Summary
DNA polymerases which duplicate cellular chromosomes are multiprotein complexes. The individual
functions of the many proteins required to duplicate a
chromosome are not fully understood. The multiprotein
complex which duplicates the Escherichia coli chromosome, DNA polymerase I11 holoenzyme (holoenzyme),
contains a DNA polymerase subunit and nine accessory
proteins. This report summarizes our current understanding of the individual functions of the accessory
proteins within the holoenzyme, lending insight into why
a chromosomal replicase needs such a complex structure.
Introduction
DNA polymerase 111 holoenzyme (holoenzyme) is the
principal replicase of the E . colz chromosome(’). 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 multiple accessory proteins.
The E . colz holoenzyme contains at least ten subunits in
It has been proposed that chromoall (Table l)(2.3).
somal replicases may contain a dimeric polymerase in
order to replicate both the leading and lagging strands
concurrently(’,‘). Indeed the 1 MDa size of the
holoenzyme and apparent equal stoichiometry of its
subunits (except which is twice the abundance of the
others) is evidence that the holoenzyme has the
z~(
following dimeric composition: ( a ~ t ) ) ~ySS’p4)&(’).
One of the features of the holoenzyme which
distinguish it a5 a chromosomal replicase is its use of
ATP to form a ti ht, gel filterable, “initiation complex”
on primed [email protected] holoenzyme initiation complex
completely replicates a uniquely primed bacteriophage
single-strand DNA (ssDNA) genome coated with the
ssDNA binding protein (SSB), at a speed of at least 500
nucleotides per second (at 30°C) without dissociating
from an 8.6 kb circular DNA even
This
remarkable processivity (nucleotides polymerized in
one template binding event) and catalytic speed is in
keeping with the rate of replication fork movement in
E. coli (1 kb/second at 37°C)“”.
The focus of this minireview is to examine the
individual functions of the accessory proteins of the
holoenzyme. Since they copurify with the polymerase
which replicates the E . coli genome, it seems likely that
they function in chromosomal replication. What then
are possible actions one may anticipate of accessory
proteins in chromosomal replication? Do they increase
the speed or processivity of DNA synthesis? Do the
polymerase accessory proteins communicate with the
replication fork helicase which separates the duplex, or
with the primase which synthesizes the short RNA
primers needed for the discontinuous synthesis of the
lagging strand? Are accessory proteins needed to hold
two polymerase subunits together for coordinated
synthesis of the two strands of DNA? Perhaps accessory
proteins help recycle the polymerase during the
multiple reinitiation events on the thousands of RNA
primers which must be extended to replicate the lagging
strand (explained in more detail later). Are accessory
proteins also involved in the process of initiation or
termination of a cycle of chromosomal replication?
lmportant to the assessment of accessory protein
function has been the availability of subassemblies of
the holoenzyme (Table 1). Subassemblies include
p011II*(~)(holoenzyme lacking only /3), pol111 core(”)
(a heterotrimer of a& which contains the DNA
polymerase (a)(”), and 3 ‘ - ~ ’exonuclease (e)(16).
activities ~ o I I I I ’ ((a
~ ~dimer
)
of mcBz subunits), the y
(composed of 5 accessory proteins:
yfiS’XW), and a yx?) complex(’’). Due to the low
abundance of the holoenzyme in cells, these subassemblies are only available in microgram quantities.
Milligrams of pure a,E , T, y and [3 subunits arc available
Table 1. The subunits of D N A polymerase 111 holoenzyme
Subunit
Mass (kda)
Functions
130
27
10
71
DNA polymerase
Proofrcading 3’-5‘ exonuclease
Unknown
Dimenzes core, DNA-dependent ATPaw
17
35
33
Binds ATP
Interacts with y to transfer /s to DNA
DNA-dependent ATPase with y
15
Unknown
12
Unknown
30
Sliding clamp on DNA, binds core
Subassembly
pol111
molecule can act catalytically to form many /3 clamps on
multiple DNA molecule^(^,^,^^). The y complex
therefore has the characteristics of a chapcronin.
Namely, it acts catalytically to couple ATP to assembly
of a complex (fi. DNA). Only the y and b subunits are
required to clamp onto primed DNA(”).
The /3 subunit, once fastened onto DNA, slides freely
(bidirectional and ATP-independent) along duplex
DXA, like a washer on a steel rod(”). However, the
clamp can not slide along ssDNA(”). The subunit is
also capable of direct interaction with a, the DNA
polymerase ~ubunit(”~~’).
