Sumber 1
Sumber 1
Sumber 1
tion. Furthermore, like cellular DNA, the viral genome is packaged into
chromatin, and its replication occurs only during the S phase of the cell
cycle.
Large T antigen, the viral initiator protein for DNA replication, is a
sequence-specific DNA-binding protein that also possesses intrinsic
DNA helicase and ATPase activity. These activities of large T antigen
are required directly for its ability to effect viral DNA replication. Large
T antigen also functions indirectly to effect viral DNA replication by
stimulating the synthesis of cellular enzymes whose activity is needed for
viral DNA replication. For example, large T antigen binds to ~ 1 0 5 ~ ~ / E 2 F
complexes, resulting in the release and activation of the transcription ac-
tivator E2F, whose target genes encode enzymes such as thymidine
kinase, dihydrofolate reductase, and DNA polymerase-cprimase.
Initiation of viral DNA replication takes place within the origin (ori),
proceeds bidirectionally, and terminates when the two replication forks
meet approximately 180° from where they started. Replication of the
leading strand occurs by a continuous mechanism, whereas that of the
lagging strand proceeds discontinuously (see Brush and Kelly, this
volume). Hence, both SV40 and PyV DNA replication are semi-
discontinuous. Large T antigen binds to repeats of its recognition ele-
ment within ori and unwinds this region to create a replication bubble.
Large T antigen also interacts with cellular replication proteins to assem-
ble the primosome at ori and functions as a helicase at replication forks.
Replication of the viral genome requires the interplay among cis-
acting sequences defining the functional ori, large T antigen, and a num-
ber of cellular proteins, including transcription activators and replication
proteins. In the sections that follow, we summarize what is known about
these three major components of the replication apparatus and suggest a
model by which their interaction results in the replication of the viral
genome.
A. SV40
250
I I
Minimal
Replication origin
Replication origin
Replication origin
6. PYV
12711 mm binding
T antigen
sites
Replication origin
Figure I (A) Structure of the SV40 ori for DNA replication. The locations of
the 72-bp repeat enhancer and the 21-bp repeat upstream promoter elements are
shown. The ori-core contains the ori of bidirectional DNA replication (OBR).
Stippled arrowheads represent the principal late (LS) and early (ES) start sites
for transcription. The locations of the three large-T-antigen-binding sites are dis-
played below the origin region. The SV40 ori-core is sufficient to constitute a
minimal ori for DNA replication in vivo; efficient replication oris are composed
of the ori-core and either the 21-bp repeats or the 72-bp repeats. ( B ) Structure of
the PyV ori for DNA replication. The positions of the a and fl elements of the
PyV enhancer are shown. A functional PyV replication ori requires the ori-core
and either the a or the p element located at the late border of ori-core.
642 J.A. Hassell and B.T. Brinton
A.
- 1,15243
SV40 origin core
5197
0.
5285
--5292l1
PyV origin core
t t
Figure 2 Sequence of the SV40 (A) and PyV ( B ) ori-cores for DNA replication.
The three principal sequence elements of the ori-core are shown: the A/T-rich
region at the late border, the central G/C-rich palindrome, and the early
palindrome at the early border. Arrowheads depict the position and orientation
of the 5 ' -GAGGC3 ' large-T-antigen-bindingmotifs.
