Vol. 266, No. 32, Issue of Novemher 15, pp. 21681-21686.1991
Printed in U.S.A.
THEJOURNAL
OF BIOLOGICAL
CHEMISTHY
(01991 by The American Society for Biochemistry and Molecular Biology, Inc.
Analysis of the ATPase Subassembly Which Initiates ProcessiveDNA
Synthesis byDNA Polymerase I11 Holoenzyme*
(Received for publication, May 13, 1991)
Rene Onrust, P. Todd Stukenberg, and Mike O’Donnell
From the Howard Hughes Medical Institute, Microbiology Department, Cornell University Medical College,
New York. New York 10021
The y complex (y66’xJ.) subassembly of DNA poly- tion complex, the holoenzyme israpid in synthesis (>500
merase I11 holoenzyme transfers the /3 subunit onto nucleotides/s) and replicates the entire templatewithout disprimed DNA in a reaction which requires ATP hy- sociating from the DNA even once (i.e.it is highly processive)
drolysis. Once on DNA, B is a “sliding clamp” which (4-6).
tethers the polymerase to DNA for highly processive
The remarkable processivity of the holoenzyme requires its
synthesis. We have examined B and the y complex to accessory proteins (4).The three subunitcore subassembly of
identify whichsubunit@)hydrolyzes ATP. We find the the holoenzyme contains the DNA polymerase subunit ( a )
y complex is a DNA dependent ATPase. The /3 subunit, (7), the proofreading 3’-5‘ exonuclease subunit (6) (8), and
which lacks ATPase activity, enhances the y complex the 6’ subunit (9). The core polymerase synthesizes DNA a t a
ATPase when primedDNA is used as an effector.
Hence, the y complex recognizes DNA and couples ATP rate of approximately 20 nucleotides/s (10) and is only prohydrolysis to clamp B onto primed DNA. Study of y cessive for approximately 11 nucleotides (4). However, the
complex subunits showed no single subunit contained highly processive character of the holoenzyme can be reconsignificant ATPase activity. However, the heterodi- stituted upon mixing the core polymerase with both the /3
y complex (y66’xll/ subunits) (3,11,
mers, y6 and yb‘, were both DNA-dependent ATPases. subunit and the 5-protein
12).
Only the y6 ATPase was stimulated by B and was funcReconstitution of the processive polymerase activity of the
tional in transferring the B from solution to primed
DNA. Similarity in ATPase activity of DNA polymer- holoenzyme can be divided into two distinct stages (3,11,12).
ase I11 holoenzyme accessory proteins to accessory pro- In the first
stage, the y complex and p subunit hydrolyze ATP
teins of phage T4 DNA polymerase and mammalian to form a preinitiation complex on primed DNA. In thesecond
DNA polymerase 6 suggests the basic strategy of chro- stage, the core polymerase assembles with the preinitiation
mosome duplication has been conserved throughout complex to form the highly processive enzyme. Thus, itis the
evolution.
preinitiation complex which confers such remarkableproces-
DNA polymerase I11 holoenzyme is the principle replicase
of the Escherichia coli chromosome (1). In common with
chromosomal replicases of phages T4 and T7, yeast, Drosophila, mammals, and their viruses, the E. coli replicase is
composed of a DNA polymerase subunit accompanied by
accessory proteins. DNA polymerase I11 holoenzyme (holoenzyme)’ contains a t least 10 subunits ( a , C, 8, T , 8, y,6, 6’, x,
$) (2). The holoenzyme hydrolyzes two molecules of ATP to
form a tightly boundinitiation complex on a primedtemplate
(3). The DNA substrate often used in holoenzyme studies is
a bacteriophage single-strand (ss) DNA circular genome (5.48.6 kb) “coated” withthe E. coli SSB protein andprimed with
a single oligonucleotide (3-6). Upon formation of the initia-
* This work was supported by National Institutes of Health Grant
GM 38839 and an Irma T. Hirschel Award (to M. O’D.). The costs of
publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18U.S.C. Section 1734 solelyto indicate
this fact.
