DNA replication: enzymology and mechanisms
Zvi Kelman and Mike O'Donnell
Cornell University Medical College, N e w York, U S A
Research into the enzymology of DNA replication has seen a multitude of
highly significant advances during the past year, in both prokaryotic and
eukaryotic systems. The scope of this article is limited to chromosomal
replicases and origins of initiation. The multiprotein chromosomal replicases
of prokaryotes and eukaryotes appear to be strikingly similar in structure
and function, although future work may reveal their differences. Recent
developments, elaborating the activation of origins in several systems, have
begun to uncover mechanisms of regulation. The enzymology of eukaryotic
origins has, until now, been limited to viral systems, but over the past few
years, enzymology has caught a grip on the cellular origins of yeast.
Current Opinion in Genetics and Development 1994, 4:185-195
Introduction
The structure of duplex DNA is so simple and elegant
that one may have thought it would be simple to duplicate. However, since the isolation of DNA polymerase I first delivered replication mechanisms into the
hands of enzymologists, the process has been shown to
be far from simple. Over 20 proteins are utilized in Escherichia coil and probably more in higher organisms
[1]. In fact, the individual functions of many of these
proteins are still unknown and even more proteins remain to be identified. In overview, the process begins
at a specific sequence called an origin upon which
proteins bind and locally unwind the duplex, allowing invasion of a helicase. The helicase couples ATP
hydrolysis to melt the duplex, and then the singlestranded DNA (ssDNA) is coated with ssDNA-binding protein (SSB). This SSB-ssDNA nucleofilament has
no secondary structure and serves as the most efficient
template for chromosomal replicases, multiprotein machineries characterized by their rapid and highly processive DNA synthesis. One strand of the chromosome
is synthesized continuously, but because of the antiparallel structure of the duplex, the other strand is copied
discontinuously as a series of fragments (Okazaki fragments). This discontinuous mode of replication necessitates frequent reinitiation of DNA chains, which are
primed by short RNA primers synthesized by a primase. Most of the above proteins are thought to work
together in one large replisome assembly for coordinated synthesis of both strands of the chromosome.
In this review, we will focus on recent developments
in the study of origin activation and the mechanism of
action of chromosomal replicases. These areas encompass only a subset of the recent advances in the enzymology of DNA replication.
Replication origins
The first (and still the only) cellular origin to have
been activated in vitro was oriC, the origin of the
E. coil chromosome [1]. Since that breakthrough, several phage and viral origins have been activated in
vitro. Origin activation requires multiple copies of
an origin-binding protein and usually additional origin accessory proteins, some of which have secondary
functions as transcriptional activators. As proposed by
Hatch Echols [2], the requirement for multiple proteins
in site-specific processes, such as activation of origins
and promoters, is imposed by the need for a high fidelity of action on specific sequences embedded in the
bulk of chromosome DNA. These multiprotein-DNA
complexes are termed specialized nucleoprotein structures (snups) [2]. Origin initiation events in E. coli,
phage ~ and simian virus 40 (SV40) are similar and
appear to apply also to the more recently characterized
systems of phage P1, bovine papilloma virus (BPV),
herpes simplex virus 1 (HSV-1), and yeast (see Fig.
1). In most systems, the origin-binding protein is pre-
Abbreviations
ACS~ARS core consensus sequence; ARS.--autonomous replication sequence; BPV--bovine papilloma virus;
HSV-l--herpes simplex virus 1; MCM--minichromosome maintenance; ORC---origin recognition complex;
PCNA--proliferating cell nuclear antigen; pol/PoI~DNA polymerase; PP2A--protein phosphatase 2A;
RF-C--replication factor C; RP-A--replication protein A; snup---specialized nucleoprotein structure;
SSB~single-stranded DNA-binding protein; ssDNA--single-stranded DNA; SV40--simian virus 40.
© Current Biology Lid ISSN 0959-437X
185
186
Chromosomes and expression mechanisms
pared for origin activation and, for this review, we define this as stage I. Multiple copies of the origin-binding
protein then assemble onto the origin with the accessory proteins to form the snup (stage II). This untwists
a section of AT-rich DNA to form an 'open complex'
(stage III), providing a point of entry for the helicase
(stage IV). After helicase entry, the chromosome can
be extensively unwound and replication forks can be
assembled. Origin activation systems differ in the details of these steps and also in their regulation. In the
following section, we will focus on these differences,
with particular attention to recent developments.
Prokaryoticorigins
Recent studies show that the activation of oriC is regulated at each stage (Fig. 1). In stage I, the DnaA
origin-binding protein is inactive when bound to acidic
phospholipids, but this repression can be relieved, and
DnaA prepared for origin binding, by the DnaK heat
shock protein or by phospholipase treatment [3]. In
stage II, the origin snup is nucleated at four DnaAbinding sites in oriC, upon which 20 or more DnaA
monomers assemble, along with several origin accessory proteins [1]. These accessory proteins include HU
and IHF, which bend DNA, and they may act by helping to wrap DNA around the DnaA subunits [4°',5].
Other accessory proteins include Fis and the recently
identified Rob protein [6"°]. They are not essential for
the activation of the origin, and how they may modu-
Stage I
Stage III
Stage I1
DNA
late the process is not yet clear. In stage III, the origin snup unwinds DNA within three AT-rich 13-mer
repeats to form the open complex and this reaction
requires a supercoiled template [1]. Formation of the
open complex is highly regulated [1]. Negative regulation is achieved by the inhibitor of cellular initiation,
IciA, which competes with DnaA for interaction with
the 13-mers and prevents the formation of the open
complex [7"°,8"°]. ATP also regulates the open complex formation. DnaA can bind either ATP or ADP
tightly, but only the ATP form is active in unwinding
the 13-mers [11. The DnaA protein becomes inactive
upon slowly hydrolyzing the ATP to ADP, and the ADP
remains bound, thus preventing subsequent reinitiations. The DnaA protein can be readily reactivated by
acidic phospholipids, which catalyze the exchange of
ATP for the bound ADP [1,9"'], but only when DnaA
is bound to or/C, this may underlie the early observations of replicon attachment to the membrane [10].
RNA polymerase provides yet another level of regulation in determining the ability of R loops (RNA
paired with one strand of the DNA duplex) to promote the open complex, and although the transcript
need not enter on'C, it must be close [11]. In stage IV,
the DnaB helicase enters the open complex, but its access absolutely requires the DnaC protein, a molecular matchmaker that delivers the hexameric DnaB into
the origin in an ATP-dependent reaction, after which
DnaC departs from the DNA [1]. Origin unwinding is
bidirectional; therefore, two hexamers of DnaB must
~
Stage IV
r - f Open
Prepare
Originbinding
System protein II +
E. coil
DnaA
Phage~.
~. O
PhagePI
RepA
origin-binding
protein
|
~
- II
II +
DnaK
Phospholipids HU. IHF,
Phospholipase
Fis, Rob
ATP
Acidic phospholipids
R loops
HU
DnaJ, DnaK,
GrpE
Yeast
ORC
SV40
T-antigen Cdc2
PP2A
BPV
E1
HSV-I
UL9
DnaA
Open complex
formation
I~
RNA pol
- II
ADP
IciA
Replication fork
proteins
Helicase
DnaB
Dna(-
Excess'~
DnaC -|
DnaB
~. p
~. p
Dnal. DnaK
Gq)E
@1994 Currenl Opinion In Genetics and Developmenl
Dna(;-Prima~e
~ SSB
~ DNA po] III hoh,enzyme
DnaB
ATP,ABFI
psi
/
-~ i)okx-Prin'=ase
ATP
Spl
banligen
E2
E!