Large T Antigen
The large T antigens of SV40 and PyV are multifunctional, nuclear
phosphoproteins (for review, see Prives 1990; Fanning 1992; Fanning
and Knippers 1992; Pipas 1992; Manfredi and Prives 1994). SV40 and
PyV large T antigen are composed of 708 and 785 amino acids, respec-
tively. These two proteins are related in primary structure (-36% se-
quence identity) and share the same biochemical activities (Fig. 3). These
SV40 and Polyomavirus DNA Replication 645
A. SV40 T antigen
1
B. PyV T antigen
Replication Protein A
RP-A, which is also known as replication factor A (RFA) and human
single-stranded DNA-binding protein (HSSB), was originally identified
as an essential single-stranded DNA-binding protein required for SV40
DNA replication in vitro (Wobbe et al. 1987; Wold and Kelly 1988;
Fairman and Stillman 1988). RP-A functions in concert with large T
antigen and DNA polymerase-a:primase to initiate DNA replication at
ori. RP-A is also needed during the elongation phase, acting to stimulate
the activity of both DNA polymerase-a:primase and DNA polymerase-8
at replication forks (for review, see Melendy and Stillman 1992). The
subunit composition of RP-A and the sequence of each of its subunits are
highly conserved among diverse species. Gene disruption experiments in
the budding yeast Saccharomyces cerevisiae demonstrate that each sub-
unit is required for viability (Heyer et al. 1990; Brill and Stillman 1991).
In this organism, and likely all eukaryotes, RP-A participates not only in
DNA replication, but also in recombination and repair (Longhese et al.
1994; Firmenich et al. 1995; Smith and Rothstein 1995).
Human RP-A is a heterotrimer of 70-kD, 32-kD, and 14-kD subunits
(Fairman and Stillman 1988; Wold and Kelly 1988). The 70-kD subunit
is independently capable of binding to single-stranded DNA (Brill and
Stillman 1989; Wold et al. 1989; Kenny et al. 1990) to DNA
polymerase-a:primase (Dornreiter et al. 1992), and to the activation
domains of several transcription activators (He et al. 1993; Li and
SV40 and Polyomavirus DNA Replication 649
Botchan 1993). No activity or function has yet been ascribed to the other
two subunits, but the 70-kD subunit is incapable of substituting for the
trimeric protein in SV40 DNA replication, implying that one or both of
them are important in this process (Erdile et al. 1991). Antibodies
specific for each of the subunits inhibit SV40 DNA replication in vitro,
suggesting that all the subunits are required (Erdile et al. 1990, 1991;
Kenny et a]. 1990; Umbricht et al. 1993). The 32-kD and 14-kD subunits
form a complex, which may serve to recruit the larger subunit (Hendrick-
son et al. 1994).
650 J.A. Hassell and B.T. Brinton
DNA Polymerase-a:Primase
DNA polymerase-mprimase is the only DNA polymerase in mammalian
cells capable of initiating DNA synthesis de novo through the synthesis
of RNA primers (for review, see Wang 1991 and this volume). Studies
with inhibitors and inactivating antibodies to the protein reveal that it is
required to replicate SV40 and PyV DNA (Melendy and Stillman 1992
and references therein). Currently, it is thought that the role of DNA
polymerase-a:primase is to initiate both leading-strand synthesis at the
ori and Okazaki fragment synthesis on the lagging-strand template at
replication forks (Waga and Stillman 1994a; Waga et al. 1994 and
references therein).
Human DNA polymerase-mprimase is composed of four subunits of
molecular mass 180, 70, 58, and 48 kD; the subunits of the mouse
protein are similar in size (180, 68, 54, and 46 kD) (Wang 1991;
Miyazawa et al. 1993). DNA polymerase activity is intrinsic to the 180-
kD subunit, whereas primase activity resides in the 48-kD subunit
(Copeland and Wang 1993). The 58-kD subunit binds to the 48-kD sub-
unit and appears to stabilize the primase activity of the smaller subunit.
The role of the 70-kD subunit is not clear. It associates with the 180-kD
subunit (Collins et al. 1993) and is phosphorylated in a cell-cycle-
dependent manner, suggesting that it may play a regulatory role
(Nasheuer et al. 1991). These attributes of the human DNA polymerase-
a:primase subunits are conserved in their murine counterparts.
The replication of SV40 and PyV DNA is highly species-specific
(Bennett et al. 1989 and references therein). SV40 DNA replication takes
place in monkey or human cells, but not in mouse cells. In contrast,
mouse cells, but not primate cells, support the replication of PyV DNA.
