THEJOURNAL OF BIOLOGICAL CHEMISTRY
Minireview
SV40 DNA Replication
Thomas J. Kelly
From the Department of Molecular Biology and Genetics,
The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205
In a growing mammalian cell approximately a million kilobases of
DNA are duplicated with great fidelity once each cell cycle.HOWthis
enormous task is accomplished is not understood, but considerable
insight into the process has been derived from studies of viruses that
multiply in animal cells. A detailed biochemical description of DNA
replication and its regulation is obviously crucial to understanding
the growth and development of complex eukaryotic organisms. Such
a description should also contribute to increased understanding of
the perturbations of growth that occur in various disease states.
SV40 as a Model Replicon
Eukaryotic cells solve the problem of replicating very large genomes
by initiating DNA synthesis a t multiple sites (1).Thus, during the S
phase of the cell cycle DNAsynthesis occurs simultaneously in many
independent units of replication or “replicons.” It follows that to
understand the replication of eukaryotic genomes one should first
understand the events that occur in single replicons. The tumor virus
SV40 has proved to be a remarkably good model for this purpose (2,
3). The viral genome consists of a circular duplex DNA molecule of
about 5000 base pairs containingone origin of DNA replication. SV40
DNA replication takes place in the nucleus of the host cell where the
viral genome is complexed with histones to form a nucleoprotein
structure indistinguishable from cellular chromatin. Since SV40 en-
codes only a single replication protein (T antigen), the virus makes
extensive use of the cellular replication machinery. As a result there
are many similarities between viral and cellular DNA replication. In
both cases initiation of DNA synthesis results in the establishment
of two replication forks that move in opposite directions. At each fork
one of the two nascent strands (the leading strand) grows continu-
ously, while the other strand (the lagging strand) grows by joining
together small segments of DNA that are repeatedly initiated with
RNA primers. Completion of replication occurs when two oppositely
moving forks meet. (In linear cellular chromosomes the two merging
forks originate in adjacent replicons, while in circular SV40 chromo-
somes they originate in the same replicon.)
Although a good deal has been learned about SV40 DNAreplication
from in uiuo studies, analysis of the molecular mechanisms involved
in the process has been accelerated by the development of an efficient
cell-free replication system (4). As a result of contributions from a
number of laboratories the basic features of the replication pathway
are becoming clear (Fig. 1).An important (and expected) dividend of
this work is the identification and functional characterization of
components of the cellular replication machinery.
Initiation of DNA Replication
The Origin-TheSV40 origin of replication isa 64-base pair
segment of the viral genome that contains all of the nucleotide
sequence elements that are required for initiation of viral DNA
replication. Recent genetic studies indicate that the origin is quite
complex, consisting of at least three functionally distinct domains
(5-7). At the center of the origin are four copies of a simple 5-base
pair sequence (GAGGC) organized as an inverted repeat. This se-
quence element is recognized bythe viral initiation protein,T antigen.
On one side of the T antigen binding domain is a 17-basepair segment
containing exclusively A and T residues. This segment is probably
the locus of the initial strand separation event in DNA replication.
On the other side of the T antigen binding site is a 15-base pair
imperfect inverted repeat of unknown function. Thereare some
striking similarities between the SV40 origin andthe origins of
prokaryotic genomes such as Escherichia coli and bacteriophage X.
Both of the latter genomes contain repeated recognition sites for the
17889
Vol. 263 No. 34 Issue of December 5, pp. 17889-17892,1988
0 1988 hy The American’Society’of Biochemistry and Molecular Biology. Inc.
Printed in U.S.A.
relevant initiator proteins(dnaA protein and 0 protein, respectively),
and both have A-T-rich regions located adjacent to therepeats. There
is growing evidence that the similarities in the genetic organization
of the SV40 and theprokaryotic origins reflect underlying similarities
in basic initiation mechanisms (Refs. 8 and 9 and see below).
