Tue JouRNAL or BioLocicaAL CHEMISTRY
Vol. 250, No. 15, Issue of August 10, pp. 5859-3865, 1975
Printed in U.S.A.

Ten Proteins Required for Conversion of #X174 Single-
stranded DNA to Duplex Form in Vitro

RESOLUTION AND RECONSTITUTION*

(Received for publication, February 13, 1975)

Ranpy Scuexman,t Jon: H. WerIner,§ ALAN WEINER,{ AND ARTHUR KorNBERG
From the Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305

SUMMARY

Protein requirements for conversion of $¢X174 single-
stranded DNA to a double-stranded replicative form with a
small gap (RF IT) have been determined by resolution and re-
constitution of the multienzyme system from extracts of
gently lysed Escherichia coli. Assays depended on: (a) com-
plementation of extracts of thermosensitive mutants and
(8) fractionation of extracts of wild type cells to divide essen-
tial components into groups, each of which was further re-
solved. These procedures have yielded eight proteins: dnaB
protein, dnaC protein, proteins i and n (two novel proteins
without a defined genetic locus), dnaG protein, DNA polymer-
ase III holoenzyme (polymerase ITI* and copolymerase III*),
and DNA unwinding protein; purification procedures for the
first four are presented here. (Closure of RF II requires as
with phage M13, DNA polymerase I and ligase.)

 

Soluble extracts of gently lysed Escherichia colt convert
$X174 single-stranded DNA to the double-stranded replicative
form (1). Enzymes involved in this reaction appear to participate
in host chromosome replication. Conditionally lethal mutants of
E. coli, defective in initiation at the chromosome origin (dnaA,
dnaC) and ongoing DNA replication (dnaB, dnaE, and dnaG)
all show in vitro defects in ¢X174 RF! formation (2, 3). Proteins
corresponding to these dna genes may be assayed and purified
using complementation of extracts prepared from mutant ceils.
Partially resolved enzyme fractions have been added to deficient
extracts and assayed at an elevated temperature at which the
mutant protein is inactive. The dnaB, dnaC, dnaK, and dnaG
gene products have been purified in this manner (4-7). However,

* This work was supported in parts by grants from the National
Institutes of Health and the National Science Foundation.

{ Present address, Department of Biology, University of Cali-
fornia at San Diego, La Jolla, California 92037.

§ Fellow of Damon Runyon Memorial Fund for Cancer Re-
search.

q Fellow of Helen Hay Whitney Foundation. Present address,
Department of Biology, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139.

‘The abbreviations used are: RF, circular double-stranded
DNA of replicative form; RFI, covalently closed RF, RF II, RF
with a discontinuity in one strand; NEM, N-ethylmaleimide.

these four proteins were insufficient for RF synthesis from single-
stranded DNA,

In another approach extracts from wild type cells are subjected
to fractionation. A procedure is adopted which generates two
fractions, each of which is required; each fraction is then sub-
divided further. This approach together with the complementa-
tion assay has been employed for complete resolution of the
components involved in the ¢X174 reaction (8, 9). This report
presents the strategy of the fractionation pattern, and the assays
and purification procedures developed for four of the essential
proteins. Information about the size, properties, abundance, and
possible functions of these proteins are suggested from these
studies.

EXPERIMENTAL PROCEDURE

Materials

Materials were from sources previously described (1, 2). Freshly
dissolved calf thymus DNA (Calbiochem) was denatured and at-
tached to cellulose by the procedure of Alberts (10) dried at 37°
and stored as a powder at —20°.

Buffer A is 50 mm Tris-HCl (pH 7.5), 0.1 m NaCl, 20% glycerol
(v/v), 1 mm EDTA, and 1 mm dithiothreitol. Buffer B is 50 mm
imidazole-HCl (pH 7.0), 20% glycerol, 1 mm EDTA, and 1 mu
dithiothreitol. Buffer C is 50 mm Tris-HCl (pH 7.5), 30% glycerol,
1mm EDTA, and 10 mo dithiothreitol. Buffer D is 50 mm Tris-HCl
(pH 7.5), 20% glycerol, and 1mm EDTA. Buffer E is 20 mm potas-
sium phosphate (pH 6.5), 20% glycerol, and 1 mm EDTA. Tris-HCl
and imidazole-HCl stock solutions (2 m) were prepared at room
temperature and diluted as required.

DNA unwinding protein was Fraction 3b (0.38 mg/ml (11));
dnaG protein (Fraction V, 0.04 mg/ml)? was prepared according to
Bouché et al.2 DNA polymerase III holoenzyme was purified as
before (12) except that Bio-Gel A-0.5m (200 to 400 mesh) replaced
Bio-Gel A-5m. Fraction IV was precipitated with an equal volume
of saturated ammonium sulfate (neutralized with ammonium hy-
droxide). After 30 min at 0° the precipitate was centrifuged at
31,500 X g for 10 min and the supernatant was discarded. Am-
monium sulfate precipitates stored at 0° were stable for at least
2 months; when redissolved in Buffer A (containing 10 mm dithio-
threitol) they retained 50% of their activity after 1 week at 0°.

Escherichia coli PC 79 (F-, his-, str®, malA, xyl-, mtl-, thi-,
pol Al, sup~, dnaD7 (now referred to as dnaC)) was provided by
Dr. P. Carl (University of Illinois, Urbana). E. coli BT1029 (dnaB,
thy~, pol Al, endo I~) was provided by Dr. H. Hoffmann-Berling
(Heidelberg). EZ. cols H560 was grown and soluble extracts were
prepared as previously described (1, 12) except that the 37° heating
step was performed with 35-ml aliquots in polyethylene centrifuge

2 J.-P. Bouché, K. Zechel, and A. Kornberg (1975) J. Biol.
Chem, 250, in press

 

5859
5860

tubes for 2 min followed by centrifugation at 48,000 X g for 30 min
at 0°. #. cols HMS-83, provided by Dr. R. L. MeMacken, of this
department, was grown as E, coli H560 (1).