Species-specific DNA replication in vivo requires large T antigen, the
652 J.A. Hassell and B.T. Brinton
cognate virus ori, and factors from permissive cells (Bennett et al. 1989).
Cell-free replication systems recapitulate this species specificity and
have proven invaluable in identifying permissive factors. The addition of
purified mouse DNA polymerase-a:primase to human cell extracts
renders them permissive for PyV DNA replication, and correspondingly,
the addition of the human enzyme to mouse cell extracts allows the
mouse extracts to support SV40 DNA replication (Murakami et al.
1986). These experiments suggest that DNA polymerase-a:primase is the
primary host determinant for species-specific viral DNA replication. Fur-
ther fractionation of the DNA polymerase revealed that the primase sub-
units (a heterodimer of the 54-kD and 46-kD subunits) from mouse DNA
polymerase-a:primase enabled human extracts to replicate PyV DNA
(Eki et al. 1991). Extension of these experiments with DNA polymerase-
a:primase, reconstituted in insect cells after infection with recombinant
baculoviruses encoding individual subunits of human or mouse origin,
showed that the small mouse 46-kD primase subunit is sufficient to
mediate species-specific replication of PyV DNA in vitro in human cell
extracts (Bruckner et al. 1995). Surprisingly, similar experiments with
these reconstituted DNA polymerase-a:primase enzymes of mixed
human-mouse origin showed that SV40 DNA replication in vitro re-
quired a different subunit, namely, the p180 catalytic subunit of the DNA
polymerase from human cells (Stadlbauer et al. 1996). Presumably, this
reflects a difference in the functional interaction between the DNA
polymerase and the large T antigen of each virus.
The large T antigens of both SV40 and PyV physically interact with
several of the subunits of DNA polymerase-a:primase in vitro (Smale
and Tjian 1986; Gannon and Lane 1987; Dornreiter et al. 1990; Moses
and Prives 1994: Schneider et al. 1994; Bruckner et al. 1995). SV40
large T antigen binds independently to both the 180-kD and the 70-kD
subunits of human DNA polymerase-a:primase (Dornreiter et al. 1992;
Collins et al. 1993), although the principal interaction appears to be with
the 70-kD subunit (Collins et al. 1993). Amino acids 195-313 in the 180-
kD subunit (Dornreiter et al. 1993) and residues 1-239 of the 70-kD sub-
unit are sufficient for interaction with SV40 large T antigen (Collins et
al. 1993). Interestingly, two discontinuous regions in SV40 large T
antigen are required to bind to the human holoenzyme (Fig. 3), but it is
not known whether each region binds to a different subunit (Smale and
Tjian 1986; Gannon and Lane 1987; Dornreiter et al. 1990). PyV large T
antigen binds to the 180-kD subunit of mouse DNA polymerase-
ccprimase, to a complex containing the murine 180-kD and 68-kD sub-
units, and to the 46-kD primase subunit (Bruckner et al. 1995). It is not
SV40 and Polyomavirus DNA Replication 653
Replication Factor C
Human RF-C (also called activator 1 or A l ) is a multisubunit DNA
polymerase accessory protein composed of five independent subunits of
656 J.A. Hassell and B.T. Brinton
units (140, 40, and 37 kD); only partial cDNAs are available for the 38-
kD and 36.5-kD species. The 140-kD subunit binds to double-stranded
oligonucleotides likely by recognizing a structure that mimics a primer/
template junction (Tsurimoto and Stillman 1991a; Burbelo et al. 1993;
Lu et al. 1993). The 37-kD subunit also is capable of binding to primer/
template junctions, but its specific DNA-binding activity is only 0.1%
that of the RF-C holoenzyme, and hence its role in primer binding is un-
clear (Pan et al. 1993; Noskov et al. 1994). Whereas all of the RF-C sub-
units bear the putative ATP-binding motif, only the 40-kD subunit is
known to bind to ATP (Pan et al. 1993).