Hence, the remarkable
processivity of the holoenzyme is rooted in a “sliding
clamp” on DNA which tethers the polymerase to the
template. As the polymerase synthesizes DNA, the /3
clamp continuously holds the polymerase to the DNA
and is pulled along the DNA by the polymerase during
DNA synthesis.
through molecular cloning of their gene^(^^^^^'"^"). The
a subunit (dnaE) contains the DNA polymerase
activity(20) and the E subunit (dnaQ,mutD) is the
The a subunit
proofreading 3’-5’ exonuclease(L6,22).
forms a tight 1:l complex with
Whereas most
DNA polymerases have 3’-5’ exonuclease activity, only
the holoenzyme relegates this activity to an accessory
protein. The functional significance of this is not
known. Three accessory proteins of the holoenzyme are
known to be required for DNA replication as they are
products of genes that are essential for cell viability: /3
(dnaN(’”)), T and 7’ (both encoded by the dnaXZ
gene(24)).The genes for the 0, h, 8,3( and W proteins
are all distinct and lie in previously genetically
unassigned reading frames (R. Onrust, Z. Dong, P.S.
Studwell and M. O’Donnell, unpublished). It has yet to
be proven whether they are essential. In the discussion
that follows, the current state of knowledge about the
functions of the accessory proteins will be summarized
and a dynamic model of the replication fork will be
proposed to explain how their individual functions may
be orchestrated to accomplish the task of duplicating
DNA.
Cycling
The lagging strand is synthesized discontinuously in
Okazaki fragments of 1-2 kb(’). Since the replication
fork advances one kb every second, the priming
apparatus must synthesize a new RNA primer to
initiate an Okazaki fragment every 1-2 seconds. Due to
the scarcity of holoenzyme (10-20 molecules per
the few polymerase molecules must be
capable of rapid transfer from the end of a completed
Okazaki fragment to a new RNA primer (see Fig. 2A).
However, in vitro, upon completing replication of a
circular ssDNA, the holoenzyme remains tightly bound
to the completed duplex circle(31).Only after several
minutes does the holoenzyme dissociate from the
completed DNA and rccycle to a fresh primed
DNA(839332).
Inability of the holoenzyme to dissociate
from a completed template poses a dilemma for the
efficient replication of the lagging strand, where the
polymerase must cycle from a completed Okazaki
fragment to a new upstream primer all within a few
seconds. As primers on the lagging strand are all on the
same template strand, one may expect the holoenzyme
to slide back along the DNA to the new primer at the
fork (Fig. 2A). In fact, the holoenzyme can slide over
duplex DNA but is unable to slide over ssDNA and thus
would be incapable of cycling to the new primer by a
sliding mechanism”).
Processivity
To determine which accessory proteins are essential to
the speed and processivity of DNA synthesis, the
holoenzyme was reconstituted from pure proteins and
subassemblies. The core polymerase has weak catalytic
efficiency and is only processive for approximately 11
nu~leotides(~).
The catalytically efficient holoenzyme
was found to be restored u on mixing core with both
the [3 and the y complex(2s’). Further study showed
reconstitution of the holoenzyme proceeded in two
stages (see Fig. l)(3.2s,26). In the first stage, the y
complex and /3 subunit hydrolyze ATP to form a tightly
bound “preinitiation complex“ clamped onto the
primed DNA. In the second stage, the preinitiation
complex binds the core and confers onto it highly
processive synthesis. ATP is only required in the first
stage.
Study of the preinitiation reaction showed the y
complex both recognizes primed DNA and hydrolyzes
ATP to clamp the /3 subunit onto DNA (R. Onrust et
al., unpublished data; L. Fradkin and A. Kornberg,
personal communication). In fact, one y complex
I:
(-J-$-yc’:--<9~+
0
primer
p clamp
core
‘b
y complex
ATP
ADP,Pi
Fig. 1. Two stage assembly of the processive polymerase. The y complcx couples ATP to clamp 13 onto a primed ssDNA circle.
The core polymerase binds the /3 clamp to yield the highly processive polymerase.
RNA primer
Leading
\\‘
Pol 111 Holoenzyme’-
Lagging
Pol 111 Holoenzyme.
Fig. 2. DNA polymerase I11 holoeiizyme must
cycle to ncw primers on the lagging strand.
Replication fork models of the leading and
lagging strands replicated by (A) separate
holoenzyme molecules, (B) a dimeric holoenzyme.
It is important to note that a dimeric polymerase does
not immediately solve the cycling problem simply by
the leading polymerase holding the lagging polymerase
near the fork and, therefore, near the new primer (Fig.
2B). Although this arrangement would speed the
association of the lagging polymerase with the new
primer (which is already extremely rapid for the
holoenzyrne(‘)), it would not increase the speed of
polymerase dissociation from a completed Okazaki
fragment, since dissociation reactions are concentration-independent. Indeed, cycling of the holoenzyme was found not to be influenced by increasing
the concentration of primed DNA templates(31).