The abbreviations used are: holoenzyme, DNA polymerase 111
holoenzyme;core polymerase, DNA polymerase I11 core (ad);
polIII*,
holoenzyme lacking p; y complex, five-subunit subassembly of the
holoenzyme containing y, 6, S’, x, $; ssDNA, single-strand DNA;
dsDNA, double-strand DNA 6x174, bacteriophage 6x174; PCNA,
proliferating cell nuclear antigen; RFI DNA, supercoiled plasmid
DNA; RFIII DNA, linear duplex DNA; SSB, E. coli single-strand
DNA binding protein; BSA, bovine serum albumin; SDS, sodium
dodecyl sulfate; DTT, dithiothreitol; dAMP-PNP, 2’-deoxy-5‘-adenyl
imidophosphate.
sivity onto the holoenzyme. Further study of the first stage
in which the preinitiation complex forms showed the y complex acts catalytically to transfer the/3 subunit from solution
to primed DNA (2, 11, 33). Only the y and 6 subunits of the
y complex are essential to transfer p onto primed DNA (20).
Once on DNA, the p subunit slides freely along the duplex
portion of the primed template (33). The /3 subunit also
directly binds toa, the DNA polymerase subunit (33). Hence,
the preinitiationcomplex is a“sliding clamp” of the p subunit
on DNA, which tethers thepolymerase to theprimed template
and thereby confers onto it highly processive synthesis.
To further our understanding of highly processive synthesis, in this report, we examine the p subunit and y complex
to identify which subunit(s) hydrolyze the ATP.
MATERIALS ANDMETHODS
Sources-Radioactive nucleotides werefrom DuPont-New England Nuclear; unlabeled nucleotides were from Pharmacia LKB Biotechnology Inc.; DNA modification enzymes were from New England
Biolabs; RNAs were from Sigma; and pure proteins were prepared as
described SSB (16), CY (7), e (S), CYC complex (17), y (IS), p (E)), yx$
(20), 6 (20), 6’ (20). The y complex was purified as described (21)
with the following modifications: chromatography on Mono Q was
performed in place of the DEAE-Trisacryl step, andthe second
heparin-agarose chromatography step was replaced by chromatography on an ATP-agarose column and a phosphocellulose column. The
concentration of p was determined by absorbance using an e2”(, value
of 17,900 M” cm“ (19). The concentration of y was determined by
amino acid analysis (Protein Microchemistry Facility, Biological
Chemistry Department, University of Michigan). Concentrations of
a, e , SSB, y complex, and yx$ were determined by the method of
Bradford (22) using BSA as a standard. The concentrations of 6 and
21681
Initiating
ATPase
21682
of Polymerase ZII Holoenzyme
6' were determined by comparison of their Coomassie Blue staining
intensity (using a laser densitometer) in a SDS-polyacrylamide gel
with standard curves of y, 8, and BSA of known concentration
analyzed in the same gel (the relative staining intensities per microgram of y, 0, and BSA were all within 30%). The concentration of
ATP was determined by absorbance at 259 nm (ezb9 = 15,400 M"
cm").
DNAs"M13mpl8 ssDNA and 6x174were phenol-extracted from
phage purified by two successive bandings (one downward and one
upward) in cesium chloride gradients as described (23). DNA oligonucleotides were synthesized on an Applied Biosystems 381A DNA
synthesizer. The M13mp18 ssDNA was primed with a DNA 30-mer
(map position 6817-6846) as described (17). MP13mp18 form I DNA
was purified by banding twice in cesium chloride equilibrium density
gradients (24).
The DNA 317-mer was excised from wild type M13 ssDNA as
follows. Two DNA 15-mers were hybridized to M13 ssDNA at the
Sau96I siteand a ClaI site (map positions5719-5733 and 6035-6049,
respectively). The two DNA 15-mers (110 pg each) were annealed to
486 pg of M13 ssDNA in 540 p1 of 10 mM Tris-HC1 (pH 7.5), 10 mM
MgCl,, 1 mM DTT by heating to 95 "C and slow cooling to room
temperature. Both Sau96I (240 units, 60 pl) and ClaI (300 units, 60
pl) were incubated with the DNA for 18 h at 37 "C, after which
agarose gel analysis showed complete digestion by both enzymes. The
digest was quenched with 12 pl of 10%SDS. The 317-mer was resolved
from the large M13 DNA remnant and theDNA oligonucleotides by
gel filtration over a 25-ml fast protein liquid chromatography Superose 6 column in 10 mM Tris-HC1 (pH 7.5), 1.0 mM EDTA, 100 mM
NaCl.