To accomplish its role in DNA replication, the RF-C subunits must
interact with each other, with PCNA, and with DNA polymerase-6 and E.
To date, two interactions among the various RF-C subunits have been de-
scribed: The 40-kD subunit associates with the 37-kD subunit in vitro
(Pan et al. 1993), and the yeast Rfc4p subunit (equivalent to 40-kD mam-
malian subunit) binds to the Rfc3p subunit (the homolog of the mam-
malian 36.5-kD subunit) during coexpression in E. coli. Interaction of
RF-C with PCNA is accomplished by direct binding of the latter to the
40-kD subunit (Pan et al. 1993). Genetic evidence in yeasts suggests that
the Rfclp subunit (homologous to the mammalian 140-kD subunit) inter-
acts with PCNA (Howell et al. 1994). There is also indirect evidence
supporting interactions between RF-C and DNA polymerase-6 and E. For
example, the isolated 37-kD subunit stimulates DNA polymerase+ ac-
tivity in vitro under high-salt conditions, and the 40-kD subunit inhibits
DNA polymerase-6 activity (Pan et al. 1993). The molecular cloning of
complete cDNAs encoding all the mammalian subunits, coupled with
more complete analyses of their interaction with each other, with PCNA,
and with the various subunits of DNA polymerase-6 and E, should lend
insight into the mechanism of action of this very important multisubunit
enzyme.
unit of unknown function (for review, see Linn 1991). Unlike DNA poly-
merase-a:primase, DNA polymerase-6 has 3 ' 4 I proofreading exo-
nuclease activity. The gene encoding the catalytic subunits of S.
cerevisiae and Schizosaccharomyces pombe DNA polymerase-6 has
been cloned and shown to be required for viability (for review, see
Campbell 1993; see also Wang, this volume). Human cDNAs encoding
the catalytic subunit also have been isolated (Yang et al. 1992).
DNA polymerase-6 binds poorly to DNA, and hence PCNA is re-
quired to mediate its interaction with the DNA template. Deletion of the
first 220 amino acids of the DNA polymerase-6 catalytic subunit from
yeast renders it insensitive to PCNA stimulation, suggesting that this
region is required for association with PCNA (Brown and Campbell
1993). Zhang et al. (1995) have provided direct evidence for an associa-
tion between human DNA polymerase-6 and PCNA that is mediated by
the amino-terminal 182 amino acids of the 125-kD DNA polymerase
catalytic subunit.
The potential that DNA polymerase-e might be required for SV40 and
PyV DNA replication has begun to receive increased attention for a num-
ber of reasons. First, the gene encoding the catalytic subunit of DNA
polymerase-e is essential, and its protein product is required for
chromosomal DNA replication in yeast (Morrison et al. 1990). Second,
in vivo (Nethanel et al. 1988) and in vitro in unfractionated mammalian
cell lysates with SV40 DNA as template, DNA polymerase-a:primase
synthesizes DNA fragments less than 40 nucleotides in length, which is
considerably shorter than Okazaki fragments (-200 nucleotides), imply-
ing that another DNA polymerase synthesizes these fragments (Bullock
et al. 1991). These findings suggest that DNA polymerase-e may be re-
quired for lagging-strand replication.
