions, eluted between 0.29 and 0.39 m KC], were combined
(Fraction III, 60 ml).

Phosphocellulose — Solid ammonium sulfate (0.3 g/ml) was
added to Fraction III. The mixture was stirred for 30 min; the
precipitate was centrifuged (30 min at 44,000 x g), suspended
in 3.5 ml of Buffer B, dialyzed for 4 h against 1 liter of Buffer
B, and clarified by centrifugation for 15 min at 15,000 x g.
The supernatant was diluted 3-fold with Buffer B (final
conductivity corresponding to 0.03 M KCl in Buffer B), applied

? dnaB protein does not require sulfhydry] reducing agents for
activity and stabilization. Dithiothreitol was added in early stages
of purification because other replication proteins were prepared
from side fractions of these steps.
264

to a phosphocellulose column (1 x 11 cm) equilibrated with
Buffer B, and eluted at 3.5 ml/h with 5-ml portions of Buffer
B containing in succession 0.05 m, 0.1 M, 0.14 M, and 0.2 m
KCl; 0.8-ml fractions were collected. Under these conditions,
dnaB protein (>95%) eluted slightly behind the bulk of
unadsorbed protein. Ammonium sulfate (0.3 g/ml) was added,
the mixture stirred for 30 min, and centrifuged at 35,000 x g
for 25 min. Precipitate was dissolved in 1.9 ml of Buffer B
containing 0.1 m KCl and clarified by centrifugation for 15
min at 30,000 x g. The supernatant was dialyzed against 1
liter of Buffer B for 2 h and again for 6 h. The dialyzed
material was applied to a second phosphocellulose column (1
x 10 cm) equilibrated with Buffer B. The column was washed
with 8 m) of the same buffer and eluted at 2 ml/h with a
linear gradient (40 ml) of KCI from 0.03 to 0.4 m in Buffer B;
0.8-ml fractions were collected. Totally adsorbed dnaB protein
activity was eluted between 0.2 and 0.32 m KCl (Fraction IV,
14 m)) (Fig. 1A).

Hydroxyapatite—A portion of Fraction IV (0.5 mg of pro-
tein) was applied to a hydroxyapatite column (0.7 x 3 cm)
equilibrated with Buffer B containing 0.25 mM KCl. The column
was washed with 4.5 ml of the same buffer solution and then
2.5 ml of the buffer solution containing 35 mm phosphate.
Elution was with a linear gradient (13 ml) of potassium
phosphate from 35 to 200 mm at 40 ml/h; 1-ml fractions were
collected. The activity peak appeared at 60 mm phosphate
(Fig. 1B). Peak fractions from identical columns were pooled
(Fraction V) and stored in liquid nitrogen.

Purification of dnaB protein on a smaller scale (100 g of
cells) was successfully completed even when the first DEAE-
cellulose and phosphocellulose chromatography steps were
omitted.

Purification of dnaB protein from E. coli H560 and
RLM3865 (pLC11-9) Cells—dnB protein was also purified
from E. coli strains H560 and RLM365 (pLCll-9). From 350 g
of H560 cells a preparation with 1,100,000 units/mg of protein
(16,000-fold purification) was obtained in 8% yield. Similar
yield and specific activity were obtained from RLM365 (pLCll-
9), the strain with a 5-fold enhanced level of dnaB protein
activity due to the presence of the dnaB gene on a hybrid
ColE1 plasmid.

Criteria of Purity and Stability

Purified dnaB protein migrated as a single band on electro-
phoresis in a 10% polyacrylamide gel slab with 0.1% SDS ina
Tris/glycine system (Fig. 2). An impurity could have been
detected if present at a level of 2%.