UL5,8,52
RP-A(SSBI p53
PP2A
ICP8
~ RP-A(SSB]
pol 8, t" (+RF-C, PCNA)
Helle ase--Primase(UL5,8.521
ICP8 (SSB)
HSV-1 Pol q+LJt42)
Fig. 1. Regulation at different stages of origin activation in a number of systems. Origin activation has been subdivided into four stages:
preparation of the origin-binding protein, assembly of the origin snup, local unwinding of origin DNA (the open complex), and entry of the
helicase. Molecules that act on each stage are divided into positive (+) and negative (-) effectors and are shown for E. colt, phage )~, phage
P1, yeast, SV40, BPV and HSV-1. A blank entry indicates that effectors of that step have not (yet) been identified. At the far right are listed
the replication proteins needed to advance the replication fork.
DNA replication: enzymolo~/and mechanisms Kelman and O'Donnell
be delivered to the open complex. Negative regulation is achieved by the presence of too much DnaC
protein, which binds to and inhibits the DnaB helicase
activity, possibly by preventing its translocation along
DNA; therefore, a 'fine balance' of DnaC and DnaB is
essential to the productivity of this stage [12].
Phage ~. uses mainly host replication proteins for origin
activation, with the exception of the phage-encoded O
and P proteins, analogs of DnaA and DnaC [1]. The ;k
origin snup is composed of four dimers of ~. O protein, which do not require pre-activation to assemble onto the origin. Formation of the open complex
at three AT-rich l 1-met repeats requires the DNA to
be supercoiled, but does not require ATP [13,14]. In
stage IV, the ~. P protein binds DnaB and delivers it
to the origin, but the P protein remains tightly associated with DnaB and prevents its helicase activity [15].
In this regard, the P protein acts as a negative regulator,
like excess DnaC, to prevent translocation of DnaB. Although ATP is not required for P protein to bring DnaB
to the origin, it is required to break the interaction
of P protein with DnaB, thus freeing the helicase for
replication; this step is mediated by heat shock proteins DnaJ, DnaK and GrpE [16,17]. Transcription from
the rightward promoter is needed for ~. origin activation in vivo, and in vitro studies show that transcription
disrupts HU-mediated negative repression (presumably
of stage II or III) [18].
Recent studies on the activation of the lysogenic origin
of phage P 1 show that the phage-encoded RepA originbinding protein is a native dimer, but the monomer
strongly associates with the origin. In stage I, RepA
is prepared for origin binding by the DnaJ, DnaK and
GrpE heat shock proteins, which couple ATP hydrolysis to monomerize RepA [19"']. The origin snup comprises RepA bound at five sites and the host DnaA protein at five other sites. At least two of the DnaA-binding
sites are essential for origin function, and in vitro studies show that the ADP form of DnaA protein is capable
of activating the origin in combination with RepA (reviewed in [20]). Although stages III and IV have not
been studied in detail, it seems likely that the open
complex forms at five slightly AT-rich 7-met repeats
and then DnaC mediates delivery of DnaB to the open
complex.
Eukaryotic viral origins
In the SV40 system (reviewed in [21]), the stage I preparation of the vitally encoded T-antigen for origin binding is regulated in both positive and negative fashion. T-antigen must be phosphorylated at Thr124 for
productive origin binding, and this modification can
be performed by the cdc2 kinase [22]. Phosphorylation at other sites can inhibit T-antigen, and protein
phosphatase 2A (PP2A) has been shown to activate
T-antigen by removing phosphates from specific serine residues implicated in DNA-binding activity [23].
The p53 tumor suppressor protein is a negative regulator of stage I; it binds to T-antigen preventing its
association with the origin [24]. Stage II, formation of
the origin snup, is facilitated by ATP binding, which
effects the assembly of two hexamers of T-antigen
onto the origin (reviewed in [25]). The two hexamers are thought to encircle the origin DNA in SV40,
because if the hexamers are pre-assembled in solution
they do not bind the origin [26"']. Moreover, in electron micrographs the double hexamer of T-antigen on
the origin does not appear to wrap DNA around it as
with other origin snups, but rather the DNA appears
to follow a straight path through the protein [27]. The
transcriptional activator Spl acts as an origin accessory
protein that perturbs the local histone distribution to
make the origin accessible for initiation proteins [28].
However, the identity of the transcriptional activator
is not important, as Spl can be exchanged for other
activators [28], but the DNA-binding domain must be
accompanied by an activation domain for origin function [29"'].
Formation of the open complex in stage III is coincident with T-antigen binding to the origin, which results
in the melting of 8 bp in the early palindrome and untwisting of an AT tract [25]. These events require binding, but not hydrolysis, of ATP and do not require a
supercoiled template [25]. In SV40, unlike the other systems discussed above, the stage IV helicase invasion is
unique in that T-antigen itself is the helicase and thus
encompasses the functions of the E. coli DnaA, B and
C proteins. Stage IV is still regulated, however; for example, it has been shown recently that PP2A-treated
T-antigen provides a cooperative interaction between
the two hexamers, which facilitates stage IV [30"]. In
addition, the human SSB, replication protein A (RP-A),
consists of three non-identical subunits (p70, p32 and
p14) and has been shown to be a positive effector of
stage IV, as RP-A is needed for T-antigen to unwind
the bulk of DNA [25]. A second point at which p53
may regulate replication has been identified recently
in an interaction of p53 with the p70 subunit of RP-A
(the ssDNA-binding subunit) that inactivates its ability
to bind ssDNA [31"']. Also, the p32 subunit of RP-A is
phosphorylated in a cell cycle dependent manner in
the G1 to S phase transition by members of the cyclin cdc2 kinase family, and addition of this kinase to
the SV40 replication system stimulates DNA synthesis
[32"']. Phosphorylation of p32 is also performed by the
DNA-activated protein kinase (GS Brush, CW Anderson, TJ Kelly, abstract 191, Eukaryotic DNA Replication
Meeting, Cold Spring Harbor, September 1993).
The BPV snup is composed of two viraUy encoded
proteins, E1 and E2. E1 is the origin-binding protein
and a helicase (like T-antigen) [33",34"'] and E2 is an
origin accessory protein (and transcriptional activator)
that binds specific sequences and helps in the delivery
of E1 to the origin via direct protein contacts [35",36]. In
this regard, E2 fulfills a stage IV function analogous to
that of E. coli DnaC in delivery of the helicase into the
origin. Regulatory aspects, exact functions of ATP and
identification of an open complex remain for future
studies, but the in vitro replication system and availability of pure E1 and E2 should yield this information
in the near future.
187
188
Chromosomesand expressionmechanisms
In the HSV-1 system, the identification of all seven of
the essential virally encoded replication proteins and
the cloning and production of each of them in quantity has enabled a number of illuminating studies, although replication in vitro of a plasmid containing the
origin has yet to be achieved. The origin snup consists
of at least two dimers of the UL9 protein, which produces the open complex in an AT-rich region even in
the absence of ATP [37]. UL9 has helicase activity, but
it does not act catalytically like the other helicases discussed thus far, Instead, it must bind DNA stoichiometrically for unwinding and requires a stretch of ssDNA
for duplex-unwinding activity [38,39]. The UL9 helicase
is stimulated by the virally encoded SSB (called ICP8)
and it seems likely they function together at the origin
to enlarge the open complex for the future replication
fork.
Yeast chromosomal origin
In the past two years, rapid and exciting advances have
been made in the study of the biochemistry of yeast
cellular replication. Yeast origins are known as autonomous replication sequences (ARS) for their ability
to confer autonomous replication on plasmids. Recent
studies [40"°] of the ARS1 origin show that it contains
four sequence elements: the A element, which contains
an 11 bp ARS core consensus sequence (ACS) common
to all ARS elements, the B3 element, which is the binding site of the ABF1 transcription factor, and the B1 and
B2 elements, which may interact with (as yet) unidentiffed origin accessory proteins. A large origin recognition complex (ORC), which binds to ARS1 at the A site,
has now been purified from Saccharomyces cerevisiae
[41"], and genomic footprinting experiments indicate
the presence of the ORC on ARS1 in vivo [42"']. The
ORC contains six subunits and requires ATP to bind
the ARS. The exact function of the ORC is not certain,
but a number of observations confirm that it plays a
central role in replication. First, the ORC does not bind
to single-site mutant forms of ACS that lack ARS activity
[41"']. Second, mutations affecting the 70 kDa subunit
of the ORC, identified because they produce a defect in
function of the HMR silencer, show that the gene is essential for cell viability, and a conditional lethal allele is
defective in chromosome replication [43"]. Third, each
of the six genes encoding the ORC subunits are essential to yeast (SP Bell, R Kobayashi, B Stillman, abstract
15, Eukaryotic DNA Replication Meeting, Cold Spring
Harbor, September 1993).