DNA polymerase-e was first isolated from budding yeast (known as
DNA polymerase 11) and later from mammalian sources. Highly purified
mammalian DNA polymerase-e is composed of two subunits of
molecular mass 215 and 55 kD, whereas the budding yeast enzyme com-
prises subunits of 255, 80, 34, 30, and 29 kD (for review, see Linn 1991;
Hiibscher and Spadari 1994). The mammalian enzyme may possess addi-
tional subunits that dissociate from the core DNA polymerase during
purification. cDNAs encoding the large catalytic subunit of human DNA
polymerase-e have been cloned (Kesti et al. 1993), as have the genes en-
coding the 255-, SO-, 34-, and 30-kD subunits of the S. cerevisiae en-
zyme (for review, see Campbell 1993). The largest subunit possesses
DNA polymerase and 3 I -5 I exonuclease activity; the function of the
smaller subunits is not known. Given the conservation of subunit com-
SV40 and Polyomavirus DNA Replication 659
Topoisomerases I and II
DNA topoisomerases are enzymes that alter the topology of DNA (for
review, see Watt and Hickson 1994; see Hangaard Andersen et al., this
volume). They have been classified as types I and I1 according to their
catalytic mechanism of action. Type I topoisomerases are nicking-
closing enzymes that transiently cleave one strand of the DNA duplex,
allowing the other strand to go through the nick before ligating the
nicked strand. These enzymes do not require ATP for their action, but
their activity is stimulated by Mg++ ions in vitro. Type I1 topoisomerases
also transiently cleave DNA, but they make double-stranded breaks so
that a double-stranded segment of DNA can go through the break before
it is sealed. Type I1 enzymes require ATP for their activity. During SV40
DNA replication, topoisomerase I or I1 is required to unwind the positive
supercoils that accumulate ahead of the replication forks (Yang et al.
1987), whereas topoisomerase I1 is needed for the decatenation (segrega-
tion) of the progeny DNA molecules after replication is completed (Yang
et al. 1987; Ishimi et al. 1991).
Type I topoisomerases are single-chain enzymes that function as
monomers. S. cerevisiae possesses two genes that encode type I topo-
isomerase; human cells have only a single gene. Neither of the yeast
genes is essential for viability. The human enzyme has a measured
660 J.A. Hassell and B.T. Brinton
fork. These authors provide evidence that DNA polymerase-b is the prin-
cipal replicative enzyme required for both leading- and lagging-strand
synthesis in vitro, and suggest that this DNA polymerase dimerizes at
replication forks. However appealing this hypothesis may be, there is
currently no evidence to support the occurrence of DNA polymerase-6
homodimers in solution. However, the possibility remains that a
heretofore undiscovered cellular protein mediates dimerization of the
DNA polymerases at the replication fork. It will be interesting to learn
whether any of these or some other protein mediates dimerization of the
DNA polymerases at replication forks.
Termination of SV40 and PyV DNA replication occurs on average
180° from the ori when the two replication forks converge; the small
genomes of these viruses do not contain replication termination signals
(for review, see DePamphilis and Bradley 1986; see Bastia and Mohanty,
this volume). When replication is about 90% completed, fork movement
is impeded and replication intermediates accumulate, indicating that the
completion of replication and the segregation of progeny molecules com-
prise a slow step in the DNA replication cycle. If DNA replication and
unwinding mediated by topoisomerase I1 continue concurrently, then the
progeny molecules separate, yielding double-stranded circular DNAs
with a small gap of about 50 nucleotides. However, should DNA replica-
tion be completed without concomitant unwinding, then catenated dimers
result whose resolution also requires topoisomerase 11. Examination of
the structure of SV40 and PyV replication intermediates suggests that
both pathways can be used to complete replication of these viral
genomes.
FUTURE PROSPECTS
The last decade has witnessed a dramatic increase in our understanding
of SV40 and PyV DNA replication, which can be attributed primarily to
the development of a cell-free system for the replication of these viral
genomes. Biochemical fractionation and complementation assays have
led to the isolation and characterization of many cellular replication
proteins from several mammalian sources, and to the molecular cloning
of cDNAs and genes that encode these proteins from diverse organisms.
Genetic studies in yeasts have validated the role of these proteins in cel-
lular DNA replication. This progress notwithstanding, much remains to
be learned. For example, we are only now beginning to appreciate the
link between cell-cycle control and the regulation of viral DNA replica-
tion. Both large T antigen and several of the cellular replication proteins
SV40 and Polyomavirus DNA Replication 667
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