 

dnaB Gene Product of E. coli

A single precipitin line was produced between anti-dnaB
antibody and purified dnaB protein in an Ouchterlony double
immunodiffusion analysis (Fig. 3). Antibody had been devel-
oped against the purest peak fractions of dnaB protein
(900,000 units/mg) derived from the second phosphocellulose
step (Fraction IV). A single precipitin line with no spur in
response to the antibody given by all dnaB protein samples
from the various stages of purification (Fractions II, IV, and
V) indicates that the preparation used for immunization was
relatively pure and that there is a single and common antigen
at various stages of purity.

dnaB protein at the DEAE-cellulose step or beyond was
stable for at least 6 months when stored in liquid nitrogen.
At 0°, the protein (40 pg/ml) lost 50% of its activity after a
month; at 30°, 50% was lost after 10 min.

Physical Properties

Molecular Weight of Subunit—The single protein band in
SDS-polyacrylamide gel electrophoresis indicates that dnaB
protein is made up of one or more polypeptides of molecular
weight about 55,000 (Fig. 4); the same molecular weight was
obtained for the protein purified from H560 cells.

Glycerol Gradient Centrifugation—The native molecular
weight of dnaB protein was estimated by glycerol gradient
centrifugation from a single peak which sedimented slightly
faster than a catalase marker (Fig. 5A) indicating a sedimen-
tation coefficient of about 11.5 S and corresponding to a
molecular weight, for a globular protein, near 250,000. This
agrees with values reported by Wright et al. (9) and Schekman
et al. (11). Under these conditions, dnaB protein appears to
behave as a tetramer. When sedimentation analysis was
performed without MgCl,, essentially no dnaB activity was
recovered from the gradient (Fig. 5B), suggesting that Mg?*
is needed to stabilize the active tetrameric structure under
these conditions.

Bio-Gel A-1.5m Filtration —The behavior of dnaB protein
on gel filtration suggests multiple forms depending on MgCl.
In its presence, dnaB protein emerged with hemoglobin,
suggesting a monomeric or an asymmetric form penetrating
the gel. In the absence of MgCl, dnaB activity appeared
in two peaks corresponding to molecular weights of 220,000
and 110,000 (Fig. 6B), suggestive of tetrameric and dimeric
forms.

Functional Properties

Requirement of dnaB Protein for Conversion of 6X SS to
RF—The requirement for dnaB protein (6, 9, 11, 18) was

 

 

 

 

 

100 T 7772.0 2.0 T t 0.25
(A) PHOSPHOCELLULOSE (B) HYDROXYAPATITE
Qa Protein
a) | = -{ 0.20
% 1.5 gnsB
= x 2 2
™ x . 2
2 9 2 protein |g 45 9
2 m = m
= z = 10h F4
> z > z Fic. 1. Chromatography of dnaB
5 > 04 5 > protein on phosphocellulose and hy-
= = - = droxyapatite.
g 0.3 g 0.2
a 3 mo 05h
3| 02 . é 5
2 =z 7 or =
0.1 z=
0 oF to Q

 

FRACTION NUMBER

 
dnaB Gene Product of E. coli

  

Fic. 2 (left), SDS-polyaerylamide gel electrophoresis of daaB
protein. A, Fraction V from HMS83 (6 ug); B, Fraction V from H560
(4 pg).

Fic. 3 (right). Double immunodiffusion analysis of dnaB protein.
Wells 1, 7, and 4 contained y-globulins; J, anti-dnaB protein 14 wg
(1 pl}; 7, anti-dnaB protein 70 ug (5 wl; ¢, control 40 pg (6 pl).
Other wells contained dnaB protein preparations: 2, Fraction Ili 30
units (5 zl); 3 and 6, Fraction V 30 units (5 pl; 5, Fraction TV 35
units (4 yl).

 

 

 

 

— 20 T T T 1 T
So
2
= 10 |
& sr 1 of
GS $e WW ty dns “4
a
= 4 I 2 4
« | 3
3 |
2

2F -
a | 4
ad
g t 1 il r i !