T Antigen-The 95-kDa SV40 T antigen plays the central role in
initiation of viral DNA replication (2, 3, 10). Binding studies suggest
that a T antigen molecule binds to each of the four pentamer repeats
in the origin to form an organized nucleoprotein structure that is
competent for initiation (11).At physiologic temperatures the for-
mation of this T antigen-origin complex is greatly facilitated by ATP
(12,13). Once bound to theorigin the T antigen is capable of entering
the duplex and catalyzing the ATP-dependent unwinding of the two
DNA strands (14-16). The unwinding reaction, which is an expression
of a recently discovered helicase activity that is intrinsic to the T
antigen molecule (17), appears to represent a critical step in the
replication process. The reaction establishes the two replication forks
and generates the substrate for assembly of the proteins required for
the priming and elongation of nascent strands.
The unwinding reaction requires the A-T-rich region adjacent to
the T antigen binding site, consistent with the idea that this is the
initial site of entry by T antigen (15, 18).While the reduced stability
of A-T base pairs would presumably facilitate strand separation in
this region, recent genetic studies indicate that theprecise nucleotide
sequence of the A-T domain, not just its base composition, is an
importantdeterminant of the efficiency of unwinding (18). One
possible explanation for this observation is that T antigen makes
sequence-specific contacts with the A-T-rich domain during the
course of the unwinding reaction. A second, perhaps more interesting,
possibility is suggested by the finding that the A-T domain causes a
significant bend inthe helix (6). Anumber of single base substitutions
within the domain that reduce the efficiency of unwinding and
replication also changes the degree of net bending. Thus, theconfor-
mation of the DNA in this domain may be critically important. For
example, bending could contribute to further destabilization of the
duplex in the A-T domain or could facilitate interactionof the domain
with T antigen during entry or unwinding.
Cellular Proteins-In addition to specific nucleotide sequence ele-
ments, the T antigen-mediated unwinding reaction requires accessory
proteins contributed by the host cell. One such protein, replication
protein A(RP-A or RF-A), has recently been purified to homogeneity
(19, 20). RP-A is absolutely required for SV40 DNA replication in
vitro. It consists of three subunits, the largest of which binds specif-
ically to single-stranded DNA.’ It is likely that one role of RP-A is
simply to prevent the reassociation of the single strands exposed
during the unwinding reaction. This view is supported by the well
documented observation that T antigen can catalyze the ATP-de-
pendent unwinding of origin-containing DNA molecules in the pres-
ence of heterologous single-stranded DNA binding proteins such as
E. coli SSB (14-16). However, E. coli SSB cannot replace RP-A in
the complete DNA replication reaction, indicating that RP-A must
play other roles in DNA replication (15). Although T antigen and
RP-A are the only proteins that are absolutely required for origin-
dependent unwinding, recent evidence indicates that optimal un-
winding efficiency requires at least one additional cellular protein
(CF-IC).’ Although it seems likely that this protein participates in a
complex with T antigen and the DNA during the early stages of the
reaction its precise function is not yet clear.
Replication and Transcription
Although the 64-base pair core origin of DNA replication contains
all of the sequence elements that are absolutely required in cis for
initiation of DNA synthesis, it is apparent that sequences outside of
the core can profoundly influence the efficiency of initiation. The
largest effects have been seen with elements previously associated
with activation of transcription, such as the SV40 enhancers or the
’ M. S. Wold, D. H. Weinberg, D. M. Virshup, J. J. Li, and T. J.
Kelly (1988) J. Biol. Chem., in press.
17890 Minireview: SV40 DNA Replication
RECOGNITION I UNWINDING
\
T antigen
Replication Protein A
CF-IC
‘0
I
TERMINATION \ INITIATION
7 Polymerase a
Primase
-
Polymerase a - Prirnase
Polymerase S
PCNA
Topolsomerase I / II
? T antigen
7 Replication protein A
FIG. 1. Stages in SV40 DNA replication. The figure summa-
rizes the proteins currently implicated in each stage of SV40 DNA
replication.
binding sites for the transcriptional factor Sp-1 (22-26). The presence
of these sequence elements adjacent to the core origin increases the
efficiency of DNA replication in vivo a t least 10-fold. Activation of
DNA replication by enhancers and other transcriptional elements is
not limited to SV40 and, in fact, appearsto be a quite general feature
of the replication of eukaryotic viruses. The phenomenon was first
seen with polyoma virus (27) but has since been observed for adeno-
virus, Epstein-Barr virus, and bovine papillomavirus, as well as SV40
(28-30).