Recent developments in the study of the genetics of
yeast replication have given researchers clues as to the
roles of important replication proteins. Several genes of
S. cerevisiae have been isolated, the products of which
are needed for minichromosome maintenance (MCM)
of ARS-containing plasmids. Three of these, MCM2,
MCM3 and MCM5, are homologous in sequence and
each is essential for cell viability [44"]. The intracellular location of MCM2, 3 and 5 is cell cycle regulated,
they are moved into the nucleus upon completion of S
phase and then moved into the cytoplasm at the start of
S phase [44",45"']. It is hypothesized that in the nucleus
the MCM proteins activate the ARS and are then transferred to the cytoplasm to prevent reinitiation [44",45"'];
however, whether they act on origins directly and in a
positive fashion must await further studies. In another
exciting development, the cdcl8 gene of Scbizosaccharomycespombe has been shown to suppress mutations of cdclO, a transcriptional activator that is needed
for entry into S phase [46"']. The cdcl8 gene may be a
major target of cdclO and, consistent with this, cdcl8
mutants fail to enter S phase [46"']. Further, cdcl8 may
play a role in checkpoint control, as cdcl8 mutants are
unable to prevent mitosis and rapid cell division even
though the chromosomes are not fully duplicated. The
cdcl8 gene sequence reveals a nucleotide-binding site,
but whether the encoded protein plays a direct role
(e.g. helicase) or an indirect role in replication must
await future isolation and characterization of the gene
product.
Events after helicase entry
Once the helicase has entered the chromosome, it presumably nucleates assembly of replication forks conmining the primase and two replicative polymerases,
one for each strand of DNA [1]. The leading and lagging strand polymerases in eukaryotes appear to be
different polymerases (polymerases 8 and e), whereas
prokaryotes use two copies of the same polymerase.
The mechanism of replicase function is the second
topic of this essay.
Chromosomal replicases
Replicasesof E. coli, phage T4, yeast and humans
Organisms that span the evolutionary spectrum have
been shown to possess replicases that appear to be
similar in function and also in their actual structure.
These are the E. coli DNA polymerase III holoenzyme (Pol III holoenzyme), the phage T4 replicase,
and the polymerase 15 (pol 8) of both yeast and humans. In each of these systems, the replicase encompasses several proteins that use ATP to initiate rapid
and highly processive DNA synthesis. It can be thought
of as having three components (see Table 1): the
catalytic component, which contains the DNA polymerase and proofreading 3"-5" exonuclease activities,
and the two categories of polymerase accessory proteins, a complex of accessory proteins and a single
subunit processivity factor. The accessory proteins are
needed to confer high processivity onto the catalytic
polymerase. Over the past two years, a deeper understanding of the action of these accessory proteins has
been developed and appears to apply generally to all
four systems. Studies have shown that the single subunit accessory protein is a DNA-sliding clamp, which
tethers the polymerase to DNA for high processivity.
DNA replication: enzymology and mechanisms Kelman and O'Donnell
The accessory protein complex functions as a 'clamp
loader' that couples ATP to assemble the clamp protein
on DNA in a two-step assembly process (see Fig. 2).
The clamp loader recognizes the primed template and
couples ATP to assemble the sliding clamp onto DNA
to form a pre-initiation complex. This is followed by
association of the polymerase to form the initiation
complex (reviewed in [47]).
The best studied system, at least for this assembly reaction, is the Pol IIl holoenzyme of E. colL The processivity factor of Pol III, the [3 subunit, is a dimer that
freely slides along duplex DNA and is topologically
linked to the DNA, in as much as it binds tighdy to a
nicked circular plasmid, but upon linearization freely
slides off over the ends [48]. These results have been
explained by the hypothesis that [3 encircles DNA like
a doughnut [48]. The crystal structure of the [3 dimer
shows that it is indeed in the shape of a ring capable
of completely surrounding duplex DNA and reveals the
unexpected feature that [3, although only a dimer, has a
sixfold appearance (Fig. 3) [49*']. This symmetry is the
result of the three globular domains that comprise each
monomer and the polypeptide chain backbone structures of these domains are nearly superimposable. The
[3 dimer cannot assemble onto DNA independently,
but in fact requires the five-protein y complex clamp
loader, a molecular matchmaker that hydrolyzes ATP
to assemble the [3 dimer around DNA. The catalytic
component of the Pol Ill holoenzyme, termed Pol III
core, assembles with the [3 ring, which tethers the Pol
III core to the DNA and continues to slide with it for
rapid and highly processive synthesis. The 7 complex
acts catalytically to assemble multiple ~ clamp pre-initiation complexes on different primed templates. The
Table 1. Three part structure of chromosomal replicases of E. co~i, phage T4, yeast and human.
Component
E. colt
Phage T4
Eukaryotic
I. Polymerase,3'-5' exonuclease
Pol III core
a (pol)
¢ (exo)
gp43
pol 8
p125
p50
B
pol¢
II. Accessorycomplex
(clamp loader)
DNA-dependent
ATPase
Binds clamp
Binds SSB
Unknown function
y-complex
7
8'
8
X
v2
gp44-gp62 complex
gp44
III. Processivity factor
(sliding clamp)
13
81>45
gp62
RF-C (A~ivato~l)
p12B
p37
p40
p38, p36
PCNA
Replicase subunits and subassemblies are isolated as three pieces: the polymerase/exonuclease, the accessory complex (clamp loader),
and the processivity factor (sliding clamp). For complexes, the individual subunits are listed along with the present knowledge of its
associated function. The E. coli Pol III holoenzyme contains one further subunit called ~ that binds one 7 complex and two Pol III cores.
The components of the yeast and human replicases have the same names and very similar structure; therefore, the yeast and human
components are grouped under the heading 'Eukaryotic', but the subunit masses listed are particular to the human replicase.
Pol !11 core
(polymerase)
[3 subuni,~e4....,~AT P
(slidingclanlp)~s'~3'DNA
7 complex
ADP, Pi
~
(clamp loader)
© I 994 Current Opinion in (,enelit • and l~.,vc, lol)n~.-nl
"i~-~)
Pre-initiation
complex
Iniliation
complex
Fig. 2. Assembly of a processive chromosomal replicase. In the E. colt system,
the 7 complex clamp loader (accessory
complex) recognizes the ssDNA-dsDNA
junction, binds the [3 subunit sliding
clamp (processivity factor), and then couples hydrolysis of ATP to assemble the
ring-shaped clamp around DNA forming
the pre-initiation complex. The 7 complex
dissociates from the pre-initiation clamp
and is capable of assembling other I],
clamps on other DNA molecules. The
Pol III core associates with the 13 sliding clamp to form the initiation complex,
which is capable of highly processive
polymerization. This mechanism may apply generally to other replicases.
189
190
Chromosomes and expression mechanisms
7 c o m p l e x can b e r e m o v e d from ttle reaction prior
to adding Pol III core without effect on the rate and
processivity of DNA synthesis (for recent reviews, see
[47,50,511.