Methods

General Procedures

Ammonium Sulfate Precipitation—Solid ammonium sulfate was
added at 0° over a 10-min interval, the solutions were stirred for
another 20 min and centrifuged for 10 min at 23,000 X g. Solutions
for extracting ammonium sulfate precipitates contained varying
amounts of ammonium sulfate added to Buffer A (e.g. solution
AS 0.20 was made up by adding 20 g of ammonium sulfate to 100
ml of Buffer A). A glass homogenizer was used manually for these
extractions.

Assay of dnaC Protein

Preparation of Mutant Enzyme Fraction—E. coli PC79 was
grown at 30° in Hershey broth, harvested, and resuspended as
described before for E. coli H560 (1). The soluble extract (Fraction
I) was prepared as before (1, 12) except that spermidine was
omitted and lysis was for 1 hour at 0°. Fraction I was adjusted to
0.2 m NaCl and applied to a column of DEAE-cellulose (equili-
brated in Buffer D + 0.2 m NaCl) 4th the volume of Fraction I.
The flow-through was collected and solid ammonium sulfate was
added to 40% saturation (0.242 g added/ml). The solution was
stirred for 30 min at 0°, and then centrifuged at 39,000 X g for 10
min. The pellet (Fraction II) was taken up in Buffer 1), oth the
volume of Fraction I (about 10 mg of protein/ml) and dialyzed
against 100 volumes of Buffer D + 0.05 m NaCl for 4 hours. The
dialyzed fraction divided into aliquots was stored in liquid nitro-
gen and remained stable for at least 3 months.

Assay—Assays were performed in 25 ul at 30° for 20 min. Com-
ponents were added in the following order at 0°: 5 to 10 ul of dilu-
tion buffer (10% sucrose, 50 mm Tris-HCl] (pH 7.5), 20 mm dithio-
threitol, 0.2 mg/ml of bovine serum albumin, and 50 mm NaCl),
40 we (4 ul) of mutant Fraction II, 1 to 6 wl of the fraction to be
assayed, and 10 ul of a reaction mixture containing 0.45 nmol of
[a-*PJdCTP (200 to 600 cpm/pmol), 1.25 nmol each of dATP,
dGTP, and TTP, 125 nmol of MgCl., 100 nmol of spermidine-HC},
20 nmol of ATP, 2.5 nmols each of GTP, UTP, and CTP, 0.33 nmol
of ¢X174 DNA, and 100 ng of rifampicin. DNA synthesis was
measured by incorporation of the labeled deoxynucleotide into an
acid-insoluble form. One unit is defined as 1 pmol of total deoxy-
nucleotide incorporated in 1 min; the value for (CMP incorpora-
tion was multiplied by 4.

Preparation of DNA-cellulose Binding and Nonbinding Fractions

£. colt H560 (150 g of cell paste in 750 ml of 10% sucrose and 50
mM Tris-HC] (pH 7.5)) was lysed and centrifuged to yield Fraction
I (500 ml) (Table I) (1, 2). Ammonium sulfate (0.226 g/ml) was
added and the precipitate was collected as described above. The
precipitate was resuspended in 50 ml of AS 0.24 and the insoluble
proteins were collected. This procedure was repeated with 10 mi of
AS 0.20. The precipitate was resuspended in Buffer D to a final
volume of 11 ml] and applied to a Sephadex G-25 column (170 ml,
3-cm diameter) equilibrated in and eluted with Buffer D. The void
volume fractions of column buffer conductivity (1.8 mmho) were
pooled (42 ml, Fraction II) and solid NaCl was added to a final
concentration of 0.2m. This fraction was then applied to a DEAE-
cellulose (DE52) column (10 ml, 2-cm diameter) equilibrated in
Buffer D + 0.20 m NaCl, followed by a 10-ml wash with the same
buffer. Fractions containing the bulk of the protein were pooled
(50 ml), ammonium sulfate was added (0.272 g/ml), and the pre-
cipitate was collected and resuspended in 5 ml of Buffer B. This
fraction was then applied to a Sephadex G-25 column (45 ml, 2-cm
diameter) equilibrated in and eluted with Buffer B.

The void volume fractions of column buffer conductivity (1.7
mmho) were pooled (15 mJ, Fraction III) and applied to a
DNaA-cellulose column (12 ml, 3.5-em diameter) also equilibrated
in Buffer B. The column was then washed with 100 ml of Buffer B.
Nonbinding (flow-through) fractions (5 ml) were collected and
assayed as described below. The DNA-cellulose binding proteins
were eluted with 40 ml of Buffer B + 2m NaCl, precipitated with
ammonium sulfate (0.226 g/ml), resuspended in 2.5 ml of Buffer B,
dialyzed against 500 ml of Buffer D + 0.2 m NaCl, and stored in

aliquots in liquid nitrogen for at least 6 months with no loss of
activity. Ammonium sulfate precipitates of Fractions IJ or ITI
may be stored at —20° for several days and subsequently carried
through the remaining steps.

Resolution of DNA-cellulose Nonbinding Proteins

The nonbinding fractions from the above preparation were
pooled (35 ml), precipitated with ammonium sulfate (0.272 g/ml),
collected, and resuspended in 1.5 ml of Buffer A. Part of the sample
(1.2 ml) was applied to a Sephadex G-150 column (42 ml, 2-cm
diameter) equilibrated in Buffer A and eluted by this buffer. Two
essential proteins were separated (Fig. 1); one was in the void vol-
ume (dnaB protein) and the other was included at Kay = 0.25
(protein i). Peak fractions of each were pooled and aliquots stored
in quid nitrogen. Quantitative determination of the preparation
through this stage is presented in Table I.