The 317-mer (4 pg/ml) was primed using a 20-fold molar excess
each of a synthetic DNA 40-mer (map position 5753-5792) and a
synthetic DNA 30-mer (map position 5975-6004) as described (17)
followed by gel filtration to remove excess DNA oligonucleotides.
Poly(dA) .oligo(dT) was prepared by annealing oligo(dT)20with
(dA),, (Midland Certified Reagent Co.) at concentrations of 811 pg/
ml and 268 pg/ml, respectively.
ATPase Assays-ATPase assays were in 10 pl of 20 mM Tris-HC1
(pH 7.5), 8 mM MgC12, 50 p M [y-"PIATP (2-4 X lo6 cpm) and
contained 145 ng of DNA (unless noted otherwise). When present,
reactions contained 22 ng of y complex, 115 ng of y, 6.7 ng of 8, 7.1
ng of 8 ' , 20 ng of yx$. Proteins were added to reactions on ice, shifted
to 37 "C for 30 min, then 0.5 pl was spotted on aplastic-backed thin
layer chromatography sheet coated with Cel-300 polyethyleneimine
(Brinkmann Instruments Co.) and developed in 0.5 M lithium chloride, 1 M formic acid. Free phosphate at the solvent front and ATP
a t the origin were quantitated by liquid scintillation counting. Pi
released (pmol/pmol of protein/min) was calculated assuming the
concentration of protein complex equaled the concentration of the
limiting protein component (as monomer) added to theassay. Control
reactions showed ATPase activity was linear for over 30 min at which
time less than 15% of the ATP had been hydrolyzed.
Replication Assays-Replication assays contained 140 ngof primed
M13mp18 ssDNA, 1.5 pg of SSB, 560 ng of ae, and 129 ng of 0 in 20
mM Tris-HC1 (pH 7.5), 8 mM MgC12,0.5 mM ATP, 60 pM dCTP, 60
p~ dGTP, 40 pg/ml BSA, 18 mM NaC1,4% glycerol, 5 mM DTT, 0.1
mM EDTA at a final volume of 25 pl (after addition of the y complex
or other subassembly). Complexes of 78, yd', and 786' were formed
upon incubating 0.92 pg of y with either 24 ng of 6,24 ng of a', or 12
ng each of 6 and 8' for 20 min at 15"C in 20mM Tris-HC1 (pH 7.5),
0.1 mM EDTA, 2 mM DTT, 20% glycerol. Various amounts of these
protein mixtures were added to theassay on ice and shifted to 37 "C
for 8 min to allow reconstitution of the processive polymerase on the
primed ssDNA. The dCTP and dGTP in the reaction mixture prevents the 3'-5' exonuclease activity in the e subunit of the ae polymerase from digesting the DNA oligonucleotide primer. A pulse of
DNA synthesis was initiated upon rapid addition of dATP and [a'*P]TTPto 60 and 20 p ~ respectively,
,
then quenched after 20
seconds by spottingonto Whatman DE81 filters. The radiolabel
incorporated into M13mp18 DNA was quantitated as described (25).
RESULTS
The y Complex Is a DNA-dependent ATPaseStimulated by
@-In the absence of DNA, neither the y complex (Table I),
p (not shown), nor a mixture of the two (Table I) demonstrated ATPase activity. This lack of ATPase activity in the
absence of DNA is consistent with previous studies, which
showed the holoenzyme hydrolyzes ATP upon binding to a
singly primed G4 bacteriophage ssDNA genome coated with
the E. coli SSB protein (17). Therefore, we added primed
DNA to the ATPase assays. To mimic templates known to
serve as substrates for the holoenzyme we used ssDNA of
natural sequence and coated it with SSB. Since only a few
molecules of ATP are hydrolyzed per primed DNA in the
holoenzyme initiation reaction (17), we increased the primer
density of the substrate by doubly priming a 317-mer ssDNA
excised from the M13 genome.
The y complex displayed ATPase activity in the presence
of the primed SSB-coated 317-mer (Fig. 1) and even showed
slight ATPase activity with the unprimed 317-mer (Fig. 1).