Rapid cycling of the polymerase from a completed
DNA circle to a new primed circle was achieved upon
endowing the newly primed circle with a /? clamp (Fig.
3)(26).Cycling of the polymerase, from the replicated
DNA to the new template containing the /3 clamp, is
complete within 10 seconds. Only the core polymerase
was found to cycle to the “new” p clamp on DNA,
leaving the “old” /3 behind, as shown in Figure 3(33).
The rate-limiting step in the cycling reaction appears to
be a bimolecular collision between the DNA containing
the new B clamp and the polymerase bound to the
completely replicated template (intermediate complex
To reflect this,
in the middle diagram of Fig. 3)(26,33).
the care polymerase is depicted in the middle diagram
of Fig. 3 as swinging off the primer terminus upon
completing the first DNA circle. In terms of the
replication fork. this feature would ensure full replication of the entire Okazaki fragment before the
polymerase cycled from it.
Cycling to new primers on the lagging strand needs to
be even faster than 10 seconds. The rate-limiting step in
the cycling reaction is not dissociation of the polymerase but rather the bimolecular collision between the
polymerase and the y ‘ m e d template containing the
new B clamp (Fig. 3)( ’). At the replication fork, these
moieties would only be separated by 1-2 kb of DNA
(Okazaki fragment), thus speeding the cycling reaction
considerably.
Structure
The holoenzyme is purified from E. coli as a
multiprotein particle(2). In this section, the probable
orientations of the subunits within the holoenzyme will
be proposed from the known interactions among
subunits.
Both y and T are produced from the same gene
(dnaXZ) (see Fig. 4, top)(24).The y subunit is produced
by a frameshift event which occurs after approximately
two thirds of the gene has been tran~lated(’~”~).
The
frameshift i s followed within two amino acids by a stop
codoa. The z subunit is the full length product of the
dnaXZ gene. Approximately equal amounts of 5 and y
are produced in E. coZicz4).
One of the roles of the T subunit is to serve as a
scaffold to dimerize the polymerase subunits, as shown
on the left side of Figure 4. The first indication that z:
dimerizes the polymerase was from the purification and
characterization of the 4-protein subassembly of
new 0
MI3
Fig. 3. Scheme of polymerase cycling to a
new primer in the experimental cycling
system. After the polymerase completes the
4x174 DNA circle, it collides with a new /3
clamp on the primed M13 DNA circle. The
polymerase cycles to the new /3 clamp on
M13 DNA, leaving behind the old @ clamp
on the replicated 4x174 DNA.
RateLlmiting
Step
-
+dna XZ gene
,T
m
I
I
2 core
core
B
Fig. 4. Putative structure of DNA polymerase 111
holoenzyme.
holoenzyme called polI11’ ( a ~ B.tPolIII’ appeared to
be a dimer of all four subunits\)”). Since pol111 core
( a d )appcared to contain only one of each subunit, the
dimeric structure of polIII’ was hypothesized to be due
to the T subunit. Study of pure N and t subunits has
shown the isolated N subunit (the polymerase) is only a
monomer, even at high concentration. However, the t
subunit, which is a dime^-(^^), binds two molecules of N
(P.S. Studwell-Vaughan and M. O‘Donnell, in press).
Hence, t appears to be the agent of polymerase
dimerization. The rsubunit also increases the affinity of
the core polymerase for the preinitiation complex(3)and
is a DNA-dependent ATPase, although the function of
its ATPase activity is u n k n ~ w n ( ~ The
~ , ~ core
~ ) . polymerase (ad) appears to form a dimer when it is
sufficiently concentrated(“).Since a 1:l complex of
,
Q subunit
shows no tendency to dimerize to ( N E ) ~ the
has also been proposed to aid polymerase dimerization(’). To reflect this, the 0 subunits of the core dimer
in Figure 4 are shown touching. The /J subunit also
interacts with the ore(^"'^) specifically by direct
The ysubunit does appear to bind
interaction with d19).
the polymerase subunit (P.S. Studwell-Vaughan and LM.
O’Donnell, unpublished) implying it is the C-terminal
portion of r (lacking in 11) that binds N.
The 5 subunits of the y complex may be arranged as
shown on the right side of Figure 4. Isolation of a y q
complex suggests x, I) or both bind directly to y‘ .
Neither nor li,have been found in association with t.
Possibly in y, the omission of the C-terminal portion of t
x
&
x
yields a surface unique to y which specifically binds
and W . It is not yet clear how the y complex contacts the
polymerase. However, the ssubunit can interact in vitro
with either rT or 6‘ to form a complex which can lock /3
onto primed DNA(19’.Thus, zis depicted in Figure 4 to
contact the y complcx by interaction with 6 and 6’.