Assay of DNA Binding and Nonbinding Fractions

Activity of the binding fraction was determined by assaying
varying amounts in the presence of dnaC protein and the nonbind-
ing fraction. Assays were in 25 ul at 30° for 20 min. Components
were mixed as follows: 10 1 of dilution fraction, 2 #l of nonbinding
fraction, 0.2 to 2 ul (1 to 10 ug) of binding fraction, 2 to 5 ug of
Fraction ITI dnaC protein, and 10 ul of the reaction mixture de-
scribed for assay of the dnaC protein. DNA synthesis was meas-
ured as above. When the resolved nonbinding fractions were puri-
fied and became available, protein i (Fraction V, 4 to 40 ng/assay)
and dnaB protein (Fraction V, 20 ng/assay) were used (see ‘‘Re-
sults’’).

Assay for Proteinn, NEM-sensitive DN A-cellulose Binding Protein

The binding fraction contains two essential components which
are inactivated by the sulfhydryl-blocking agent, NEM: DNA
polymerase ITI holoenzyme and another component, called protein
n. The binding fraction was treated with 10 mm NEM (freshly dis-
solved in water) at 30° for 15 min and excess NEM was removed
with 20 mm dithiothreitol at 0°. Assays for protein n were mixed
as follows: 10 ul of dilution buffer, 0.1 to 2 wl of the fraction to be
assayed, 5 ul of a mixture of NEM-treated binding fraction (1 to 10
ug), protein i (Fraction V, 40 ng), dnaB protein (Fraction V, 20
ng), DNA polymerase IIT holoenzyme (Fraction IV, 0.33 ug), and
dnaC protein (Fraction III, 2 to 5 ug), followed by 10 ul of the
reaction mixture used for assay of the dnaC protein. Alternatively,
purified DNA unwinding protein (700 ng) plus dnaG protein (20
ng) can replace the NEM-treated binding fraction.

 

 

 

 

10 T T T T
EXCLUDED INCLUDED
sk FRACTION FRACTION _
t 9
o oe
7 . a
«x ! .
z L- i ‘ 4
5 ° i 4
r i 9
2 : 5
& 4- e e 9 b 4
a : .
5 ;
2
> 2b \ 2 4
i ’Q PQ
ods One + ot
_ “@c@. boone) b.
0 eo i e-@ !
0 10 20 30 40 50
FRACTION

Fie. 1. Sephadex G-150 chromatography of the DNA-cellulose
nonbinding fraction. The DNA-cellulose flow-through fraction
(see ‘“‘Methods’’) (1.2 ml, 13 mg, 470 units/mg) was applied to a
Sephadex G-150 column (42 ml, 2-cm diameter) equilibrated in and
eluted with Buffer A. Fractions of 1.25 ml were collected at a flow
rate of 20 ml/hour. Aliquots (2 ul) of the void volume protein com-
bined with portions (2 ul) of the column fractions were used to
assay for the included fraction (see ‘‘Methods’’). The excluded
fraction was then assayed by combining 2-ul aliquots of the in-
cluded peak with 2-4] portions of the void volume region.
TaBLe I

Resolution of DN A-cellulose binding fractions,
dnaB protein, and protein i

Assays were performed as under “Methods.” Activity was
measured with 2- to 4-ug additions of dnaC protein (Fraction
TIT). Stage II was assayed with dnaC protein and an excess of
binding or nonbinding fractions. Stage III was assayed with
dnaC protein, DNA-cellulose binding fraction, and either an
excess of Sephadex G-150 excluded fraction or included fraction
to permit titration of the component to be assayed.

 

5861

TaBLe II
Stages in reconstitution

Assays were performed as described under ‘“‘Methods.’’ Where
indicated, reactions contained 2 to 5 ug of dnaC protein (Fraction
TIT), 20 ug of Fraction II (depleted of dnaC protein), 10 ug of
DNA-cellulose binding proteins, 10 ug of DNA-cellulose non-
binding proteins, 40 ng of protein i (Fraction V), 20 ng of dnaB
protein (Fraction IV), 10 hg of NEM-treated DNA-cellulose
binding proteins, 0.3 ng of DNA polymerase III holoenzyme
(Fraction IV), 0.13 ug of protein n (Fraction V), 40 ng of dnaG
protein (Fraction V), and 0.73 ue of DNA unwinding protein
(Fraction 3b).

 

 

 

 

Total
Stages in fractionation
Protein Units Specie
mg/ml x 107% untils/mg
Extract....000000000 000.0000 ccc ee 19.7
Stage I
Ammonium sulfate................ 2.7 90 790
DEAE-cellulose................... 6.1 124 1,360
Stage IT
DNA-cellulose binding fraction.... 5.3 121 9,100
Nonbinding fraction............... 11.7 119 6,800
Stage IIT
Sephadex G-150 excluded fraction
(dnaB protein).................... 1.5 8.6 2,300
Included fraction (protein i)...... 0.6 40.2 | 13,400

 

 

Glycerol Gradient Sedimentations

These were performed in an SW 56 rotor of the Beckman L2-65B
ultracentrifuge. Fractions were collected through a puncture at
the bottom of the tube. Hemoglobin and myoglobin markers were
located by measuring Ao.

RESULTS

Strategy of Fractionation

The fractionation pattern of an extract of wild type cells can
be divided into five stages (Table II, Fig. 2).

Separation of dnaC Protein by Ammonium Sulfate—Fraction II
(see “Methods” for preparation of DNA-cellulose binding and
nonbinding fraction) was deficient in dnaC protein (Table II,
Stage I). The latter could be assayed with the dnaC-depleted
ammonium sulfate fraction from wild type cells or from dnaC
mutant cells (Table II, Footnote a).

Separation of DN A-cellulose Binding and Nonbinding Proteins
—Fraction II (depleted of dnaC protein) was applied to a column
of DNA-cellulose and the nonbinding (flow-through) and binding
fractions were collected. At this stage (Table II, Stage II), assays
required dnaC protein and both the DNA-cellulose binding and
nonbinding fractions.