The ATPase activity was not likely a contaminant in the y
complex preparation as indicated by the coincidence of thermal inactivation rates of y complex ATPase activity and y
complex replication activity (with @ and at) (Fig. 2). Furthermore, ATPase activity was present in every y complex preparation and comigrated with the y complex during its purification (not shown). The y complex hydrolyzed dATP at a
rate similar to thatfor ATP and was unable to hydrolyze any
of the other (deoxy)ribonucleoside triphosphates (Table 11).
This is the same nucleoside triphosphate specificity (ATP
and dATP) as displayed by the holoenzyme in initiation of
processive DNA synthesis (4).
The /3 subunit showed no ATPase activity on the primed
317-mer (Fig. 1) or on the unprimed 317-mer (not shown).
However, addition of /3 to the y complex resulted in approximately 3-fold more ATPase activity (Fig. 1).We presume the
p subunit stimulated the ATPase activity of the y complex.
However, it remains possible that a latent ATPase activity
intrinsic to p becomes active upon interaction with the y
complex and DNA. The p stimulation of the y complex
ATPase was specificto theprimed 317-mer (Fig. 1); p did not
stimulate the y complex ATPase on the unprimed 317-mer
(Fig. 1). Stimulation by ,6 of the y complex ATPase was
observed over a wide range of y complex concentration (over
a 60-fold range, not shown).
Steady state analysis of the y complex ATPase on the
primed 317-mer showed p elevated the V,, of the ATPase
(from 25 to 80 mol of ATP hydrolyzed/min/mol of y complex)
and increased the K,,, for ATP 2-fold (from 26 to 49 p ~ (Fig.
)
3). The y complex ATPase was as active at 2 nM primed 317mer as at 20nM primed 317-mer (not shown). Hence the K ,
of the y complex ATPase for primed DNA is lower than 2
nM. The concentration of 3' primer ends used in these studies
was above 15 nM.
Various nucleic acids were examined for their ability to
induce the y complex ATPase (Table I). Although poly(dA)
was a poor effector of the y complex ATPase, both poly(dA).
oligo(dT) and oligo(dT) were good effectors, suggesting the
importance of 3' ends whether hybridized to ssDNA or not.
Linear duplex DNA (RFIII) was also an effector of the y
complex ATPase. Unprimed ssDNAs of natural sequence were
all effectors of the y complex ATPase. Secondary structure
within ssDNA was probably the inducer of the y complex
ATPase since coating natural sequence ssDNAs with SSB
diminished their effectiveness to near the level of poly(dA).
Addition of SSB to the primed 317-mer resulted in a %fold
enhancement of y complex ATPase. (However, SSB did not
enhance y complex ATPase activity on poly(dA).oligo(dT).
Perhaps on natural sequence ssDNA the SSB prevents some
y complex molecules from interacting nonproductively with
the DNA.) RNAs were not effectors of the y complex ATPase.
The p subunitstimulated the y complex ATPase on
poly(dA).oligo(dT), but not on poly(dA) alone or oligo(dT)
Initiating ATPase of Polymerase 111Holoenzyme
21683
TABLEI
ATPase activity of the y complex on various nucleic acid effectors
ATPase assays were as described under “Materials and Methods” except the 10-pl reactions contained 22 ng of
y complex. 42 ng of @ (when present), 980 ng of SSB (when present), and72.5 ng of nucleic acid. The primed and
umrimed 317-mer DNAs were Dresent at 25 ne.
Nucleic acid
y complex
y complex, 6
y complex, SSB
y complex, SSB, 6
mol P, releasedlmol y complexlrnin
No DNA
POlY(dA)
Oligo(dT)
Poly(dA). oligo(dT)
3.2
Unprimed 317-mer
13.1
Primed 317-mer
6x174 ssDNA
2.0
M13mp18 ssDNA
M13mp18 RFIII(Sma1 cut)
0.6
E. coli tRNA
Yeast RNA
a
I2O0
1000
t
0.3
0.3
4.1
6.5
6.6
4.5
6.7
6.8
3.4
0.8
1.2
0.1
0.3
2.9
10.8
9.6
10.9
14.8
13.2
9.6
0.9
0.6
0.4
0.7
1.4
11.4
3.1
39.5
0.5
0.6
11.8
0.4
0.2
0.5
0.5
1.3
4.0
2.9
4.0
0.9
TABLEI1
ycomplex. p,
primed DNA
y complex utilization of (deoxy)ribonucleoside triphosphates
Assays were as described under “Materials and Methods” except
the 10-p1 reactions contained 145 ng of poly(dA).oligo(dT), 44 ng of
y complex, 50 p~ [y-3”P](de~xy)ribonucle~~ide
triphosphate
((d)NTP).and 43 ne of R (when Dresent).