0 0.2 0.4 0.6 0.8 1.0

RELATIVE MOBILITY

Fic. 4. Determination of dnaB polypeptide molecular weight by
SDS-polyacrylamide gel electrophoresis. Protein standards were: J,
albumin (68,000); 2, ovalbumin (43,000); 3, chymotrypsinogen A
(25,700), and ¢, lysozyme (14,300). Mobilities are expressed relative
to the marker dye, bromphenol blue.

observed in the reconstituted system (Fig. 7). Similar results
were also obtained with the partially reconstituted system
(data not shown). Rate of DNA synthesis was a linear function
of dnaB protein concentration up to 8 units (Fig. 7A). A
decrease in DNA synthesis at higher dnaB protein concentra-
tions may be due to direct inhibition by excess dnaB protein.
DNA synthesis proceeded almost linearly for about 10 min at
30° and leveled off when about 70% of the added template
circles were replicated (Fig. 7B).

Complementation of dnaB~ Extract ~ Purified dnaB protein
was capable of complementing extracts from the dnaB mutant

 

  
 

 

 

 

 

265
2.0 7 1 1 142.0
1A) MgCl 1.5
dnaB
187 catalase 7 hemoglobin] s
' ‘ \ 741.0
woh ff i 41.0 B
— ( {1 EB-galactosidase! t ° 7
E ; $ i 4 zor
ie fo dos 2 3
® osbt j 405 23
x yg? f x r 2
2 } i ’ ba > oO
g i ! 3 b>
5 er ‘ a — &
= Ob CS emebrnnie ed ent e Me aknnnd g Pdo § 2
ip ' ’ B75 g 5
> , (8) NO MgClp z >
- =
9 moglobi > x
< ist \ Argalactosidase LY omonin 1.6 & 6 2
2| \ catalase A ~ Lie Pa V
* =. =
. / iy 3 3
1.0} . PY 410 z 3
! d dnaB { ‘ 2
j b ; \ 405
0.8- \ \ “4 0.5
res AoA
. Seon terh npn,
0 be Fh ——trerennrnnnntiiiisbisinn— Lf 0 wy
6 10 20 40 40 50
FRACTION NUMBER TOP

Fic. 5. Glycerol gradient centrifugation of dnaB protein. The
draB protein applied was Fraction V (1600 units), Recovery of dnaB

- activity in A was about 60%.

 

       
 

 

 

 

 

 

 

eB Ck
t q E i =z
{Al hemogiobin daaB & 4 4
s - 4
Lak catalase 8 F
B-Gatactosidate z
acre] SO Od,
oxq74 /\¢#
10 1.0 =
3
A = 2 2
a we 4
i ‘ <= a
i \ — o
E ost fT * Josh “¢ 5
= m4 he se os
t Uye | by et
= j 4 5 : ma a
x ] 2 ~ =
g ol she A 0 #1, B do > Jo 8
3 40 60 80 100 = & = 9
z T Tame 7 —J By «7 x4 2
z {B} hemagiobin 3 5 S 2
catalase = NS
g B-Galactosidase “3S 245 73 _
a = 2 5
a oxt74 ure | 3 $|
7 14.0 K+ Sy =
Sh / 4
Rg NR Pe F -
ay =
h PVARR [|
1 { =
ia 6©¢ APRA RY |
Ney se
ase ort | Ml | 205 42
piop Ay 1\t ais 4:
peo ¥ 1 =
| t Py i vi —
bj d } bay AL 3 t
ay \ =
Le deatened mveroneeenee Pel wh do Jo 49
20 30 40 so F

FRACTION NUMBER

Fic. 6. Bio-Gel A-1.5m filtration of dnaB protein, dnaB protein
(Fraction V, 3000 units) was filtered in the presence (A) or absence
(B) of 10 mm MgCl. In A, a larger column (0.7 x 30 cm) was used
and 2-drop fractions were collected. Recovery of dnaB activity was
about 40% in both cases.
266

 

 
  

dnaB Gene Product of E. coli

 

 

 

 

 

 

 

 

   

 

 

 