How similar are the 3"4 replicase and eukaryotic
pol 8 to E. coli Pol III holoenzyme? Both "I"4 and
eukaryotes have an accessory complex that is analogous to the E. coli clamp loader T complex. In yeast
and human, the accessory complex is replication factor
C (RF-C, also called Activator-l) (reviewed in [52]) and
in "I"4, it is the complex of the gene 44 protein (gp44)
and gene 62 protein (gp62) (gp44--gp62, reviewed in
[53]). These accessory complexes are DNA-dependent
ATPases that are stimulated by their respective clamp
protein, and in all cases a primed template is the best
DNA effector. Since 1992, the genes encoding the five
subunits of human RF-C have been identified, as have
the remaining four genes of the five-subunit E. coli T
complex [54"-57",58--611. Amino acid sequence comparison shows that several subunits of RF-C are homologous to the 7 and 8' subunits of E. coli ~l complex
and to the T4 gp44 [56"]. The level of homology is
sufficient to predict that they will have similar threedimensional structures. Despite these structural similarities, it remains to be established whether the T4
and eukaryotic accessory complexes are truly clamp
loaders and whether they need to remain associated
with the polymerase during elongation. Recent experiments, however, are consistent with their being clamp
loaders and indicate that they may not be needed during elongation.
In the "1"4system, the processivity factor is the gene 45
protein (gp45), and in yeast and humans, it is the proliferating cell nuclear antigen (PCNA) (Table 1). But,
do they really form rings like the E. coli ~ subunit?
None of these proteins has a significant level of sequence homology with any other, and a major difference is that PCNA and gp45 are trimers, whereas
13 is a dimer, and they are only 2/3 the size of 13.
The sixfold synmaetry of the 13 dimer held the explanation for these differences in size and aggregation
state. A trinaer of PCNA (and gp45) is of similar mass
to a dinaer of I~ so, if the PCNA and gp45 monomers
were two-domain proteins, instead of three, then the
trimer would contain a total of six domains like the
13 dimer. Indeed, a sequence alignment of gp45 and
PCNA with the first two domains of 1~, using the structure of 13as a guide, shows that the hydrophobic core
residues are positionally conserved, lending strength
to this hypothesis 149"']. As of September 1993, the
answer was determined for the yeast PCNA, and the
answer was a most definite yes, as the X-ray structure
shows it to be a trinaer in the shape of a ring with the
same outside and inside diameters as the 13 dimer
(J Kuriyan, personal communication). Further, the
affinity of human PCNA to DNA (placed there by RFC) depends on the geometry of the DNA, being tightly
retained on circular DNA, but freely sliding off upon
linearization of the DNA; therefore, PCNA exhibits the
same hallmarks of topological binding to DNA as the
E. colf ~ subunit (N Yao, Z Kelman, Z Dong, Z-Q Pan,
J Hurwitz, M O'Donnell, unpublished data).
Now, back to the issue of whether the accessory proteins of "I"4 and eukaryotes are clamp loaders, and
whether they must be present with the polymerase and
clamp protein during elongation. Three recent reports
in these systems indicate that the clamp--polymerase
unit is all that is needed for processivity. Clever experiments in the yeast system show that on linear
"A.
.
.
.
B"
D i m e r ._~
Interface
.~
3
Fig. 3. Crystal structure of the 13DNA-sliding clamp of DNA polymerase III holoenzyme. The structure on the left is viewed 'face on' looking
through the central cavity. The outside is a continuous layer of antiparallel sheet, which also forms the dimer interfaces (arrows). The central
cavity is lined with 12 o. helices, the only helices in the entire molecule. The structure on the right is the 13dimer turned on its side, with
duplex DNA modeled through the center. The two structurally distinct faces (A and B) are the result of the head-to-tail arrangement of the
subunits. Reproduced from Molecular Biology of the Cell 1992, 3:955 by copyright permission of the American Society for Cell Biology.
DNA replication: enzymology and mechanisms Kelman and O'Donnell
DNA (but not circular) PCNA confers processivity onto
pol 8 in the complete absence of ATP and RF-C [62"].
This result was interpreted as showing that the PCNA
ring threads itself onto the end of linear DNA and then
couples with pol 8 for processive synthesis, thus circumventing the need to open the ring, an action
that requires the RF-C complex and ATP. In the T4
system, evidence that the gp44-gp62 complex is not
needed during elongation has come from the observation that supply of a large excess of gp45 increases
the processivity of the polymerase in the absence of
the gp44-gp62 complex, a result that is similar to
those from previous studies in the E. coli system and
which enforces the idea of the clamp-polymerase as
the processive unit [63"]. Furthermore, an elegant electron microscopy study has shown that the T4 sliding
clamp on DNA appears as a 'hash-mark' (with similar dimensions to the ~ ring) through which DNA is
threaded, and these 'hash-marks' appear in clusters
indicating that they can slide [64"']. The size of the
'hash-mark' is insufficient to acconmlodate the mass
of both the gp44-gp62 complex and the gp45 trimer
and in light of similarity to other systems it is probably
a gp45 clamp.
The actual mechanism by which the clamp loader assembles the clamp around DNA is a fascinating issue
and is being addressed in all these systems, but detailed
mechanisms are still unknown. Most of the work has
been in the T4 and E. coli systems, in which the individual subunits of the clamp loaders are available in
pure form. No individual subunit of either of these
clamp loaders can assemble their respective clamp
onto DNA; presumably this reaction is too complicated for just one protein. The five subunits of the
E. coli ~l complex are % 8, 8", X and "q. Although only T
and 8 are essential to place ~ onto DNA, the 8' subunit
stimulates this reaction considerably [65,66]. DNA-dependent ATPase activity is produced by a mixture of
T and 8' [65] and T is the presumed site of hydrolysis,
as it is known to bind ATP [1]. The 8 subunit forms a
protein-protein complex with ~ [67]. Hence, it appears
that ~ ' recognizes the primed template, and the 8 subunit functions to bring ~ into the structure for assembly
around DNA and hydrolysis of ATP. At elevated ionic
strength, such as exists in the cell, the X and ~t subunits
of the T complex are also needed to initiate processive
synthesis [68]. This is probably rooted in the fact that
the T complex associates with SSB-coated DNA [69"],
an interaction mediated by X that may give the T complex the added grip that it needs to bind the template
in elevated salt (Z Kelman, M O'Donnell, unpublished
data).
The T4 clamp loader is composed of a tetramer of gp44
tighdy associated with one protomer of gp62 [53]. By
itself, the gp44 is a DNA-dependent ATPase (like E. coli
"#'), implying that it harbors the DNA-binding and
ATPase sites [70]. The DNA-dependent ATPase of the
gp44-gp62 complex is stimulated by gp45, whereas the
gp44 ATPase is not, implying that gp62 interacts with
gp45 (like E. coli 8) [70]. The gp44-gp62 complex interacts with gp32 (T4 SSB) bound to ssDNA (like E. coli
7,.), but it is not known whether the interaction is mediated by gp44 or gp62 [71]. Using footprinting methods,
laser-induced UV-crosslinking of protein to DNA, and
novel DNA-protein cross-linking agents, it has been
found that the 1"4 gp44-gp62 complex needs only to
bind ATP to bring the gp45 clamp to DNA, although
it is not known whether the clamp surrounds DNA
at this point [72,73"',74]. Further, these studies show
that the gp44-gp62 complex works on the primed
template junction to place the gp45 clamp on DNA,
and then ATP hydrolysis induces a movement or the
dissociation of gp44-gp62 from the DNA, presumably
to make room for the polymerase [74,75].
Much less is known about the RF-C subunits, although
with the recent identification of the genes encoding
them we can expect much more information in the
near future. The human p128 is known to bind DNA,
as it was identified in 1993 in a south-western analysis as a DNA-binding protein called PO-GA [57"]. At
the same time, a similar screen resulted in the isolation of the analogous RF-C subunit of the mouse [76"].
The p37 and p40 proteins are the only RF-C subunits
to be obtained in pure form thus far and studies have
shown that p37 binds DNA, suggesting that p128 and
p37 may be similar to ~/and 8" (and T4 gp44) [77"]. The
p40 binds ATP and PCNA, which suggests a functional
analogy to 8 (and T4 gp62) [77"]. A summary of these
individual subunit functions is included in Table 1.