One can envision a number of ways in which transcriptional
sequence elements might serve to activate DNA replication. In the
case of SV40 it is likely that the binding of transcriptional activator
proteins affects replication indirectly by perturbing the local distri-
bution of nucleosomes, so that the DNA in the adjacent core origin
region is relatively nucleosome-free (31, 32). This presumably facili-
tates the interactions of the core origin with T antigen and other
initiation proteins, thereby increasing the efficiency of unwinding
and initiation. This general idea has considerable experimental sup-
port. Direct analysis of SV40 chromatin isolated from infected cells
has revealed that the core origin region is less likely to be packaged
into a nucleosome than other regions of the viral genome (31-33). In
addition, studiesof viral mutants have demonstrated that thegenetic
determinants of the nucleosome exclusion effect reside in the SV40
enhancer elementsand the Sp-1 binding sites (34-36). Finally, recent
in uitro studies with the SV40 system indicate that a purified tran-
scriptionalactivatorprotein can reduce the inhibitory effect on
replication of packaging the template into nucleosomes?
In other viral systems the activation of DNA replication by tran-
scriptional elements may operate by different mechanisms. For ex-
ample, initiation of adenovirus DNA replication is highly dependent
upon two cellular proteins, NF-I and NF-111, that bind to specific
sequence elements in the viral origin of DNA replication. Both
proteins have been shown to function as transcriptional activators of
specific cellular genes (28, 37). In this case strong stimulation of
initiation by the two proteins can be observed in a purified cell-free
system that lacks histones, so the mechanism of activation clearly
differs from that of SV40. Since the adenovirus cell-free system also
lacks RNA polymerase activity, the effect of the proteins is not a
consequence of stimulating transcription in the neighborhood of the
origin as in the case of bacteriophage X. The most likely hypothesis
is that NF-I and NF-111 act via direct protein-protein interactions to
stabilize a specific initiation complex consisting of origin DNA and
viral initiation proteins.
* L. Cheng and T.J. Kelly, unpublished data.
Elongation of DNA Chains and Fork Movement
Fractionation of the cell-free SV40 DNA replication system has
provided a useful approach to identifying cellular proteins that are
involved in the elongation of nascent DNA chains. This work has
nicely complemented biochemical studies of isolated eukaryotic DNA
polymerases that have been proceeding for a number of years. Al-
though muchwork remains to be done, a broad outline of the
enzymology of the mammalian replication fork is rapidly emerging.
DNA Polymerase a-Mammalian cells contain four distinguishable
DNA polymerase activities designated a,@, y, and 6. Of these DNA
polymerase a (38) and probably DNA polymerase 6 (39) are required
for SV40 DNA replication in the cell-free system (4, 40, 41).’ DNA
polymerase 01 has long been considered to be the major replicative
polymerase in animal cells (38). The activity of the enzyme is highly
correlated with cell proliferation, and inhibitors of the purified en-
zyme inhibit chromosomal DNA replication in uiuo. DNA polymerase
a has been purified from a variety of sources by several laboratories,
and there is now general agreement that the enzyme is composed of
four distinct subunits (38,40).The largest subunit (180 kDa) contains
the polymerase active site. In thecase of the Drosophila DNA polym-
erase a the large subunit also harbors a cryptic 3’4’-exonuclease
that serves a proofreading function during polymerization (43). In
the intactpolymerase the exonuclease activity is normally masked by
a second subunit of 70 kDa. The proofreading exonuclease activity is
very likely an important general feature of DNA polymerase a that
contributes significantly to thefidelity of DNA replication. However,
the presence of the exonuclease activity in DNA polymerase a en-
zymes from other species has not yet been confirmed. The smallest
subunits of DNA polymerase a (50-60 kDa) constitute a primase
enzyme capable of synthesizing short RNA transcripts thatcan serve
as primers for subsequent DNA chain elongation by the catalytic
subunit (44,45). Thus,DNA polymerase a contains all of the activi-
ties required for the initiation and synthesis of nascent DNA chains
on single-stranded DNA templates. It is not, however, a highly
processive DNA polymerase as less than 100 nucleotides are polym-
erized per binding event under the usual assay conditions.