70
{A} 7
oe -o
tay! t T 7 a BQ Control Y-globulin
120+ /™. 4
* ™s, sob 4
100+ / —.,
° 40k 4
Boh 5
30+ 4
60 7 Anti-dnaB Y-globulin
20+ +
40b 6 4 _
= 3 10h ° 4
2 20h 4 5
a = 0 r 1 1 1
z 0 L L l l \ 3 0 05 1.0 1.5 2.0
2 0 10)~—20—~=:«Cti(itiSC*«S 5 Y¥-GLOBULIN (ug) Anti-protein i
s daaB PROTEIN (units) g T T T t 80 _
© 200 ~ {B) -
a T T T T fe 100b 4 =
i {B} ere Cait 9 4 €
8 - : 3 ns é
= —— erro ——— ae
2 L ° +doaB sok 7 “\y-GLOBULIN: z e9o—— oe ~~ 4
150+ 4 “6/7 oo‘ 2 re No antibody
<
Control, B wg or 16 4g c YO
sor ff = 40 7 4
100+ 4 i =
/ Anti-dnaB, 1.7 ug g
40h ° 4 2
i en 20 4
i —
i 7 i Anti-dnaB, 3.4 ug _|
20 rj _ Anti-gnaB
oe ene 0 «= —dnaB / a =
pk&= i i | i 9 bag eat | 0 5 10 15 20
0 10 20 30 40 0 10 20 #30 40 50

 

TIME (min)

Fic. 7 (left). Dependence of 6X DNA replication on dnaB protein
in the total reconstitution system. A, varying amounts of dnaB
protein (Fraction V) were added to the standard assay mixture; B, a
12-fold standard mixture containing 120 units of dnaB protein
(Fraction V) was incubated at 30°; 25-1] aliquots were analyzed at
intervals.

Fic. 8 (center). Effects of anti-dnaB antibody on ¢X DNA repli-
cation. A, the reaction (standard conditions) contained 15 units of
dnaB protein (Fraction V). Indicated amounts of y-globulin were
added and the mixtures incubated for 15 min at 0° before addition of
@X SS, spermidine, rifampicin, MgCl,, rNTPs, and dNTPs. B,

(BT1029) for #X SS to RF conversion, but dnaC protein was
occasionally also necessary for maximal restoration of repli-
cation activity, as described previously (data not shown) (11).

Inhibition of dnaB Protein Activity by Specific Antibody —
Antibody produced against purified dnaB protein inhibited
@X DNA replication (Fig. 8A). At 1 ug, anti-dnaB y-globulin
neutralized about 10 units of dnaB protein activity. The
slight activation by the control y-globulin may be due to
protection of the system from inactivation during the prelimi-
nary incubation. Inhibition by anti-dnaB antibody was par-
tially reversed by the addition of excess dnaB protein (Fig.
8B). Failure by even large amounts of dnaB protein to reverse
it fully is probably due to the inhibitory effect of excess dnaB
protein noted earlier.

Inhibition of Replicative Intermediate Activity by Anti-
dnaB Antibody — The dnaB protein becomes part of a replica-
tion intermediate required by primase in its priming action
(39, 40). Addition of anti-dnaB antibody at any point during
or after formation of the intermediate produces profound
inhibition (Fig. 9). By contrast, action of protein i, also
essential for producing the replication intermediate (19), be-
comes resistant to anti-protein i antibody once the intermedi-
ate is formed (Fig. 9). Actions of antibodies against other
participants in forming the replication intermediate fall into
two classes (39, 40): Class 1, proteins such as dnaB and DBP,
which participate stoichiometrically and appear to become
part of the intermediate complex, were inhibited by their
respective antibodies after, as well as before, formation of the

dnaB PROTEIN (units)

PREINCUBATION BEFORE
SERUM ADDITION (min)

varying amounts of dnaB protein were treated with y-globulin for
15 min at 0°; after addition of other components as in A, incubation
was for 10 min at 30°.

Fic. 9 (right). Effects of antibodies on replication intermediate
formation and activity. A standard two-stage replication mixture
(20-times scale, ATP increased 3-fold) was incubated at 30°, and, at
intervals indicated, 20-] aliquots (20 ul) were mixed with 1 pl of
either assay buffer (no antibody) or the indicated antiserum.
Mixtures were then incubated to a total of 20 min at 30° to complete
the first stage and finally supplemented with second stage compo-
nents (in 5 yl) and kept 2 min longer at 30°.

complex; Class 2, proteins i and n, which appear to act
catalytically (19), were susceptible to antibody only before the
complex was formed.