In all these systems, the three replicase components
have only weak interactions with one another, at least
in the absence of DNA. In the E. coli system, however,
there is another subunit called ~ that firmly binds one
~/complex molecule and two molecules of Pol III core,
thereby acting as ~i scaffold to hold all of these proteins tightly together in a 'holoenzyme' particle [67]. A
dimeric polymerase complete with one clamp loader
within a single molecular structure fits nicely with the
need to synthesize two strands of DNA, one of which
is synthesized discontinuously and requires multiple
clamps to be loaded onto it (lagging strand). At present,
the T4 and eukaryotic replicases have been purified as
the three separable components and whether, in these
systems, the clamp loaders maintain an association
with the polymerase and clamp or have a functional
equivalent of "t to hold them together remains to be
determined.
Replicases of HSV-1 and phage T7
The other two replicases that are highly processive are
those of HSV-1 and phage T7 [1]. These replicases require only one accessory protein for high processivity,
UL42 for HSV-1 and E. colithioredoxin for T7, and they
do not need ATP. Thus, they lack a clamp loader. The
crystal structure of thioredoxin bears no resemblance
to a ring, so the molecular basis by which the accessory protein provides processivity to the polymerase is
not clear. One simple possibility is that the polymerase
has a cleft into which the DNA fits and the accessory
protein seals off this cleft to trap DNA inside.
191
192
Chromosomesand expression mechanisms
Interaction of sliding clamps with. other proteins
The clamp proteins have recently been shown to share
a common property, in that they interact with proteins other than the replicative polymerase. The best
studied case is that of T4, in which the gp45 clamp
interacts with RNA polymerase. Phage T4, as well as
several viruses (e.g. SV40, adenovirus and HSV), has
an early-to-late switch in gene expression, so that late
g e n e s ( e g c a p s i d p r o t e i n s ) are n o t e x p r e s s e d until t h e
viral g e n o m e h a s b e e n r e p l i c a t e d . Elegant s t u d i e s b y
P e t e r G e i d u s c h e k ' s r e s e a r c h g r o u p h a v e r e v e a l e d that
the u n d e r l y i n g m e c h a n i s m o f the early-to-late s w i t c h
is a n i n t e r a c t i o n b e t w e e n the T4 r e p l i c a s e a c c e s s o r y
p r o t e i n s a n d t h e E. c o l i RNA p o l y m e r a s e ( m o d i f i e d b y
"1"4 g p 3 3 a n d gp55). In 1992, a c o n t i n u a t i o n o f t h e s e
s t u d i e s w a s p u b l i s h e d s h o w i n g that this switch is t h e
result o f a t r a c k i n g m e c h a n i s m , w h e r e b y the a c c e s s o r y
p r o t e i n s a s s e m b l e at a nick a n d slide a l o n g DNA, t h u s
acting as a ' m o b i l e e n h a n c e r ' for late g e n e a c t i v a t i o n
[78"']. P r e s u m a b l y t h e i n t e r a c t i o n is t h r o u g h the g p 4 5
c l a m p a n d t h e m o d i f i e d RNA p o l y m e r a s e , a l t h o u g h a
role for t h e g p 4 4 - g p 6 2 c o m p l e x c a n n o t b e r u l e d out,
as it w a s n o t r e m o v e d from the s y s t e m after f o r m a t i o n
o f the sliding c l a m p . This m e c h a n i s m m a y a p p l y to
e u k a r y o t i c v i r u s e s in g e n e r a l , as a b a c u l o v i r u s p r o t e i n
w i t h 42% i d e n t i t y to PCNA is also n e e d e d for a c t i v a t i o n
o f late g e n e s [79].
Use o f the s l i d i n g c l a m p for o t h e r DNA m e t a b o l i c p r o c e s s e s a p p e a r s to b e t h e rule rather than the e x c e p t i o n ,
as the c l a m p s o f E. c o l i a n d o f h u m a n also interact w i t h
o t h e r p r o t e i n s . In E. coli, t h e ~ c l a m p is utilized b y Pol
III a n d D N A p o l y m e r a s e II, a n d in h u m a n s a n d yeast,
the PCNA c l a m p is utilized b y b o t h p o l 8 a n d p o l
[1]. In fact, the h u m a n PCNA has r e c e n t l y b e e n s h o w n
to b i n d to m e m b e r s o f the cyclin D a n d c y c l i n - d e p e n d e n t k i n a s e families [80"']. T h e function o f t h e latter
i n t e r a c t i o n is u n k n o w n . W o u l d t h e PCNA ring n e e d to
b e o n o r off D N A to manifest the b i o l o g i c a l activity? It
is t e m p t i n g to s p e c u l a t e that the PCNA ring m a y t e t h e r
cyclins a n d their a s s o c i a t e d k i n a s e s to DNA for s o m e
a s p e c t o f cell c y c l e c o n t r o l (e.g. mitotic c h e c k p o i n t ) .
t e m s is r e v e a l i n g f u n d a m e n t a l diversification. T h e n e w
i n f o r m a t i o n a b o u t y e a s t origin structure, a n d the p r o teins that act o n it, l e a d us to a n t i c i p a t e future d i s c o v eries c o n c e r n i n g the m e c h a n i s m s b y w h i c h e u k a r y o t i c
cellular origins a r e activated, r e g u l a t e d a n d e v e n t u a l l y
i n t e g r a t e d w i t h signal t r a n s d u c t i o n .
References and recommended reading
Papers of particular interest, published within the annual perkxt of
review, have been highlighted as:
•
of special interest
•.
of outstanding interest
1.
KORNBERGA, BAKERTA: DNA Replication. New York: WH
Freeman; 1991.
2.
ECHOLS H: Multiple DNA-Protein Interactions Governing High-Precision DNA Transactions. Science 1986,
233:1050--1056.
3.
HWANGDS, CROOKE E, KORNBERG A: Aggregated DnaA
Protein is Dissociated and Activated for DNA Replication
by Phospholipase or DnaK Protein. J BIol Chem 1990,
265:19244-19248.
4.
HWANGDS, KORNBERG A: O p e n i n g of the Replication Origin
•.
of Escberlcbla coil by DnaA Protein With Protein HU or
IHE J Btol Cbem 1992, 267:23083-23086.
Important demonstration that either protein HU or IHF will suffice
to form the open complex on negatively supercoiled plasmid filled
with DnaA protein complexed with ATP.
5.
SCHMIDMB: More Than Just 'Histone-Like' Proteins. Cell
1990, 63:451--453.
6.
**
SKARSTADK, THONY 13, HWANG DS, KORNBERG A: A NOvel Binding Protein of the Origin of the Escherfcbla coil
Chromosome. J Btol Cbem 1993, 268:5365-5370.
Identification of a novel origin-binding protein in E. coil The Rob
protein is present at 5000 copies per cell, contains a helix-turn-helix
motif and binds at the right border of ortC.
HWANGDS, KORNBERGA: Opposed Actions of Regulatory
Proteins, DnaA and IciA, in Opening of the Replication Origin of E$cherlchla coll. J Blol Chem 1992, 267:23087-23091.
First demonstration that DnaA protein interacts with the middle and
right 13-mers of orICwith sequence specificity, and that lciA protein
blocks this interaction by binding all three 13-mers.
7.
•*
8.
H W A N G DS, THONY B, KORNBERG A: IdA Protein, a Spe-
•-
Conclusions
In s u m m a r y , k n o w l e d g e is e x p a n d i n g r a p i d l y a b o u t
r e p l i c a t i o n m e c h a n i s m s in s e v e r a l systems, t o o m a n y ,
in fact, for t h e m all to h a v e b e e n m e n t i o n e d h e r e .