(d)NTP
Y
comulex
Y
comdex
+B
mol PCreleased/mol y compkxlmin”
L””
0.3
13.8
30
20
10
MINUTES
FIG. 1. The y complex ATPase depends on primed DNA and
is stimulated by
ATPase assays were performed as described
under “Materials and Methods” except the volume was 20 p1 and
contained 44 ng of y complex, 980 ng of SSB and one of the following:
22 ng of unprimed 317-mer (+), 22 ng of unprimed 300-mer and 86
ng of @ (open circles), 22 ng of primed 317-mer (closed squares), or 20
ng of primed 317-mer and 86 ng of @ (closed circles). The open squares
were reactions that contained 20 ng of primed 317-mer, 980 ng of
SSB, and 86 ng of @.
@.
ATP
CTP
GTP
UTP
dATP
dCTP
dGTP
dTTP
0.3
I
2520
0.3
0.3
0.2
0.0
of P,
0.3
(”-1)
FIG. 3. Steady state kinetic analysisof the y complex ATPase. ATPase assays were performed as described under “Materials
and Methods” except the assays contained 22 ng of primed 318-mer,
490 ng of SSB, and the indicated amount of ATP. Squares, without
@;
circles, plus 43 ng of @.
,ATPase
15
0.3
IIATP
,Reolication
10
14.6
0.3
0.3
“ T h e levelof detection in these assays was0.1-0.3mol
released/mol of y complex/min.
-0.10.1
5
7.6
0.3
0.3
0.3
7.4
0.3
0.3
30
TIME AT 45% (MINUTES)
FIG.2. Thermal inactivation rates of they complex ATPase
and replication activities. The y complex (440 ng) was incubated
at 45 “C in 80 pl of20mM Tris-HC1 (pH U ) , 0.1 mM EDTA, 2 mM
DTT, and 20% glycerol. At the times indicated, 5 p1 was withdrawn
and assayed for ATPase activity at 37 “C on poly(dA) .oligo(dT)
(circles) as described under “Materials and Methods,” and 0.5 p1 was
assayed for replication activity (squares) as described under “Materials and Methods” except for use of 2.2 pg of LYE, 17 ng of @,and a 2min incubation at 37 “C before the 20-s pulse of DNA synthesis.
alone, indicating p needs a 3’ end hybridized to ssDNA to
stimulate they complex ATPase. Interestingly, the
y complex
ATPase was stimulated by p on bacteriophage ssDNAs in the
absence of SSB but not in the presence of SSB, indicating
secondarystructurewithinthe
ssDNA was thesubstrate
effectorfor p to stimulate the y complex ATPase. p also
stimulated the y complex ATPase in the presence of RFIII
duplex DNA (Table I) (0 was not an ATPase in thepresence
of duplex DNA, not shown).
The y Complex Contains at Least Two ATPases-To identify the ATPase subunit(s) of the y complex, the y, 6 , and 6‘
Initiating ATPase of Polymerase III Holoenzyme
21684
12
10
6
W
+
6
3
z
4
1
2
U
W
10
a
w
6
r
lo
6
6
4
2
but was nearly as active on oligo(dT) (Fig. 4C).In theabsence
of DNA as well as in the presence of poly(dA) the ATPase
activity of the y6 was negligible (Fig.4,B and 0).
In our earlier work we found that 76' was not active in
reconstitutinga processive polymerase with the a€ and p
subunits (20). Hence, we were somewhat surprised to find
ATPase activity upon mixing y with 6' (6 and 6' are distinct
polypeptides'). The 7 6 ' ATPase was most active on poly(dA).
oligo(dT) (Fig. 4A), about one-third as active on oligo(dT)
(Fig. 4C),and was without activity on poly(&) (Fig. 4B)and
in the absence of DNA (Fig. 40).
A mixture of y, 6, and 6' subunits yielded more ATPase
activity on poly(dA). oligo(dT) than summation of the y6 and
76' activities, indicating formation of a $6' subassembly (Fig.