Resolution of DN A-cellulose Nonbinding Proteins on Sephadex
G-150-—-Filtration resolved this fraction into excluded and in-
cluded activities (Fig. 1). Both were needed in addition to the
DNaA-cellulose binding proteins and dnaC protein (Table I;
Table II, Stage III). The excluded activity was identified as the
dnaB gene product by further purification (see below) and com-
plementation assays (Table III); the included activity, called
protein i, also appeared to be a single component upon purifica-
tion (see below), but no genetic locus has yet been found for it.

NEM-sensitive DN A-cellulose Binding Proteins—Reconstitu-
tion at Stage III (Table IT) was inactive when the DNA-cellulose
binding fractions were treated with NEM (Table II, Stage IV).
The NEM-sensitive components could be resolved by Sephadex
G-150 filtration into excluded and included fractions. The ex-

 

 

Stages
Component omitted
I? | II | Ill | IV Vv
pmol DNA synthesis
None (compiete mixture)......} 88 52 47 37 120
dnaC protein.................. 4.0/ 9.1] 0.4] 6.4 7.9
Fraction II (depleted of dnaC

protein).................... 2.0
DNA-cellulose binding pro-

teins..... eee 2.0} 1.1
DNA-cellulose nonbinding

proteins..................... 1.9
Proteini...............00.005, 6.7] 8.7] 13.9
dnaB protein.................. 4.0| 7.6| 24.9
NEM-treated DNA-cellulose

binding proteins............ 6.7
DNA polymerase III holoen-

ZYME... eee 1.8 2.2
Protein n.................205. 6.6 5.4
dnaG protein................. 8.0
DNA unwinding protein....... 1.9

 

 

 

 

 

 

* A complementation assay of the dnaC protein (Fraction ITI)
gave a value of 60 pmol with the dnaC mutant Fraction II (40
ug), but only 4 pmol without it; the value for Fraction II by itself
was 2.0 pmol.

SOLUBLE EXTRACT
AMMONIUM SULFATE

 

1

SOLUBLE

ELUATE

NEM TREATMENT

RESISTANT

INSOLUBLE

 

ONA-CELLULOSE

 

FLOW-THROUGH
SEPHADEX (G—150)

EXCLUDED INCLUDED

 

 

SENSITIVE
[sos BproTein | | PROTEIN: | [goss PROTEIN | G-180
alent ala EXCLUDED INCLUDED

Fie, 2. Scheme for resolution of the ¢X174 replicative enzymes.

cluded fraction was identified as DNA polymerase III holoen-
zyme (12). The other NEM-sensitive activity, called protein n,
could then be assayed upon the addition of NEM-treated DNA-
cellulose binding proteins, DNA polymerase III holoenzyme,
DNA-cellulose nonbinding proteins, and dnaC protein. Protein
n has been purified to near homogeneity (see below) but has not
been identified with any of the known genetic loci.
NEM-resistant DN A-cellulose Binding Proteins—Complete re-
constitution was achieved by replacing the NEM-treated DNA-
cellulose binding fraction by the E.coli DNA unwinding protein
(11) and dnaG protein (Table II, Stage V). Purification of
5862

Tasie III
dnaB Complementation assay

Escherichia coli BT1029 was grown and soluble extracts were
prepared as described for dnaC mutant enzyme fraction. Am-
monium sulfate was added to Fraction I (0.240 g/ml) at 0°. The
suspension was centrifuged at 10,000 X g for 20 min. The pellet
was dissolved in 9 volume of Buffer A without NaCl. Assays
were performed as described for dnaC complementation. Addi-
tions of dnaC protein were 9 units of Fraction III.

 

dnaC protein
Protein added to dnaB mutant fraction

None Present

 

bmol DNA synthesis

None..........0... 000. c eee ee eee 8.0 11.8
dnaB protein..................... 13.5 54.6
DNA unwinding protein.......... 12.2 11.4
Protein i... 6.6.0.0... eee eee 11.3 17.3
Protein n.............0.0.2.-.-.05. 12.3 12.3
dnaG protein. .............0.2.... 12.2 9.0
DNA polymerase ITI holoenzyme. . 12.7 9.4

 

 

these proteins based on their role in the conversion of phage G4
single-stranded DNA to RF are presented elsewhere (11)?

The product at Stage V was RF II with a nearly full length
linear synthetic strand (8).

The procedures described above and outlined in Fig. 2 were
guides to the existence and general properties of the several pro-
teins and provided assays for them. The endogenous (over-all)
replication activity of the extract was 800 to 1000 units/g of cell
paste. On this basis, the activity of the individual components,
expressed as units/g of cell paste, was: dnaB protein, 7000; dnaC
protein, 3000; dnaG protein, 8000; DNA polymerase III holo-
enzyme, 5000; protein n, 1400; protein i, 1100 units. In recent
experiments with protein i activity levels of 5000 units/g of cell
paste have been observed. Thus, each of the activities was present
at a level equal to or greater than the endogenous activity. Inde-
pendent purification procedures had to be developed to obtain
each protein in optimal yields. The purification procedures for
dnaC protein, protein i, dnaB protein, and protein n are given
below.

Purtfication of dnaC Protein

This protein is the activity most readily identified as specific
for ¢X174 and also the most labile. Although dnaC activity can
be isolated from E. colt H560, only 165 units/g of cell paste were
recovered in extracts compared to 2600 to 3000 units/g for E.
coli HMS-83. Beyond the enrichment of about 70-fold (Table IV)
further attempts at purification have been frustrated by loss of
activity.

Glycerol gradient sedimentation of dnaC protein (Fig. 3) gave
an § value of 2.0 corresponding to a molecular weight of about
20,000.