C h r o m o s o m a l r e p l i c a s e s a p p e a r to b e quite similar in
their m e c h a n i s m for attaining high processivity. H o w ever, litde is k n o w n a b o u t h o w t h e y a s s e m b l e at the
origin o r c o m m u n i c a t e with the h e l i c a s e a n d p r i m a s e ,
o r h o w t h e s e p r o c e s s i v e e n z y m e s release DNA r a p i d l y
and rebind new primers during discontinuous synthesis o f t h e l a g g i n g strand. Likewise, the outline o f e v e n t s
in a c t i v a t i o n o f origins has b e e n c o n s e r v e d in e v o l u t i o n
f r o m p r o k a r y o t e s to e u k a r y o t e s , b u t i m p o r t a n t d e t a i l s
in t h e m a n i p u l a t i o n o f t h e s e s e q u e n c e s b y proteins, t h e
roles o f ATP, a n d r e g u l a t i o n o f initiation in different sys-
cific Inhibitor of Initiation of Escberlcbla coil Chromosomal
Replication. J Btol Cbem 1992, 267:2209-2213.
Elevation of the intracellular concentration of IciA protein produces
a lag in cell growth upon transfer to fresh medium. This correlates
with a fourfold increase of IciA protein in the stationary phase.
9.
•-
CROOKEEC, CASTUMACE, KORNBERGA: T h e Chromosome
Origin of Escberlcbla coil Stabilizes DnaA Protein During Rejuvenation by Phospholipids. J Bfol Cbem 1992,
267:16779-16782.
First demonstration that not all of the 20 or more DnaA molecules
bound to orfC need to be in the ATP-bound form for origin activation; some can remain complexed with ADP.
10.
JACOBF, BRENNERS, CtJZBN F: On the Regulation of DNA
Replication in Bacteria. Cold Sprtng Harb Syrup Quant BIol
1963, 28:329-348.
11.
BAKERT, KORNBERG A: Transcriptional Activation of Initiation of Replication from the E. coil Chromosomal Origin:
an RNA-DNA Hybrid Near orlC. Cell 1988, 55:113-123.
12.
ALLENGC, KORNBERGA: Fine Balance in the Regulation
of DnaB Helicase by DnaC Protein in Replication in Escherlchla coll. J Btol Cbem 1991, 266:22096-22101.
DNA replication: enzymology and mechanisms Kelman and O'Donnell
13.
SCHNOS M, ZAHN K, INMAN RB, BI.A'FI'NER FR: Initiation
Protein Induced Helix Destabilization at the ~. Origin: a
Prepriming Step in DNA Replication. Cell 1988, 52:385-395.
14.
ALFANOC, MCMACKENR: Ordered Assembly of Nucleoprorein Structures at the Bacteriophage ~. Replication Origin
During the Initiation of DNA Replication. J Blol Chem 1989,
264:10699-10708.
15.
MALLOTYJB, ALFANO C, MCMACKEN R: Host Virus Interactions in the Initiation of Bacteriophage ~. DNA Replication.
Recruitment of Escherichla coil DnaB Helicase by ~. P Replication Protein. J Btol Chem 1990, 265:13297-13307.
16.
ALFANOC, McMACKEN R: Heat Shock Protein-Mediated Dis.
assembly of Nucleoprotein Structures Required for the Initiation of Bacteriophage ~. DNA Replication. J Btol Chem
1989, 264:10709-10718.
17.
LIBEREKK, GEORGOPOULOS C, ZYLICZ M: Role of the Escherlchla coil DnaK and DngJ Heat Shock Proteins in the
Initiation of Bacteriophage ~. DNA Replication. Proc Nail
A c a d Set USA 1988, 85:6632--6636.
18.
MENSA-WILMOTK, CARROLLK, MCMACKENR: Transcriptional
Activation of Bacteriophage ~. DNA Replication In Vitro:
Regulatory Role of Histone-Like Protein HU of Escberichla
coll. EMBO J 1989, 8:2393-2402,
19.
WICKNER S, SKOWYRA D, HOSKINS J, MCKENNEY K: DnaJ,
DnaK, and GrpE Heat Shock Proteins are Required in orlP1
DNA Replication Solely at the RepA Monomerization Step.
Proc Nail Acad Sol USA 1992, 89:10345--10349.
Urea treatment monomerizes RepA and completely bypasses the requirement for heat shock proteins for in t~tro replication of an oriP1
plasmid, showing that heat shock proteins are required only at the
monomerization step.
ss
20.
BAKERTA, WICKNER SH: Genetics and Enzymology of DNA
Replication in Escherlchla coll. A n n u Rex, Genet 1992,
26:447-477.
21.
CHALLH,
ERG MD, KELLY TJ: Animal Virus DNA Replication.
A n n u Rev Blochem 1989, 58:671-717.
22.
MCVEY D, BRIZUELA L, MOHR l, MAILSHAKDR, GLUZMAN
BEACH D: Phosphorylation of Large Tumor Antigen
by cdc2 Stimulates SV40 DNA Replication. Nature 1989,
341:503-507.
Y,
23.
SCHEII)MANN KH, VIRSHUP DM, KELLY TJ: Protein Phosphatase 2A Dephosphorylates Siman Virus 40 Large T
Antigen Specifically at Residues Involved in Regulation of
DNA-Binding Activity. J Virol 1991, 65:2098-2101.
24.
WANG EH, FRIEDMANPN, PRIVE.';C: The Murine p53 Protein
Blocks Replication of SV40 DNA in Vitro by Inhibiting the
Initiation Functions of SV40 Large T Antigen. Cell 1989,
57:379-392.
25.
lk.)ROWIECJA, DEAN Fig, BULLOCK PA, HURWI'I'ZJ: Binding
and Unwinding - - How T Antigen Engages the SV40 Origin
of DNA Replication. Cell 1990, 60:181-184.
DEAN FB, BOROWIEC JA, EKI T, HURWITZ J: The Simian
Virus 40 T Antigen Double Hexamer Assembles Around
the DNA at the Replication Origin. J Blol Chem 1992,
267:14129-14137.
In the absence of DNA, ATP promotes hexamerization of T-antigen.
This hexamer is stable to isolation on a glycerol gradient, but cannot
bind the origin. Incubation without ATP monomerizes T-antigen and
activates it for proper assembly onto the origin upon adding ATP and
DNA. This suggests that the hexamers assemble to encircle the origin
DNA.
29.
,,.
CHENGL, WORKMANJL, KINGSTON RE, KEI.LYmJ: Regulation
of DNA Replication In Vitro by the Transcriptional Activation Domain of GAL4-VP16. Proc Nail Acad Scl USA 1992,
89: 589--593.
It is hypothesized that transcription fhctors either interfere with assembly of chromatin near the origin or facilitate binding of initiation
factors at the origin. Whatever the mechanism, it may be similar to
the role these factors play in transcription activation.
30.
ss
VIRSHUPDM, RUS.¢,OAAR, KELLYTJ: Mechanism of Activation
of Siman Virus 40 DNA Replication by Protein Phosphatase
2A. Mol Cell Btol 1992, 12:4883--4895.
The two hexamers of T-antigen assemble on the origin in two distinct stages. This paper demonstrates that this is regulated by protein phosphatase 2A. The major influence of protein phosphatase
2A treatment is that it enhances co-operative binding of the .second
hexamer to the origin.
31.
ss
DLrITAA, ROPPERTJM, A.q'I'ERJC, WINCHF.STER E: Inhibition
of DNA Replication Factor RPA by p53. Nature 1993,
365:79--82.
The authors show that p53 can bind both T-antigen and RP-A at
the .same time and, consistent with this, a different region of p53
is needed to bind replication protein A than to bind T-antigen.
32.
•.
DtfITA A, S'nLLMANB: cdc2 Family Kinases Phosphorylate
a Human Cell DNA Replication Factor, RPA, and Activate
DNA Replication. EMBO J 1992, 11:2189-2199.
Stimulation of SV40 replication by cdc2 kinase was performed in
cell extracts. The origin unwinding reaction was "also stimulated. An
unidentified factor in the extract is required for stimulation.
33.
s.
SEO Y-S, MULLERF, LUSKYM, HORWITZJ: Bovine Papilloma
Virus (BPV)-Encoded E1 Protein Contains Multiple Activities
Required for BPV DNA Replication. Proc Nail Acad Set USA
1993, 90:702-706.