Association of °H-labeled dnaB Protein with Intermediate —
Direct demonstration of dnaB protein in the replication inter-
mediate was made with *H-labeled dnaB protein. The protein
by itself was fully retained upon filtration through a column
of Bio-Gel A-5m; after formation of the intermediate the
radioactive protein was excluded from the gel along with the
replicative activity indicative of the intermediate (Fig. 10A).
Without incubation to produce the intermediate, only one-
fourth as much labeled protein was found in the excluded
fraction (Fig. 10B).

Inhibition of Primer RNA Synthesis by Anti-dnaB Anti-
body—The dnaB protein bound in replication intermediate
participates in primer RNA synthesis as suggested by experi-
ments with anti-dnaB antibody (Table II). Primer synthesis
by primase was profoundly inhibited; by contrast, anti-protein
i antibody had no effect. Incorporation under these conditions
shows extent rather than rate of RNA synthesis.

DNA Elongation Step Not Inhibited by Anti-dnaB Aniti-
body — Although anti-dnaB antibody inhibited formation of
the replication intermediate (Fig. 9) and its function in the
synthesis of primer (Table II), it had no significant effect on
the final stage of DNA synthesis (Table III). Anti-protein i
antibody, which inhibited the first stage, had no effect on the
next two stages. Thus dnaB protein is not required for DNA
elongation once the primer is formed. The fate of the bound
dnaB Gene Product of E. coli

 

 
   
      

 

  

 

T T T T
(a) INCUBATED
60 44
(3H) dnap
43
40+
8
42 xz
20} z
41.0
§ replic. 1 2
intermed. 35
Zz a
Eo wl §
a T T T 3
{ NOT INCUBATED 42
40- 3
2
FH] daas 7?
20/--
. 41
replic.
intermed.
e

 

 

0 10 20 30 40
FRACTION NUMBER

Fig. 10 (left). Association of [[H]dnaB protein with the replica-
tion intermediate. *H-labeled dnaB protein (2000 cpm, about 4
units) was mixed with other components of the first stage mixture
(7-times scale, 91 yl). After incubation for 15 min at 30° (in A) and 0
min (in B), 13 yl were analyzed for replicative activity. The rest of
the mixture was applied to a Bio-Gel A-5m column (0.7 x 8 cm),
then eluted at 24° as described (“Materials and Methods”). Replica-
tive activity was assayed by incubating (3 min at 30°) with second
stage components containing [a-**P]dNTPs. Recoveries of *H and

TABLE II
Inhibition of primer RNA synthesis by anti-dnaB antibody

Primer RNA was synthesized on the replication intermediate
(formed by incubating the first stage components for 15 min at 30°)
by further incubation with the second stage components for 10 min
at 30°. Conditions are given under “Materials and Methods,” except
that NTP concentrations were: 80 4m ATP, 40 um each of GTP and
CTP, and 4 uM [5,6-"HJUTP (10 Ci/mmol). y-Globulins were added
in the second stage as specified.

267

 

  
 

 

 

70 T T
dnaB activity ——
+4250
60
= >
£ 3
2 50r +200 &
= >
io ATPase 3
£ 40h <
§ 4150 4
> =
E30 5 04
E phosphate _Jio9 x
< 20h 3 1°3
g 2 5
| dso 2 02 =
10 ~ =
0.1
0 ee _ 0
9 10 20 30

FRACTION NUMBER
replicative activity were >90%.

Fia. 11 (right). ATPase activity of dnaB protein. dnaB protein
(Fraction IV, 750 zg) was applied to a hydroxyapatite column (0.7
x 2.6 cm), washed with 6 ml of 20 mm potassium phosphate (pH 6.5)
containing 0.25 m KCl, 20% glycerol, and 1 mm EDTA, and eluted
with a linear gradient (13 ml) of phosphate from 20 to 260 mm.
Fractions (0.9 ml) were collected and assayed for dnaB protein and
ATPase activity. Phosphate was determined by conductivity.