DNA Polymerase 6-DNA polymerase 6 (39) was originally distin-
guished from DNA polymerase a because it contains areadily detect-
able 3’+5’-exonuclease activity (46). The two polymerases also differ
from each other in their template preferences, chromatographic prop-
erties, and responses to various antibodies and inhibitors. Moreover,
polymerase 6 appears to lack an associated primase activity. Thus, it
seems likely at thispoint that DNA polymerases a and 6 are distinct
molecular entities; however, since the structural features of DNA
polymerase 6 are not yet well understood, the possibility remains that
the two polymerases share similar or identical subunits.
A 37-kDa protein that dramatically increases the activity of DNA
polymerase 6 with template/primers containing long single-stranded
regions was recently purified to homogeneity (47). Interestingly, the
37-kDa protein proved to be identical to a previously described
polypeptide found specifically in proliferating cells and referred to as
proliferating cell nuclear antigen (PCNA)3 or cyclin (48, 49). In the
presence of PCNA, DNA polymerase 6 is a highly processive enzyme
catalyzing the polymerization of more than 1000 nucleotidesfiinding
event. PCNA has no effect on the activity or processivity of DNA
polymerase a, providing further evidence that the two polymerases
are biochemically distinct.
It hasbeen demonstrated that PCNA is required for efficient SV40
DNA replication in the cell-free system (50). In theabsence of PCNA
initiation of DNA replication at theviral origin can occur normally,
but only short nascent strands, containing a maximum of a few
hundred nucleotides, are synthesized (41, 50). The observation that
PCNA is required for extensive chain elongation provides strong
circumstantial evidence that DNA polymerase 6 is involved in DNA
replication, a view that is also supported by inhibitor studies(51,52).
More direct evidence on this point has come from recent experiments
indicating that both DNA polymerase a and DNA polymerase 6
activities are required to reconstitute efficient DNA replication in the
SV40 cell-free system?
The Two-polymeraseModel-If both DNA polymerase a and DNA
polymerase 6 are involved in DNA replication, how do they divide the
labor? At this point there areseveral possible answers to thisquestion,
but one particularly intriguing possibility is that DNA polymerase 6
The abbreviation used is: PCNA, proliferating cell nuclear anti-
gen.
‘D. H. Weinberg and T. J. Kelly, unpublished data.
 Minireview: S V40 DNA Replication
carries out DNA synthesis on the leading strand andDNA polymerase
a carries out DNA synthesis on the lagging strand (53). This model
is consonant with the known biochemical properties of the two
polymerases. The leading strand polymerase would be expected to be
highly processive and would derive little advantage from close asso-
ciation with a primase activity. The lagging strand polymerase, on
the other hand, would require only moderate processivity but would
benefit enormously from a tightly associated primase activity. The
model is also supported by recent studies indicating that PCNA, the
accessory factor for polymerase 6, is required for leading strand
synthesis in areconstituted SV40 DNA replication system (54).
Although direct evidence for the model is currentlylacking, it provides
a satisfying solution to thebiochemical problems posed by the differ-
ing modes of DNA synthesis on the two sides of the replication fork,
and it also represents a good working hypothesis for the design of
future experiments.
Fork Movement-As a eukaryotic replication fork advances the
parental DNA strands are unwound at a rate of about 1000 base
pairs/min. By analogy with prokaryotic systems the key enzymatic
player in this process is likely to be a DNA helicase. DNA helicases
are enzymes that utilize ATP to move undirectionally along a DNA
strand, opening any regions of duplex DNA that are encountered.
The identity of the helicase activity required for fork movement
during SV40 DNA replication has not been established with certainty.