This paper shows that the E1 helicase is most efficient on a forked
DNA template and is supported by all eight nucleoside triphospates.
34.
•*
WANGL, MOHR I, Fom.x E, LIM DA, NOHAILE M, BOTCHAN
M: The E1 Protein of Bovine Papilloma Virus 1 is an ATPDependent DNA Helicase. Proc Naa Acad Sol USA 1993,
90:5086-5090.
Important demonstration that at high levels of El, and in the absence
of E2, plasmids lacking the origin are unwound and replicated. This
paper discusses how this promiscuous origin-independent replication relates to earlier observations in some eukaryotic systems (e.g.
Xenopus), which implied that cellular chromosomes do not have defined origins.
35.
•
LUSKYM, HURWrlXJ, SEO Y-S: Cooperative Assembly of the
Bovine Papilloma Virus El and E2 Proteins on the Replication Origin Requires an Intact E2 Binding Site. J Blol Chem
1993, 268:15795-15803.
The authors demonstrate that proper spacing between the El and E2
sites is required for cooperative binding of these proteins to DNA.
36.
WANG L, MOHR l, LI R, NOTI'OLI T, SUN S, BOTCHANM:
Transcription Factor E2 Regulates BPV-1 DNA Replication
in Vitro by Direct Protein-Protein Interaction. Cold Spring
Harb Syrup Quant Biol 1991, 56:335-346.
37.
KOFFA, SCHWEDI-2SJF, TFGTMEYER P: Herpes Simplex Virus
Origin-Binding Protein (UL9) Loops and Distorts the Viral
Replication Origin. J Vtrol 1991, 65:3284-3292.
38.
BRUCKNERRC, CRUTEJJ, DODSON MS, LEHMANIR: The Herpes
Simplex Virus 1 Origin Binding Protein: a DNA Helicase. J
Blol Chem 1991, 266:2669-2674.
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FtERERD, CHALLBERGMD: Purification and Characterization
of ULg, the Herpes Simplex Virus Type 1 Origin-Binding
Protein. J Virol 1992, 66:3986-3995.
26.
s.
27.
28.
MAS'I'RANGELO1A, HOUGH PVC, WALLJS, DOt)SON M, DEAN
FB, HURWI'IXJ: ATP-Dependent Assembly of Double Hexamer of SV40 T Antigen at the Viral Origin of DNA
Replication. Nature 1989, 338: 658-662.
CHENGL, KELLYTJ: Transcriptional Activator Nuclear Factor
I Stimulates the Replication of SV40 Minichromosomes In
Vlvo and in Vitro. Cell 198% 59:541-551.
40.
MARAHRENSY, STILLMAN13: A Yeast Chromosomal Origin of
DNA Replication Defined by Multiple Functional Elements.
Science 1992, 255:817-823.
An important study in which thirty-four overlapping linker insertion
mutants were used to define the precise boundaries of the four eless
193
194
Chromosomesand expression mechanisms
ments of ARSl. The multi-element nature of 0J~S1 was correlated to
the known complexity of transcriptional promoters.
41.
•.
BELL SP, SllLLMANB: ATP-Dependent Recognition of Eukaryotic Origins of DNA Replication by a Multiprotein Complex.
Nature 1992, 357:128-!34.
This paper describes the purification of the origin recognition complex (ORC) through preparation of yeast nuclear extract. DNA-binding activity was detectable only after ion exchange cl~'omatography,
and even then ATP was needed.
42.
DIm.EYJFX, COCKERJH: Protein-DNA Interactions at a Yeast
•*
Replication Origin. Nature 1992, 357:169-172.
The genomic footprint of ARS1 indicates that ORC is situated on the
A element and ABF1 on B3. An additional footprint over element B2
may indicate that another protein is present. The results also suggest
that ORC may remain bound to AKS1 throughout the cell cycle.
43.
•
MICKLEMG, ROWLEY A, HARWOODJ, NA.SM~'I'H K, DIFFLEY
JFX: Yeast Origin Recognition Complex is Involved in
DNA Replication and Transcriptional Silencing. Natttre 1993,
366:87-89.
This study provided the first genetic evidence that ORC is needed
for replication.
44.
•
YAN H, MERCHANTAM, TYE BK: Cell Cycle-Regulated Nuclear
Localization of MCM2 and MCM3, which are Required for
the Initiation of DNA Synthesis at Chromosomal Replication
Origins in Yeast. Genes Dev 1993, 7:2149-2160.
This report describes studies showing that defective MCM2 and
MCM3 proteins result in defects in origin-specific replication.
45.
•-
CHENY, HENNESSYKM, BO'I~'rEtN D, TYE B-K: CDC46/MCM5,
a Yeast Protein whose Subcellular Locafization is Cell CycleRegulated, is Involved in DNA Replication at Autonomously
Repficating Sequences, Proc Natl Acad Set USA 1992,
89:10459-10463.
MCM5 is shown to be identical to CDC46, and mutants are shown to
be defective in maintaining minichromosomes. The paper describes
the cell cycle regulated subcellular localization of MCM5.
46.
•*
KELLYTJ, MARTIN GS, FORSBURG SL, S'IEPHEN RJ, RUSSO A,
NURSE P: The Fission Yeast cdcl8 • Gene Product Couples
S Phase to START and Mitosis. Cell 1993, 74:371-382.
Checkpoint control is a process that prevents mitosis until after the
completion o r s phase. Thus, the cdc18 product functions at initiation
and termination of S phase.
47.
O'DONNELL M, KURIYAN J, KONG X-P, STUKENBERG PT,
ONRUSTR: The Sliding Clamp of DNA Polymerase llI Hoioenzyme Encircles DNA. Moi BIol Cell 1992, 3:953-957.
48.
STUKENBERG PT, STUDWEI.L-VAUGHAN PS, O'DONNELL M:
Mechanism of the ]3-Clamp of DNA Polymerase IIi Holoenzyme. J Biol Chem 1991, 266:11328-11334.
49.
••
KONGX-P, ONRUST R, O'DONNELL M, KURIYANJ: Three Dimensional Structure of the ]3 Subunit of Escherlchla coil
DNA Polymerase III Holoenzyme: a Sliding DNA Clamp. Cell
1992, 69:425-437.
This report shows that the [3 subunit is in the shape of a ring, as
predicted [48], and the authors present the new hypothesis that proliferating cell nuclear antigen (PCNA) and gp45 are trimers with a
similar shape to that of ]3, based on amino acid sequence comparisons and the structure of ~.
50.
O'DONNELL M, KUPJYAN J, KONG X-P, STUKENBERG PT,
ONRUST R, YAO N: The ]3 Sliding Clamp of E. coil DNA
Polymerase Ill Balances Opposing Functions. Nucleic Acids
Mol Bfol 1993, 8:in press.
51.
KURIYANJ, O'DONNELL M: Sliding Clamps of DNA Polymerases. J Mol BIol 1993, 234:915-925.
52.
DOWNEYKM, TAN C-K, SO AG: DNA Polymera~ Delta:
a Second Eukaryotic DNA Replicase. Bide•says 1990,
12:231-236.
53.
YOUNGMC, REDDY MK, YON HIPPLEPH: Structure and Function of the Bacteriophage T4 DNA Polymerase Holoenzyme.
Biochemistry 1992, 31:8675--8690.
~i.
•
CHENM, PAN Z-Q, HUR~aTZJ: Studies of the Cloned 37-kDa
Subunit of Activator 1 (Replication Factor C) of Hela Cells.
Proc Nail Acad Sol USA 1992, 89:5211-5215.
The sequence of p37 of RF-C has a high degree of homology to that
of p40 of RF-C.
55.
•
CHEN M, PAN Z-Q, HURWI'I-LJ: Sequence and Expression
in Escberlchla coil of the 40-kDa Subunit of Activator 1
(Replication Factor C) of Hela Cells. Pro(: Naa Acad Sol
USA 1992, 89:2516-2520.