Purification of dnaB Protein

The purification of dnaB protein is summarized in Table V.
This protein was initially identified as the excluded factor on
Sephadex G-150 gel filtration and subsequently shown to comple-
ment a crude fraction prepared from a dnaB temperature-sensi-
tive mutant (Table III). In order to complement this mutant it
was necessary to add dnaC protein in addition to dnaB protein.
Similar pleiotropic requirements seen with other mutant extracts

TaBLe IV
Purification of dnaC protein

Escherichia coli HMS83 (335 ml, 65 g) prepared as for H560
(see ‘‘Methods’’) was thawed at 10° and diluted with 150 ml of
0.05 m Tris-HCl (pH 7.5)/10% sucrose. To the suspension was
added 1.2 ml of 0.4 m dithiothreitol, 18.5 ml of 4m KCl, 4.8 ml of
lysozyme (20 mg/ml), 4.8 ml of 10% Brij 58, 19.5 ml of 0.5 m
EDTA, and 9.7 ml of 1 Mspermidine-HCl. The pH was adjusted
to 8.5 with 1.5 g of solid Tris base. The suspension was incubated
for 25 min at 4°, then centrifuged at 48,000 X g for 30 min to
produce Fraction I (415 ml). Ammonium sulfate (0.240 g/ml)
was added; the precipitate collected as described under ‘‘Meth-
ods’’ was resuspended in 33.5 ml of AS 0.24. The undissolved
material was collected and dissolved in Buffer B containing 0.2
m KCl (Fraction II, 16 ml). Fraction II (5.5 ml) was dialyzed
against two 1-liter changes of Buffer B plus 0.2 m KCI for a total
of 2.5 hours. The sample was diluted 1 to 5 in successive 1-ml
portions in Buffer B and applied to a phosphocellulose column
(45 ml; 2.5-cem diameter) equilibrated in Buffer B, the column
was washed with 45 ml of Buffer B and dnaC activity was eluted
with a linear KCl gradient (250 ml, 0.05 to 0.4 m in Buffer B).
Pooled fractions (40 ml) precipitated with ammonium sulfate
(0.39 g/ml) were dissolved in 4 ml of Buffer B containing 0.2 u
KCI (Fraction IIT) and stored in aliquots in liquid nitrogen where
it was stable for at least 3 months. Fraction II may be stored at
—20°, but is unstable at 0°.

 

 

Fraction Total Protein Specific Yield oe
X 10° | me/mi | “mits! | oe
I. Extract ................. 284 | 17 40
II. Ammonium sulfate ¢.....| 214 | 78 170 | 75 4.3
III. Phosphocellulose’.......| 156 4.5 | 2650 | 55 | 70

 

@ Calculation of yield and purification was based on the dnaC
activity recovered in all of the ammonium sulfate fractions,
assays in Fraction I were not reliable.

+ This step was carried out in a portion of Fraction IT (5.5 ml)
but is calculated for the entire preparation.

 

60 T T T T
WHALE
HEMOGLOBIN MYOGLOBIN

50 }- ' } 4

UNITS / FRACTION
s
T
1

20 -- 4

10 =

 

 

 

0 i J
Qo 5 10 15 20 25 30 35

FRACTION

Fic. 3. Glycerol gradient sedimentation of dnaC protein. dnaC
protein (75 ug, 300 units) was applied to a 3.8-ml 10 to 25% glycerol
gradient in 0.05 m imidazole-HCl (pH 7.0)/0.2 m KCI/1 mm
EDTA/1i mo dithiothreitol. Centrifugation was at 55,000 rpm for
24 hours at 0°. Thirty-four fractions were collected, assayed, and
marker positions were determined as described under ‘‘Methods.”’
TaBLe V
Purification of dnaB protein

Escherichia colt H560 (1.5 liters, 300 g of frozen cells) was
lysed to yield 1180 ml of Fraction I. Ammonium sulfate was
added (0.226 g/ml); the precipitate was collected and resuspended
in 118 ml of AS 0.20; the pellets were treated successively with
24-ml aliquots of AS 0.16, AS 0.15, and AS 0.14. The supernatant
fluids from these last three washes were combined and the dnaB
activity reprecipitated with ammonium sulfate (0.1 g/ml). The
precipitate was collected and resuspended in Buffer D containing
0.25 m NaCl (Fraction II, 10 ml). The sample was applied to a
column of Bio-Gel A-1.5m (200 to 400 mesh, 475 ml, 3.8-cm diam-
eter) equilibrated in and eluted with Buffer D containing 0.25 m
NaCl. Fractions with dnaB activity (100 ml), eluted at a Kay of
0.36 to 0.64, were treated with ammonium sulfate (0.24 g/ml);
the precipitate was dissolved in 3 ml of Buffer D containing 0.05
mM NaCl (Fraction III, 3.2 ml). The sample was applied to a
column of Sephadex G-25 (40 ml, 2-cm diameter) equilibrated in
Buffer D containing 0.05 m KCl and the void volume fractions
were pooled (10 ml) and applied to a column of DEAE-cellulose
(20 ml, 2-em diameter) equilibrated in Buffer D (0.05 m NaCl).
The column was then washed with 10 ml of the same buffer fol-
lowed by 30 ml of Buffer D containing 0.15 mM NaCl; dnaB activity
was eluted with a linear NaCl gradient (120 ml, 0.15 to 0.40 m in
Buffer D) at about 0.35 m Cl- and concentrated on DEAE-cellu-
lose. Aliquots (2.5 ml) of the pooled activity (82 ml) were each
diluted with 3 volumes of 20% glycerol/1 mm EDTA and applied
sequentially to a column of DEAE-cellulose (2 ml], 1-em diameter)
equilibrated in Buffer D. The activity was then step eluted with
10 ml of Buffer D + 0.40 mM NaCl and 1-ml fractions were collected.
The dnaB activity was pooled (Fraction IV, 2.8 ml) and dialyzed
against three 500-ml changes of Buffer E for a total of 5 hours.
The sample was applied to a column of phosphocellulose (10 ml,
l-cm diameter) equilibrated in Buffer E. The column was then
washed with 20 ml of Buffer E and dnaB activity eluted with a
linear KCl gradient (30 ml, 0 to 0.40 m KCl in Buffer E) at about
0.3 m CI-. The pooled activity (Fraction V, 6.2 ml) was stored in
aliquots in liquid nitrogen where it was stable for at least 3
months.