One reasonable possibility is that T antigen fulfills this function in
addition to its role in mediating unwinding of the origin region prior
to initiation. In the presence of a single-stranded DNA binding
protein the intrinsic helicase activity of T antigen is capable of
unwinding long segments of duplex DNA ina highly processive
manner (55, 56). Moveover, it has been reported that monoclonal
antibodies that inhibit the helicase activity of T antigen also inhibit
ongoing DNA synthesis in subcellular
a SV40 DNA replication system
(57). Studies with model substrates indicate that the T antigen
helicase translocates in the 3' to 5' direction on single DNA strands
(55, 56). Thus, if T antigen is the helicase responsible for fork
movement it would be expected to move along the leading strand. In
contrast, all of the prokaryotic DNA helicases that are known to be
directly involved in DNA replication move along the lagging strand
(21).
At this time it remains possible that a cellular DNA helicase, rather
than T antigen, is responsible for unwinding the parental strands a t
SV40 replication forks. It is evident that there must exist a cellular
helicase(s) that operates a t chromosomal replication forks, and such
an enzyme could mediate SV40 fork movement. While helicase activ-
ities have been identified in extracts of eukaryotic cells, it is not yet
clear whether any of these activities are involved in DNA replication
(58, 59). It has also been reported that highly purified DNA polym-
erase d is capable of some degree of strand displacement during DNA
synthesis, suggesting that helicase activity may be intrinsic to this
enzyme (53). Alternatively, the helicase activity required for chro-
mosomal replication may reside in an as yet unidentified cellular
protein.
In addition to a DNA helicase the rapid advancement of the
replication fork requires the activity of a topoisomerase to relieve
superhelical tension that would otherwise hinder the unwinding of
the parentalstrands. This is most obvious in the case of a covalently
closed circular genome where the unwinding of one turn of the
parental helix results in the introduction of one superhelical turn.
However, a similar phenomenon probably occurs with long linear
genomes, especially those containing multiple replication or tran-
scription units thatwould beexpected to present considerable resist-
ance to rotation in a viscous medium. In both cases a topoisomerase
can act as a swivel, thus reducing the energetic barrier to unwinding.
There are two major mammalian DNA topoisomerases. Topoisom-
erase I introduces transient single-stranded breaks in duplex DNA,
while topoisomerase I1 introduces transient double-stranded breaks.
Reconstruction experiments in the SV40 cell-free system have pro-
vided evidence that either topoisomerase I or topoisomerase I1 can
provide the swivel necessary for efficient fork movement (60).
Segregation-Termination of SV40 DNA replication takes place
when the two replication forks meet approximately halfway around
the genome. As pointed out above, this is equivalent to the merger of
forks from adjacent replicons during chromosomal DNA replication.
There appear to be no special nucleotide sequences that signal the
termination of SV40 DNA replication, but it is not yet clear whether
termination requires special protein factors. In general, the links
between the parental strands are not completely removed prior to
termination, so the immediate products of DNA replication consist
17891
of two daughter molecules that are multiply intertwined (61). The
final act of DNA replication is the segregation of these intertwined
daughter duplexes into two separate unlinked molecules. Reconstruc-
tion experiments in uitro suggest that this reaction is catalyzed by
topoisomerase I1 (60). Thus, there are two distinct roles for topo-
isomerases during SV40 DNA replication. One role, which can be
played by either topoisomerase I or topoisomerase 11, is to provide a
swivel during chain elongation. The second role, which can only be
fulfilled by topoisomerase 11, is to mediate segregation of the newly
synthesizedviral chromosomes. Genetic experiments in yeast strongly
suggest that thetopoisomerases play these same two roles during the
replication and segregation of cellular chromosomes (62,63,71).
Regulation of DNA Replication
Although viruses have evolvedhighly efficient mechanisms for
producing many copies of their genomes in a relatively short period
of time, this does not mean that viral DNA replication is an uncon-
trolled process. In fact, SV40 DNA replication appears to be subject
to regulation at several different levels. Although most of these
regulatory mechanisms are not well understood, they are objects of
considerable interest because of the likelihood that they may shed
some light on cellular regulatory mechanisms.