This paper reports limited .sequence homology between the p40 subunit of RF-C, "1"4gp44 and E. cull ~/:rod 3.
56.
•
O'DONNELLM, ONRUS'I" R, DEAN FB, CHEN M, HURWI'IZJ:
Homology in Accessory Proteins of Replicativ¢ Polymerases
E. coil to Humans. Nucleic Acids Re• 1993, 21:1-3.
The amino acid sequences of E. coital, "¢and 8', T4 gp44 and human
RF-C subunits 1340, p38, 1337 and p36 are all homologous, implying
a common ancestor.
57.
•
Lu Y, ZEVl"AS, RIEGELT: Cloning and Expression of a Novel
Human DNA Binding Protein, PO.GA. Blocbem Btophys Re•
Cumin 1993, 193: 779-786.
The PO-GA protein (p120 of RF-C) sequence has homology to E. colt
and yeast DNA ligases.
58.
CAR'IER JR, FRANDEN MA, AEBERSOLD R, MCHENRY CS:
Molecular Cloning, Sequencing, and Overexpression of the
Structural Gene Encoding the 8 Subunit of Escherlchla
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59.
CARTER JR, FRANDEN MA, AI-'BERSOLD R, MCHENRY CS:
Identification, Isolation, and Characterization of the Struo
rural Gene Encoding the 8' Subunit of Escherichla
coil DNA Polymerase Ill Holoenzyme. J Bacteriol 1993,
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60.
DUNGZ, ONRUST R, SKANGALISM, O'DONNELLM: DNA Polymerase III Accessory Proteins. i. holA and holB Encoding
8 and 5'. J BIol Cbem 1993, 268:11758-11765.
61.
XIAO H, CROMBIE R, DONG Z, ONRUSW R, O'DONNELL M:
DNA Polymerase Iii Accessory Proteins. 111. holC and h o l d
Encoding X and ~. J Btol Chem 1993, 268:11758--11765.
62.
•*
BURGERSPM, YODER BL: ATP-Independent Loading of the
Proliferating Cell Nuclear Antigen Requires DNA Ends. J BIol
Chem 1993, 268:19923-19926.
PCNA was shown to be capable of loading omo the end of a long
stretch of duplex DNA (0.55 kb), implying that it can slide long distances, but it could not load over ssDNA coated with SSB. These are
similar mobility characteristics to those of the E. c-off ]3 sliding clamp.
63.
•
REDDYMK, WEITZELSE, VON HIPPLE PH: Assembly of a Functional Replication Complex without ATP Hydrolysis: a Direct
Interaction of Bacteriophage "1"4 gp45 with T4 DNA Polymerase. Proc Natl Acad Set USA 1993, 90:3211-3215.
An interesting observation that use of a large amount of gp45 results in clamp assembly on a circular DNA molecule without the
gp44-gp62 complex or ATP.
64.
GOGOLEP, YOUNG MC, KUBASEKWL, JARVISTC, VON HIPPEL
PH: Cryoclectron Microscopic Visualization of Functional
Subassemblies of the Bacteriophage T4 DNA Replication
Complex. J Mol Blol 1992, 224:395--412.
The observed T4 DNA replication complex structures (hash-marks)
were not formed on supercoiled DNA, but only on DNA with a nick
or a gap. In some instances several hash-marks were observed in
clusters on one DNA molecule. It is not yet certain whether the
observed hash-mark structure is the gp45 trimer or the gp44--gp62
complex.
•.
65.
ONRUST R, O'DONNELL M: DNA Polymerase IlI Accessory
Proteins. H. Characterization of 8 and 8". J BIol Chem 1993,
268:11766-11772.
66.
ONRUSTR, STLIKENBERG PT, O'DONNELL M: Analysis of the
ATPase Subassembly which Initiates Processive DNA Synthesis by DNA Polymerase III Holoenzyme. J BIol Chem 1991,
266:21(-~1-21686.
DNA replication: enzymology and mechanisms Kelman and O'Donnell
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68.
ONRUSTR: The Structure and Function of the Accessory
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XIAOH, DUNG Z, O'DONNELL M: DNA Polymerase III Accessory Proteins. VI. Characterization of c and y. J Btol Chem
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69.
•
FRADKINLG, KORNBERG A: Prereplicative Complex of Cornponents of DNA Polymerase III Holoenzyme of Escherlchla
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This study identifies an association between y complex and ssDNA
that requires SSB. The paper also shows that the "ycomplex can place
[3 onto DNA without SSB.
zyme at a Primer-Template Junction. J Biol Chem 1991,
266:20034--20044.
76.
•
BURBELOPD, UTANI A, PAN Z-Q, YAMADAY: Cloning of the
Large Subunit of Activator 1 (Repfication Factor C) Reveals
Homology with Bacterial DNA I igases. Proc Nail Acad Scl
USA 1993, 90:11543--11547.
This paper describes the cloning of the gene encoding RF-C. The authors note regions of homology between the carboxy-terminal portion of the large subunit of murine RF-C and the four smaller subunits
of human RF-C.
70.
RUSH J, LIN T-C, QUINONES M, SPICER EK, DOUGLAS I,
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PAN Z-Q, CHEN M. HuRwrrz J: The Subunits of Activator 1
(Replication Factor C') Carry Out Multiple Functions Essential for Proliferating-Cell Nuclear Antigen-Dependent DNA
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The authors show that under some conditions p37 stimulates pol 8,
but not pol 8. They also show that p40 inhibits pol 5, suggesting a
p40-pol 8 interaction.
71.
CHA T-A, ALBER'I~BM: In Vltro Studies of the T4 Bacteriophage DNA Replication System. Cancer Celb 1988, 6:1-10.
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MUNH MM. ALBER'I.'S BM: The T4 DNA Polymerase
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HOCKENSMrI'HJ'~/, KUBASEKWL, EVER'I~Z EM, MESNER LD,
VON HIPPEL PH: Laser Cross-Linking of Proteins to Nucleic
Acids. II. Interactions of the Bacteriophage T4 DNA Replication Polymerase Accessory Proteins Complex with DNA.
J B~ol Chem 1993, 268:15721-15730.
This paper shows that UV-induced cross-links between DNA and
gp62 are increased upon adding gp45 to the gp44-gp62 complex,
indicative of a conformational change of gp44--gp62 relative to DNA
upon placing gp45 on DNA. The authors present models by which
gp45 is placed on DNA by gp44-gp62 complex.
77.
•
HERENDEENDR, KASSAVE'nSGA, GEIDUSCHEKEP: A Transcriptional Enhancer whose Function Imposes a Requirement that Proteins Track Along DNA. Science 1992,
256:1298-1303.
The nicked site for assembly of the T4 sliding clamp was placed on
one DNA ring of a two ring catenane; the other ring contained the T4
late promoter. The sliding clamp on one ring was unable to enhance
transcription on the other ring.
• •
74.
CAPSONTL, BENKOVIC SJ, NOSSAL NG: Protein-DNA CrossLinking Demonstrates Stepwis¢ ATP-Dependent Assembly
of T4 DNA Polymerasc and its Accessory Proteins on the
Primer-Template. Cell 1991, 65:249-258.
75.
MUNNMM, ALBERTS BM: DNA Footprinting Studies of the
Complex Formed by the T4 DNA Polymerase Holoen-
79.
O'REILLYDR, CRAWFORDAM, MILLER LK: Viral Proliferating
Cell Nuclear Antigen. Nature 1989, 337:606.
80.
•-
XIONGY, ZHANG H, BEACH D: D Type Cyclins Associated
with Multiple Protein Kinases and the DNA Replication and
Repair Factor PCNA. Cell 1992, 71:505-514.
It is proposed that PCNA is a member of multiprotein complexes
containing p21 and combinatorial variations of cyclins D1 and 3 and
cyclin-dependent kinases 2, 4 and 5.
Z Kelman and M O'Donnell, Department of Microbiology, Comell
University Medical College, 1300 York Avenue, New York, New York
10021, USA.
195