TaBLe III
Effects on DNA replication of anti-dnaB antibody added at three
stages
See “Three-stage DNA Replication” under “Materials and Meth-
ods;” y-globulins were added at stages specified.
Added at stage

 

 

 

y-Globulin |SH]UMP incorporated

pmol
None 0.362
Nonimmune (8 yg) 0.368
Anti-protein i (8 ug) 0.357
Anti-dnaB (4 yg) 0.072
Anti-dnaB (14 pg) 0.059
Anti-protein i (8 ug) + anti-dnaB 0.050

(14 yg)

 

dnaB protein has not been determined.

Requirement for dnaB Protein in E. coli DNA Replication
in vitro—E. coli dnaB mutants are unable to elongate DNA
at the replication fork (1-5). In lysates of the mutants, using
the cellophane-disc system, semiconservative DNA replication
at pre-formed forks can be measured (29) and the.requirement
for dnaB gene product shown. Our preparation of pure dnaB
protein and its specific antibody has permitted us to demon-
strate directly the place of dnaB protein in the replication of
the bacterial chromosome. Antibody against dnaB protein
profoundly inhibited DNA synthesis in the cellophane-disc
lysate system, but control y-globulin did not (Table IV). The
inhibition was partially reversed when purified dnaB gene
product was mixed with anti-dnaB y-globulin prior to adding
it to the lysate.

ATPase in dnaB Protein —Ribonucleoside triphosphatase
activity of dnaB protein, partially dependent on single-
stranded DNA, has been previously reported (9, 14). We

y-Globulins* Pre 1 an 2 Replica- synthesis
ing Priming tion
pmol

Experiment 1

None 54.9
Experiment 2

Anti-dnaB + 0.6

Anti-protein i +

Anti-dnaB + 0.9

Anti-protein i +

Anti-dnaB + 41.7

Anti-protein i +
Experiment 3

Anti-protein i + 37.7

Anti-protein i + 47.1
Experiment 4

Nonimmune + 45.5

Anti-protein i +

Nonimmune + 39.3

Anti-protein i +

Nonimmune + 47.2

Anti-protein i +

 

* Amounts of y-globulins used were: nonimmune, 8 yg; anti-
protein i, 8 wg; and anti-dnaB, 14 yg.

confirmed these observations by examining purified dnaB
protein and the effects of antibody. Throughout purification
of dnaB protein from HMS83 as well as H560 cells, NTPase
activity was found associated with dnaB protein activity.
268

TaBLe IV
Inhibition of Escherichia coli DNA replication by anti-dnaB
antibody in cellophane-dise system
See “Materials and Methods.” y-Globulins and dnaB protein were
added to the lysate 10 min before initiation of DNA synthesis.

Normalized DNA

Addition Incubation

 

synthesis
cpm ‘Hiepm 4C
Experiment 1
None ~ 3.7
+ 22.5
Anti-dnaB y-globulin (4 ~ 2.5
ug) + 5.5
dnaB protein (55 units) - 4.4
+ 23.5
Anti-dnaB y-globulin (4 - 3.9
ug) + dnaB protein (55 + 13.3
units)
Experiment 2
None + 15.2
Nonimmune y-globulin + 16.7
(4.5 ug)
Anti-dnaB y-globulin
(0.4 pg) + 9.7
(1.7 peg) + 7.4
(4.2 wg) + 5.8
dnaB protein (12 units) + 24.2
Anti-dnaB y-globulin (1.7 + 24.8

ug) + dnaB protein (40
units)

 

After the phosphocellulose step, the ratio of NTPase (with SS
DNA present) to dnaB protein activity reached 2.5 and there-
after remained nearly constant. In the elution profile of
highly purified dnaB protein from hydroxyapatite, the ATP-
ase coincided with dnaB activity (Fig. 11).