Fraction II may be stored at —20°. The activity is not stable
at low ionic strength (<0.1 m Cl-) at 0° for more than 1 day.

 

 

Fraction ira Protein Specific Yield 7 ‘urib-
1073 | mg/ml unitsime %
I, Extract............... 2000 | 20 0.087
II. Ammonium sulfate*. ..; 1720 | 13.2 13 81 150
III. Bio-Gel A-1.5m........ 555 | 8.8 25 34 290
IV. DEAE-cellulose....... 295 | 0.3 | 350 14 | 4020
V. Phosphocellulose...... 73 | 0.02 | 590 4 | 6780

 

« Calculation of yield and purification was based on the activity
recovered in all of the ammonium sulfate fractions; assays in
Fraction I were not reliable.

have complicated the use of this approach to purify the deficient
protein.

Preliminary characterization of dnaB protein indicates a native
molecular weight of about 250,000 in agreement with the pub-
lished value (4). The function of dnaB protein has not yet been
elucidated.

Purification of Protein +

Protein i was purified 30,000-fold by the procedure summarized
in Table VI. Sodium dodecy] sulfate gel electrophoresis indicated
a single major band which migrated slightly slower than DNA
unwinding protein and thus had a monomer molecular weight of

5863

Tass VI
Purification of protein ¢

Fraction I (2130 ml) prepared from a lysate of Escherichia coli
H560 (3.0 liters, 600 g) was treated with ammonium sulfate as
described for dnaB protein (Table V) except that 213 ml of AS
0.30 and 43 ml of the AS 0.16, AS 0.15, and AS 0.14 were used.
The final precipitate (AS 0.14 pellet) which contained the bulk
of protein i activity (as well as DNA polymerase III holoenzyme
(12)) was resuspended in 25 ml of Buffer A (Fraction II, 30 ml),
clarified, and applied to a column of Sephadex G-25 (250 ml, 3-
em diameter) equilibrated in and eluted with Buffer C. The
Sephadex G-25 void volume fractions of conductivity equivalent
to Buffer C were pooled and applied to a column of DEAE-cellu-
lose (50 ml, 2-cm diameter) equilibrated in Buffer C. Protein i
activity was eluted with a linear NaCl gradient (500 ml, 0 to 0.3
m NaCl in Buffer C) at about 0.15 m Cl- (Fraction III, 50 ml),
concentrated with ammonium sulfate (0.24 g/ml) and resuspended
in 5 ml of Buffer D. This sample was applied to a column of
Sephadex G-150 (250 ml, 2.5-em diameter) equilibrated with
Buffer D. Protein i activity was eluted at K,, = 0.25 (Fraction
IV, 30 ml). This fraction was concentrated on a column of DEAE-
cellulose (2.5 ml, 1.2-cem diameter). After equilibration in Buffer
D, protein i activity was step eluted with 10 ml of Buffer D con-
taining 0.2 m NaCl; protein i (6 ml) was dialyzed against two
500-m1 changes of Buffer E for a total of 5 hours and immediately
applied to a column of phosphocellulose (5 ml, 1-em diameter)
equilibrated in Buffer E. The column was washed with 4 ml of
Buffer E and protein i activity eluted with a linear KCl gradient
(50 ml, 0 to 0.4 m KCl in Buffer E) at about 0.2 m Cl-. The pooled
activity (Fraction V, 5 ml) was stored in aliquots in liquid nitro-
gen where it was stable for at least 6 months.

Fraction II may be stored at —20°, but once dissolved should
be chromatographed on DEAE-cellulose immediately.

 

 

Fraction Pati Protein Speciic Yield sal
X 108 | ma/me | MRiime|
I. Extract... ............ 690 | 25 0.013
II, Ammonium sulfates. .| 420 1.9 7.3 60 590
TI. DEAE-cellulose......| 138 0.23 | 12 21 980
IV. Sephadex G-150...... 113 0.14 | 27 17 2,180
V. Phosphocellulose..... 80 0.043/370 12 | 29,600

 

 

* Calculation of yield and purification was based on the ac-
tivity recovered in all of the ammonium sulfate fractions; assays
in Fraction I were not reliable.

about 20,000.? Glycerol gradient sedimentation of protein i (Fig.
4) gave an 8 value of 3.0 corresponding to a molecular weight of
about 35,000 to 40,000 indicating protein i is either an asymmetric
monomer or a dimer. From the data of Table VI we estimate
about 100 to 150 molecules of protein i per cell. No function for
protein i has yet been determined. Based on similarities in the
purification procedure and physical properties it appears that
protein i corresponds to the Factor X recently reported by
Wickner and Hurwitz (9).

Purification of Protein n

Protein n was purified about 2,000-fold by the procedure in
Table VII. Sodium dodecyl sulfate gel electrophoresis indicated
a single major band at 82,000 daltons and minor bands at 25,000
and 50,000 daltons. The activity appeared to be associated with
the major band. Fraction VI was estimated to be about 80% pure
by Coomassie blue staining. From the data of Table VII about

3 R.McMacken and A. Kornberg, unpublished work.
5864

 

 

 

 

25 T T
HEMOGLOBIN MYOGLOBIN
{
20 7
2
°
5 15 7
<
ae
w
+
nn
5 10h 4
2
5 =
: L i i
10 20 30 40 50
FRACTION

Fic 4. Glycerol gradient sedimentation of protein i. Protein i
(2.4 ng, 900 units) was applied to a 3.8-ml, 10 to 30% glycerol gradi-
ent in: 0.05 m Tris-HCI (pH 7.5)/0.20 m NaCl/1 mm EDTA. Cen-
trifugation was at 55,000 rpm for 16 hours at 0°. Forty-three frac-
tions were collected, assayed, and marker positions were deter-
mined as described under ‘‘Methods.”’