One of the most interesting properties of the T antigen is its ability
to activate the infected cell for DNA synthesis by perturbing the
normal mechanisms that regulate cell proliferation (10). It is well
known that resting or quiescent mammalian cells do not express
many of the gene products that are required for DNA replication.
These include DNA polymerase a, PCNA, topoisomerase 11, and
others. It follows that such cells would represent an unproductive
environment for SV40 infection. However, the SV40 T antigen in
some manner induces quiescent cells to enter S phase so that they
express all of the required replication proteins. The biochemical basis
of the phenomenon is not clear, but further work on the problem may
well provide considerable insight into the mechanisms that control
cellular proliferation.
At another level, SV40 regulates the replication of its own genome
by controlling the amount of available T antigen. This is accom-
plished by an autoregulatory mechanism that controls the frequency
of initiation of transcription of the T antigen gene (64). The essential
elements comprising the promoter for the T antigen gene lie within
and adjacent to the minimal origin of DNA replication. The binding
of T antigen to recognition sites in the origin region represses tran-
scription from this promoter. One possible function of this regulatory
mechanism is to optimize the fraction of the cell's energy resources
that are devoted to genome replication uersus production of virion
proteins.
There is also some evidence that certain biochemical activities of
T antigen may be controlled by post-translational modification. For
example, T antigen is a phosphoprotein with numerous potential sites
of phosphorylation. In infected cells the degree of phosphorylation of
T antigen appears to increase significantly with time after its synthe-
sis (65,66). Several studies have suggested that phosphorylation of T
antigen may reduce its ability to support SV40 DNA replication in
uitro, perhaps by decreasing the affinity of the protein for its specific
recognition sites (67-69). However, the biological relevance of these
observations has not been established, so at present the notion that
SV40 DNA replication may be controlled in part by covalent modi-
fication of the initiator protein remains an intriguing but unproven
possibility.
Similarity of the Basic Replication
Mechanisms in
Prokaryotes and Eukaryotes
It is apparent from what has been learned so farabout DNA
replication in prokaryotes and eukaryotes that many of the most
fundamental features of the process have been highly conserved
during evolution. As has been pointed out, the replication origins of
SV40 and other animal viruses, while not identical to those of
prokaryotes or even to each other, clearly share anumber of common
structural features with their prokaryotic counterparts. The initial
stages of DNA replication in prokaryotes and eukaryotes appear to
involve recognition of a specific nucleotide sequence followed by a
localized unwinding of the duplex (70). It is this step that determines
the specificity of DNA replication, andit is also this step that
generates the structure upon which the enzymatic machinery neces-
sary for rapid chain elongation is assembled. The general organization
of the replication forks and the processes that occur on the leading
and lagging strands are fundamentally similar in prokaryotes and
17892 Minireview: SV40 DNA Replication
eukaryotes. Finally, there is growing evidence that inboth eukaryotic
and prokaryotic systems the replication machinery at the fork is
asymmetric, containing one DNA polymerase activity with biochem-
ical properties suitable for continuous DNA synthesis and another
with biochemical properties suitablefor discontinuous DNA synthesis
(70).
In spite of these profound similarities, it seems likely that impor-
tant differences exist in the DNA replication mechanisms of eukar-
yotic and prokaryotic cells. This is suggested by the much greater
complexity of the eukaryotic genome, the presence of multiple repli-
cation units within a single chromosome, and theexistence of unique
genetic elements, such as telomeres and centromeres, that play im-
portant roles in chromosome segregation and stability. Moreover,
DNA replication in eukaryotes, unlike prokaryotes, is confined to a
specific portion of the cell cycle, the S phase, during which every
segment of DNA must be duplicated once and only once. Finally,
eukaryotic cell proliferation must be responsive to a variety of exter-
nal stimuli that occur during the normal development and growth of
the organism. Thus, theregulatory mechanisms that control eukary-
otic DNA replication are likely to be more complex and diversified
than those thatoperate in prokaryotes.
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