4A). Mixture of 6 and 6' (without y) did not yield ATPase
activity (Fig. 4A). The 766' ATPase was quite active on
oligo(dT) (Fig. 4C)but was less active on poly(dA) (Fig. 4B)
and in the absence of DNA (Fig. 40).
The yx$ complex exhibited very slight DNA-dependent
ATPase activity (Fig. 4A), although the preparation has not
been studied for a slight ATPase contaminant (i.e. the yx$
preparation is limited in availability). Nevertheless, significant ATPase activity was only gained upon mixing the yx$
complex with either 6 or 6' (Fig. 4A). Also, mixture of both 6
and 6' with yx$ produced more ATPase activity than summation of yx$6 and yx$6', indicating formation of a yx$66'
complex (Fig. 4A). The reconstituted 5 subunit y complex
was approximately 1.4 times more active than y66', indicating
the x$ subunits (also distinct genes)3 stimulate the ATPase
activity of y66' or have ATPase activity of their own. The
ATPase activity of the reconstituted y complex on poly(dA).
oligo(dT) was approximately 30% more active than the y
complex purified intact (Fig. 4A). This is within experimental
error of the protein concentration measurements.
The ATPase activities of all the subunit combinations were
as low on poly(dA) as in the absence of DNA (Fig. 4, B and
0,respectively). Oligo(dT) induced nearly the same level
(-7040%) of ATPase activity as poly(dA)-oligo(dT) (Fig.
4C).The exceptions were the 76' and yx$6' ATPases which
were only 20-30% as active on oligo(dT) relative to poly(dA).
oligo(dT). Perhaps y6 interacts with ssDNA ends more efficiently than 76'.
p Specifically Stimulates the ATPase Activity of the y6
Subassembly-The 766' ATPase was stimulated by @ (Fig.
5A) much like p stimulated the y complex (Fig. 1).Hence,
the x+ subunits are not required for p to stimulate the y
complex ATPase. The p stimulation of the 766' ATPase was
specific to thepoly(dA).oligo(dT) template and was not supported by poly(dA) alone or by oligo(dT) alone (Fig. 5A).
Furthermore, /3 did not stimulate the 76' ATPase on any of
these DNA substrates (Fig. 5 B ) . However, @ stimulated the
y6 ATPase 10-fold specifically on the primed template,
poly(dA).oligo(dT) (Fig. 5C). The greater /3 stimulation of
the y6 ATPase was due to thehigher amount of @-independent
ATPase in y66'. The /3 subunit affected the yx$6, yx$6', and
yx$66' ATPases in similar fashion as the 7 6 , yb', and $6'
ATPases, respectively (not shown). Furthermore, 6 did not
stimulate the low level ATPase of yx$ or of y (not shown).
Nor did @ reveal an ATPase upon mixture with 6, b', or 66'
(not shown).
The 6' Subunit Stimulates y6 in Replication-Previous
studies showed y6 was capable of reconstituting a fully pro-
I
I
6
6 M' I
I
I
nvnvnvnv
I
SUBUNITS
FIG. 4. Reconstitution analysis of y complex ATPases on
DNA templates. ATPase assays were as described under"Materials
and Methods" using the following as effector D N A A, poly(&).
oligo(dT); B , poly(&); C, oligo(dT); D, no DNA.
subunits and yx$ complex were assayed alone and in combinations in the presence of poly(dA)-oligo(dT) (Fig. 4A). The
individual y, 6, and 6' subunits were not ATPases (although
use of a large amount of y in the assay showed slight DNAdependent ATPase activity'). In earlier studies we showed a
y6 complex could be reconstituted upon mixing the y and 6
subunits (20). The y6 complex was active in reconstituting a
processive polymerase with the at and p subunits (20). We
also identified a DNA-dependent ATPase activity within y6
(26). The ATPase activity of y6 is characterized in Fig. 4.The
y6 ATPase was most active on poly(dA) .oligo(dT) (Fig. 4 A )
The DNA-stimulated ATPase in the y preparation (99%pure)
was only 0.05 mol of ATP hydrolyzed/mol of y/min on poly(&).
oligo(dT); no ATPase activity was observed on poly(&). Its identity
as y was suggested by comigration with y upon gel filtration and
during salt gradient elution of Mono Q, heparin-agarose, and AfiiGel Blue A columns. The ATPase was not contaminating y complex
(as Mono Q resolves y complex from y ) or 7 (which resolves from y
on heparin). @ did not stimulate the putative y ATPase.