200 molecules of protein n are present per cell. Protein n aggre-
gates at protein concentrations greater than 0.1 mg/ml and salt
concentrations less than 0.25 m making estimation of the native
molecular weight inaccurate. The protein binds specifically to
single-stranded DNA dramatically altering the contour length.‘

DISCUSSION

What seemed to be a simple operation of copying the small,
single-stranded DNA circle of ¢X174 to form the duplex requires
a multienzyme system. The many proteins which make up this
system hold great interest because they appear to be the compo-
nents which the uninfected Escherichia colt uses to initiate and
sustain the replication of its own chromosome. Thus, the resolu-
tion and reconstitution of this system made possible by the sim-
plicity of the phage DNA template has implications for under-
standing replication of duplex DNA of £. coli. In an analogous
way the filamentous phage M13 has been a useful probe for
another replicative system in E£. colt, one which appears to Le
reserved for replication of extrachromosomal elements (13).

It simplifies discussion to consider three major stages in the
synthesis of a DNA chain: initiation, elongation, and termina-
tion. Both the @X174 and M13 replication systems are similar
in their requirement for an RNA-primer fragment synthesized
in the initiation stage, for the DNA polymerase III holoenzyme
and DNA unwinding protein to sustain the elongation stage, and
for DNA polymerase I and ligase to carry out the termination
stage (e.g. RF II conversion to RF I) (8). The two systems differ
in the mechanism used for synthesis of the RNA primer. Initia-
tion on M13 DNA depends on a form of the multisubunit RNA
polymerase and so is sensitive to inhibition by rifampicin. The
@X174 system, on the other hand, is resistant to rifampicin and
depends on a novel RNA synthetic enzyme.”

Eight proteins have been resolved from extracts of E. colt
that are needed for reconstituting the ¢X174 single strand con-
version to RF II. Available information about their physical
and functional properties, summarized in Table VIII, is incom-

4J.H. Weiner and A. Kornberg, unpublished work.

Tasue VII
Purification of protein n

Escherichia colt H560 (700 ml, 140 g) was lysed to yield Fraction
I (575 ml). Ammonium sulfate was added (0.226 g/ml) and the
precipitate collected and resuspended in 58 ml of AS 0.24. The
precipitate was collected and the procedure repeated with 11-ml
aliquots of AS 0.20 and AS 0.18. The final precipitate was col-
lected, dissolved in 8.5 ml of Buffer D containing 1 mm dithio-
threitol, clarified and applied to a Sephadex G-25 column (150 ml,
8-cm diameter) equilibrated in and eluted with Buffer D (1 mm
dithiothreitol).

To the pooled void volume fraction NaCl was added to a con-
centration of 0.2 mM. Fraction II (84 ml) was applied to a DEAE-
cellulose column (10 ml, 2-cm diameter) equilibrated in and
eluted with the same buffer. The protein fractions in the flow-
through were pooled, precipitated with ammonium sulfate (0.313
g/ml), and the precipitate was collected and resuspended in 3.3
ml of Buffer B. This fraction was then applied to a Sephadex
G-25 column (45-ml, 2-cm diameter) equilibrated in and eluted
with Buffer B. The pooled Sephadex G-25 void volume protein
(Fraction III, 7.5 ml) was applied to a DNA-cellulose column
(12 ml, 4-cm diameter) equilibrated in Buffer B. The column
was washed with 100 ml of Buffer B and the protein n activity
was eluted with 40 ml of Buffer B containing 2m NaCl. The eluate
was precipitated with ammonium sulfate (0.226 g/ml) and dis-
solved in Buffer D (1 mm dithiothreitol) (Fraction IV, 4 ml).
This sample was dialyzed against 1 liter of Buffer D (1 mm dithio-
threitol) for 12 hours and the precipitate which formed was col-
lected by centrifugation at 41,000 X g for 60 min and suspended
by homogenizing it in 4 ml of Buffer B (Fraction V). The pre-
cipitate was again collected and the wash was repeated. The
final protein pellet was resuspended in 2 ml of Buffer B contain-
ing 0.25 mM NaCl, clarified, and the supernatant (Fraction VI)
withdrawn. This fraction was stored in aliquots in liquid nitrogen
where it was stable for at least 6 months. At 0° Fraction VI is
stable for at least 2 months. Ammonium sulfate precipitates of
each of the fractions were stable on storage at 0° for at least 2
weeks.

 

 

Fraction Total | Protein | SP&IBS | yield a
x 10-8 | mg/ml uaiteime %

I. Extract.............. 190. | 16.7 0.016
II. Ammonium sulfates. .} 128 2.9 1.3 87 90
III. DEAE-cellulose. ..... 48 6.4 1.0 34 70
IV. DNA-cellulose........ 35 2.3 3.8 24 250

V. Low ionic strength

precipitation......... 29 0.36 | 20 20 | 1,320
VI. Wash................. 12 0.17 | 34 8 | 2,160

 

@ Calculation of yield and purification was based on the ac-
tivity recovered in all of the ammonium sulfate fractions; assays
in Fraction I were not reliable.

plete in most instances. It seems clear that the polymerase IJI*
and copolymerase III* (6) subunits of the DNA polymerase III
holoenzyme are needed for DNA synthesis and that binding of
the DNA template by the DNA unwinding protein is required
both for initiation and elongation.

A strong clue to the function of the dnaG protein has been
furnished recently by its action in the conversion of the viral
single-stranded DNA of G4, a phage related to $X174.5 In this
case, the RNA primer is synthesized by the dnaG protein in the
presence of DNA unwinding protein;? the other proteins needed

5K. Zechel, J.-P. Bouché, and A. Kornberg (1975) J. Biol.
Chem. 250, in press.
 