Replication Fork Mechanics
Tn this section, the findings from the biochemical
studies of the holoenzyme and from those on its
putative structure will be synthesized into a speculative
hypothesis of how these subunits work together to
accomplish the task of replicating duplex DNA. In
Figure 5A, the structure of the holoenzyme is fitted into
the mold of the replication fork. Above the holoenzyme
are the priming and helicase proteins. The top core is
the leading strand polymerase and the bottom core is
the lagging strand polymerase. Replication of the
leading and lagging strands are two very different
processes and therefore one may expect that the two
polymcrases would have distinct biochemical properties. The y complex is hypothesized to impart special
properties only to the lagging strand polymerase.
As the lagging strand polymerase (lower core)
extends an Okazaki fragment, the y complex hydrolyzes
ATP to clamp a new /3 onto a lagging strand RNA
primer (A + B). After the lagging strand polymerase
completes the Okazaki fragment, it dissociates from the
old [j clamp at the end of the Okazaki fragment and
Fig. 5. Hypothetical model of DNA polymerase
111holoenzyme action at a
replication fork. (A) The
lagging polymerase (bottom core) is extending an
Okazaki fragment. (B) A
new fi is clamped onto a
fresh lagging strand RNA
primer by the y complex.
Also, the lagging polymerase finishes an Okazaki
fragment. (C) The lagging
polymerase cycles to the
on the lagging
new
strand primer. The old /?is
shown on the end of the
completed Okazaki fragment.
binds the new @clampon the upstream primer (B + C).
as suggested by the in vitro two-circle cycling reaction
described earlier (Fig. 3). Now the cycling event is
complete, with the lagging strand core polymerase
positioned on the new /3 clamp on the fresh RNA
primer. In going from C + A, extension of the RNA
primer generates a new Okazaki fragment. This model
predicts a new p subunit is required for each lagging
strand RNA primer, consistent with the large excess of
p subunit relativc to a/ subunit in E . coli (300 $“’) and
10-20 ax3”) pcr cell).
Concluding Remarks
This review has summarized the known activities of the
E . coli holoenzyme accessory proteins. The limited
availability of the ,)I 8, 6 and S’subunits has so far
precluded intensive study of their function. However,
the recent identification of their genes should make
possible their biochemical and genetic characterization,
and ultimately the X-ray structure of the entire
holoenzyme. Further qucstions which must now be
addressed include: How does the @ clamp cycle?
Exactly which, if any, of the accessory proteins are
distributed asymmetrically with respect to the polymerase subunits? What are the functions of the S’, and
I) subunits of the y complex? Do some of the y complex
subunits act to regulate /3 clamp formation? Do the
accessory proteins also interact with other replication
fork proteins such as helicase, primase. topoisomerase,
and ligase, or are they needed to assemble or
dissassemble a fork during initiation or termination of a
cycle of replication? It is also conceivable that the
polymerase accessory proteins could interact with the
protein machinery of other areas of DNA metabolism
such as repair, recombination, transposition, mutagenesis. or transcription. These and other exciting
questions await future studies of holoenzyme structure
and function.
x,
x
Acknowledgements
This work was supported by National Institutes of
Health Grant GM 38839.
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Institute, Microbiology Department, Cornell
University Mcdical College, 1300 York Avenue, New
York, NY 10021, USA.
Conference on The Melanotropic Peptides
September. 6 to 9. 1992
Palais des Congres, Place de la Cnthedrale, Rouen. France
The conference will evaluate the recent findings on biochemistry, physiology and pharmacology on
melanocyte-stimulating hormones (IMSH) and melanin-concentrating hormone (MCH). The current
knowledge of hormonal and neuromodulator/neurotransmitter functions of melanotropins will be
presented. The conference will cover all recent developments concerning the melanotropic peptides, from
basic research to clinical perspectives.
There will be contributed poster sessions in conjunction with this conference. The deadline for submission of
poster abstracts is April 15, 1992.
Confeuezce Chairmen:
Hubert Vaudry, Ph.D., D.Sc.
Alex N. Eberlk, Ph.D., D.Sc.
Laboratory of Molecular Endocrinology
Department of Research (ZLF)
University of Rouen. B.P. 118
University Hospital
76134 Mont-Saint-Aignan
Hebelstrasse 20
France
CH-4031 Basel, Switzerland
For abstract specifications and for further information please contact:
Conference Department, New York Academy of Sciences, 2 East 63rd Street, New York, NY 10021, USA
TEL: 212-838-0230, FAX: 212-888-2894