' We have recently cloned and mapped the genes for b , 6 ' , x, and $
on the E. coli chromosome. They are all distinct and lie in previously
unidentified open reading frames(Z. Dong, R. Onrust, and M. O'Donnell, unpublished data).
Initiating
ATPase
of Polymerase III Holoenzyme
21685
DNA 30nnr,
W
5
3
K
W
P
5
1 51 0
20
25
30
$8’. $, -$’
fmol of 01th.r ycomp~or,
FIG. 6. The 6’ subunit stimulates yb in reconstitution of a
processive polymerase with fl and at. Replication assays were
performed as described under “Materials and Methods.” The scheme
above the plot summarizes the replication assay. The primed
M13mp18 ssDNA was preincubated with m , p, and y complex (or
subassembly) for 8 min to allow reconstitution of the processive
polymerase prior to initiating a 20-9 pulse of synthesis. Assays contained either y complex (circles),y66‘ (closed squares), yb (triangles),
or 7 6 ’ (open squares). The molar amount of subassembly added to
the reaction was taken as the total femtomoles of 6 added to the
assay. In the case of yb’ the ferntomoles of 6‘ were used. In the case
of y complex the femtomoles of 6 were calculated assuming one 6
subunit per 200-kDa y complex as determined in Ref. 21.
w4 1
W
K
1
h
W
6
=
20
40
60
BETA(ng)
FIG. 5. fl specifically stimulates the y6 ATPase of the y
complex. ATPase assays were performed as described under “Ma-
catalytic ability of the y complex to assemble multiple @
clamps on primed DNA circles (33). Once on DNA, the @
clamp and at polymerase are fully capable of processively
replicating the primed DNA circle within 20 seconds (33).
Hence, this assay actually measures the efficiency of y complex subassemblies in ability to transfer @ onto primed DNA.
DISCUSSION
The studies of this report show the y complex is a DNAdependent ATPase. The best DNA effector was primed DNA
terials and Methods” using the indicated amount of and either 766’ of natural sequence and coated with SSB. The @ subunit
( A ) ,yb’ (B),
or y6 (C). The DNA effectors were as follows: squures,
showed no detectable ATPase with or without DNA. However,
poly(dA).oligo(dT); triangles, poly(dA); circles, oligo(dT).
the y complex and primed DNA yielded approximately 3-fold
more
ATPase activity in the presence of @ than in its absence.
cessive polymerase with /3 and the at polymerase (20). The
Presumably,
/3 stimulated the ATPase activity of the y com76’ was not active in the reconstitution assay with /3 and at
plex, although the possibility that @ becomes an ATPase in
(20). We have found here that y6’ is a DNA-dependent
the presence of the y complex and primed DNA can not be
ATPaseand 6‘ stimulates the y6 ATPase. This led us to
ruled out. Study of the y, 6,6’subunits and y x J .subassembly
examine whether the 6’ subunit would stimulate y6 in the of the y complex showed none of these alone were significant
replication assay with @ and ae. To test this, the y6, y6’, y66’, ATPases. However, mixing experiments showed both y6 and
and y complex were individually titrated into reactions con- y6’ were DNA-dependent ATPases. Only the y6 ATPase was
taining @,
at polymerase, and primed M13mp18 ssDNA coated
stimulated by @.Furthermore, only y6 was active in reconstiwith SSB (Fig. 6). The subunits were preincubated with the tution of the processive polymerase with @ and at,although
primed DNA to allow reconstitution of the processive poly- 6’ stimulated y6 in the replication assay.