 

 

 

 

 

 

 

Taste VIII
Summary of protein properties
DNA |NEM-

Protein bind- sensi- hon Moe Function
dnaC............. + + | dnaC | 20,000 pb
dnaB............. _- — | dnaB | 250,000¢ ?
Protein i.......... ck - ? 40,000 ?
Proteinn......... +]+ ? 80,000 | DNA binding
DNA polymerase

IIT holoenzyme? | + | + | dnaE | 330,000¢| DNA synthesis
dnaG............. + | — | dnaG | 65,000/; RNA synthesis
DNA-unwinding

protein......... +i - ? 76,000?| DNA binding

« Denatured calf thymus DNA-cellulose and 50 mm imidazole-
HCi (pH 7.0).

> ?, unknown.

¢ Ref. 4.

4 DNA polymerase III* (dnaE locus) + copolymerase III*
(77,000-dalton subunit, gene locus unknown).

* Ref. 12.

t Footnote 2.

° Ref. 11.

for ¢X174 are dispensable (8). It seems reasonable that dnaG
protein would perform the RNA synthetase function of ¢X174
DNA as it does for G4 DNA and that the four additional proteins
may serve by orienting regions of the ¢X174 DNA into a form
suitable as an origin (“‘promoter”’ for RNA synthesis).

Current studies of a complex of protein n with @X174 DNA
show a dramatic change in conformation of the DNA. It is re-
duced 6- to 7-fold in contour length and appears as a string of
beads, represented by about 25 molecules of protein n. As for the
functions of protein i, dnaB protein, and dnaC protein nothing
is known as yet. Protein i, like protein n and the DNA unwinding
protein, is required in the reaction at a level of approximately 25
molecules/DNA circle; the dnaB protein may be needed at a
lower ratio and is known to have an ATPase activity stimulated
by DNA (14). Probably some or all of these proteins interact
physically to form the assembly unit required for the initiation
events.

Among the eight proteins discussed, four are still unassigned
to genetic loci. Mutants defective in proteins i or n, copolymerase
III*, or the DNA unwinding protein have yet to be discovered
or may be identified with one of the recently reported dnaH (15),
dnal (16), or dnaZ (17) thermosensitive replication mutants. The
product of the dnaA gene of the established loci for DNA replica-
tion, is not among those needed for reconstituting the ¢X174
conversion. Nevertheless a dependence on a dnaA gene product
was clearly implied in earlier complementation studies with un-
fractionated cell extracts (2). Its function may prove to be

5865

indirect, such as the neutralization of an inhibitory effect of the
defective dnaA product contributed by the mutant extract.

The requirement for spermidine, as well as for the DNA un-
winding protein (8) is puzzling in view of the antagonistic func-
tions these molecules generally perform. Binding of spermidine
to DNA, which favors helix formation may be essential for main-
taining a certain conformation at a particular stage in the reac-
tion, whereas the unwinding protein may serve by binding the
single-stranded part of the molecule for other operations in the
reaction. Whether the participation of either the polyamine or
the unwinding protein in this enzyme system is an accurate guide
to their physiological roles must remain undecided. The fully
single-stranded DNA template provided in the in vitro system is
not found in the cell. Instead decapsidation of the phage particle
and penetration of the DNA into the cell is tightly coupled to its
replication. Current studies designed to reconstitute the molecu-
lar details of these early stages of infection may clarify some of
these questions.

Note Added in Proof—Procedures for stabilization and purifi-
cation of the dnaC protein now yield a preparation about 20%
pure with a specific activity of 80,000 units/mg (see Table IV
for comparison).

REFERENCES

1. WickneR, W., Bruttaa, D., ScHEKMAN, R., AnD KORNBERG,
A. (1972) Proc. Nail. Acad. Sci. U. 8. A. 69, 965-969

2 Scnexman, R., Wickner, W., Wesrercaarp, O., Bruriaa,
D., Gerper, K., Bertscu, L., anp Kornsere, A. (1972)
Proc. Natl. Acad. Sct. U.S. A. 69, 2691-2695

3. Wickner, R. B., Wrient, M., WiIckNER, S., anp Hurwitz, J.
(1972) Proc. Natl. Acad. Sci. U. 8. A. 69, 3233-3237

4. Wricat, M., Wickner, S., anp Hurwitz, J. (1973) Proc. Natl.
Acad. Set. U. 8. A. 10, 3120-3124

5. Wickxner, 8., Berkower, I., Wricut, M., anp Hurwitz, J.
(1973) Proc. Natl. Acad. Sct. U. S. A. 70, 2369-2373

6. Wickner, W., ScHEKMAN, R., GEIDER, K., anD KoRNBERG, A.
(1973) Proce. Nail. Acad. Sct. U. 8S. A. 70, 1764-1767

7. Wickner, S., Wrient, M., anp Hurwitz, J. (1973) Proc. Nail.
Acad. Sct. U. 8. A. 70, 1613-1618

8. ScHEKMAN, R., WEINER, A., AND KoRNBERG, A. (1974) Science
186, 987-993

9. Wicxner, S., anp Hurwitz, J. (1974) Proc. Natl. Acad. Sci.
U.S.A. 71, 4120-4124

10. Atserts, B., awn Herrick, G. (1971) Methods Enzymol. 21,
198-217

11. Weiner, J. H., Bertscu, L. L., anp Kornpere, A. (1975) J.
Biol. Chem. 260, 1972-1980

12, WickNER, W., AnD Kornpera, A. (1974) J. Biol. Chem. 249,
6244-6249

13. Gerper, K., anp Kornpera, A. (1974) J. Biol. Chem. 249,
3999-4005

14. Wickner, S., Wricut, M., anp Hurwitz, J. (1974) Proc. Nail.
Acad. Sci. U. 8S. A. 11, 783-787

15. Komano, T., anp Saxar, H. (1972) in Molecular Biology Meet-
ing of Japan, Kyoritsu Shuppan Co., Tokyo

16. Borersmann, D., Messer, W., ano Scuuicut, M. (1974) J.
Bacteriol. 118, 783-787

17. Fine, C. C., ALuen, J. S., Gustarson, R. A., ALLEN, R. G.,
AND WALKER, J. R. (1974) J. Bacteriol. 119, 443-449