ATPase showed an 8- to 10-fold stimulation by SS DNA.
The K,, for ATP in the presence of 6X SS DNA was 50 pM.
Both DNA-independent and -dependent ATPase activities
were inhibited by about the same percentage by anti-dnaB
antibody (14 ug of antibody inhibited 50 units of dnaB ATPase
activity 76% and 86%, respectively); antibody effectiveness
was only about half as great as against the dnaB protein
replicative activity. ATPase activity was present in the
replication intermediate and inhibited by anti-dnaB y-globulin,
indicating that dnaB ATPase activity is fully functional in the
intermediate (39).

Cellular Content of dnaB Protein

Approximately 20 dnaB oligomers were calculated to be
present per HMS83 or H560 cell, and about 100 in the plasmid-
bearing cell (RLM365 (pLClI-9)). The greater abundance of
dnaB protein in the latter had no apparent effect on its
growth, unlike the inhibition observed by excess protein in
vitro (Fig. 7A).

DISCUSSION

Purification of the dnaB protein was undertaken to enlarge
our understanding of its crucial role in DNA replication. It
had been known from studies in vivo that temperature-sensi-
tive dnaB mutants raised to a restrictive temperature imme-
diately stopped making DNA (1-5), but it was unclear at
which stage in DNA synthesis dnaB gene product acted.

With the pure protein in hand and with a highly specific
and active antibody against it, we have conducted studies

dnaB Gene Product of E. coli

reported here and elsewhere (39) which place the dnaB protein
in a pivotal role in the initiation of DNA chains. Synthesis of
a replication intermediate of the X viral circle requires the
participation of DNA-binding protein to coat the single strand,
and dnaC protein, proteins i and n, and ATP to fix a dnaB
molecule in the intermediate (19, 39). We show here that
anti-dnaB antibody prevents formation of the intermediate
and also neutralizes the activity of the intermediate in sup-
porting primer formation by primase. Once primer is pro-
duced, subsequent elongation by DNA synthesis is not affected
by anti-dnaB antibody. This indicates that dnaB protein does
not participate in elongation (whether attached to the DNA
or dissociated from it) or, less likely, that it has become
unavailable for interaction with the antibody.

We have presented a model (39) in which dnaB protein acts
as a “mobile promoter” for primer synthesis by primase to
initiate the growth of a DNA strand at the chromosome
origin as well as at the replicating fork. In view of the small
number of dnaB protein molecules in a cell and the complex
nature of generating the nucleoprotein replication intermedi-
ate, it seems plausible that dnaB protein would not dissociate
upon promoting primase action but rather remain attached to
the template. It could move along the template in the direction
of replicating fork movement to provide fresh loci for primase
action. Energy to propel the dnaB protein along the template
may be provided from hydrolysis of ATP which the protein
itself can manage.

In addition to the demonstration that dnaB protein partici-
pates in EF. coli DNA replication by the lysate-cellophane disc
system, much remains to be learned about the physical and
functional properties of dnaB protein in vitro and how they
are correlated with events inferred from in vivo studies.
Defects attributed to dnaB mutants range from failure in
DNA replication (1-5, 15, 16, 41) to changes in membrane
structure (42) and possibly altered incompatibility with F-
factor (48, 44). Multiple allelic forms of this oligomeric protein
may very well account for the large variety of responses
observed with different mutants (15, 16). The relationship
between dnaB protein and the deficiency of a protein of
similar subunit size in membranes of dnaB mutants (42) also
needs to be explained.

The complex formed between dnaB and dnaC proteins in
the presence of ATP would appear to be an important step in
formation of the replication intermediate but neither this
reaction nor the functions performed by proteins i and n are
understood. The mission of dnaB protein in the replication
intermediate would appear to be to signal primase action,
possibly by creation of secondary structure in the template
strand or through direct protein-protein interactions. Thus
clarification of what dnaB protein does must encompass its
interactions with other replication proteins and with the
template, and its inherent capacity to utilize ATP.

Acknowledgments — We wish to thank Dr. B. M. Olivera
(University of Utah) for his collaboration in cellophane-disc
experiments and Dr. E. Berger (Stanford University) for
advice on immunodiffusion.

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