merase followed by a 20-s pulse of DNA synthesis. Previous
These results suggest the function of the y complex is to
studies have shown that even the minimal polymerase, recon- recognize DNA and couple ATP hydrolysis to clamp @ onto
stituted using 76, @,
and a c , is fully processive and yields the DNA as shown in Fig. 7. In the first diagram of Fig. 7,
completed duplex circles within 20 s (20). The y6 was active the y complex is shown on the ss/dsDNA-primed junction
in reconstituting a processive polymerase with @ and at;the with both ATP and @ bound to it. Evidence for ability of the
76‘ was inactive (Fig. 6 ) ,consistent with the previous studies y complex to bind ATP and primed DNA is its ATPase
(17). However, the y66‘ subassembly was much more active activity which depends on DNA and is maximally stimulated
than y6 in the reconstitution assay (Fig. 6). In fact, y66‘ was by primed DNA. Evidence the y complex on primed DNA
as effective as the entire y complex. Hence 6‘ stimulates both binds @ is the stimulation of the y complex ATPase by @ in
the ATPase activity and the replication activity of y6. The the presence of primed DNA. These experiments do not
greateramount of DNA circles replicated relative to the
address whether the y complex first binds DNA and then p,
amount of y complex (and y66’) added is explained by the or whether the y complex first binds @ and then DNA. These
Initiating ATPase of Polymerase 111 Holoenzyme
21686
the polymerase (31). The phage T4 gene44/62accessory
protein complex, like 76, is a DNA-dependent ATPase stimulated by 3' ends and is further stimulated by the T4gene 45
accessory protein (like @)(13). The T4system appears to lack
,
the ATPase analogous to y6' and the proteins analogous to x
y comileplex
y complex
and $. Likewise the human 6 polymerase is stimulated in
FIG. 7. Role of the y complex in transfer of @ to primed processive synthesis by its accessory proteins, the multiproDNA. The y complex binds the ATP, primed DNA, and 0 subunit,
then hydrolyzes ATP to clamp onto the duplex portion of the tein activator 1 (RF-C complex), and the PCNA protein (14,
32). Activator 1(RF-C), like the y complex, has five subunits
primed DNA.
(15), is aprimed DNA-stimulated ATPase, and is stimulated
early steps are under investigation. Furthermore, the natural additionally by PCNA (accessory protein with analogous
primed template for the y complex is a RNAaDNA hetero- function to @)(14,15). Theapparent conservation in function
duplex made by primase during replication of the lagging of polymerase accessory proteins spanningthe spectrum from
strand. Thus, studies of the exchange of an RNA primer E. coli to humans suggests the basic solution to problems of
terminus from primase to the y complex are necessary to replicating duplex DNA have gone unchanged throughout
further understandthe role of the y complex in lagging strand evolution.
DNA synthesis.
Acknowledgment-We are grateful to Maija Skangalis for the cell
In the second diagram of Fig. 7, the y complex hydrolyzes lysis
and initial ammonium sulfate fractionations of the several
ATP to clamp @ onto DNA. The subunit that binds the ATP kilograms of E. coli cells that made this work possible.
coupled to the @ clamp reaction must be y, 6, or @ since only
these three areneeded to form the preinitiation complex (20).
Addendum-Lee Fradkin and Arthur Kornberg have also studied
The apparentlack of ATPase activity in @ implies either y or the ATPase properties of the y complex and its interaction with 05
6 is the ATP-binding subunit. It is known that y binds ATP Their conclusions are similar to those described here?
tightly, making y a favorite candidate (30). Similar studies of
REFERENCES
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2. Maki, S., and Kornberg, A. (1988)J. Biol. Chem. 263,6561-6569
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Further evidence that ATP is coupled to formation of the preinitiation complex only at the point of the ternary complex of 0.y
complex.DNA as indicated by the following experiment. ATP was
incubated with either 1)y complex and primed M13mp18 ssDNA, 2)
0 and primed M13mp18 ssDNA, or 3) y complex and p. Then the
ATP was removed using hexokinase and glucose. Upon addition of
the missing component (either 0, y complex, or DNA) as well as core,
dCTP,dGTP, [a-"'PldTTP, anddAMP-PNP (a dATP analogue
which supports DNA synthesis but not the holoenzyme initiation
reaction (Ref. 5)) no replication of the M13mp18 ssDNA was observed. Hence the ATP was not utilized in the preincubation of two
out of three components prior to hexokinase treatment. Negative
results were also obtained when the experiment was performed by
preincubating just one component with ATP followed by removal of
ATP then addition of the other components. In the positive control,
preincubation of all the components (p, y complex, and primed
M13mp18 ssDNA) with ATP, followed by removal of ATP, yielded
replication of the M13mp18 ssDNA upon addition of core, dCTP,
dGTP, [a-"PIdTTP, and dAMP-PNP.
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