From the Department of Biochemistry and Cancer Center, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
Many recent reviews of eukaryotic DNA
replication have emphasized our current understanding of either the
initiation and regulation of eukaryotic nuclear DNA replication (1, 2),
the DNA polymerases and other proteins involved (3-11), or the
entire range of knowledge of the replication process (12-17). Here, we
will focus on recent findings concerning specific enzymatic reactions
necessary for the growth of the eukaryotic replication fork.
Synthesis of the Leading Strand The separation of parental DNA strands determines the direction of
movement of the replication fork. Because of the antiparallel structure
of DNA, one new DNA strand is synthesized continuously in the direction
of fork movement and is designated the leading strand. The other, or
lagging strand, grows in the direction away from fork movement. On this
strand, short discontinuous segments of DNA, called Okazaki fragments,
are synthesized from RNA primers. During replication, the RNA primers
are removed, and each fragment is joined to complete lagging strand
synthesis.
Knowledge of the reactions of the leading strand is derived largely
from studies in vitro of the replication of simian virus 40 (SV40)1 (1, 5, 12), a circular dsDNA virus
with a single origin of replication. The viral protein large T antigen
binds the origin and utilizes a 3 Recent structural analysis of DNA pol III holoenzyme from the
homologous Escherichia coli system indicates that the
sliding clamp ( Synthesis of the Lagging Strand Priming by DNA pol
Numerous RNA primers must be removed. The action of two nucleases is
sufficient to perform this task (33). RNase H1 endonucleolytically cleaves the initiator RNA one nucleotide upstream of the RNA-DNA junction. The initiator RNA is removed intact leaving a single ribonucleotide on the downstream DNA portion of the Okazaki fragment. RNase H1 cleavage leaves the junctional ribonucleotide irrespective of
the length of the initiator RNA. The position of cleavage is not
affected by either the RNA or DNA sequence around the cleavage site. In
fact, cleavage remains specific even if mismatched nucleotides are
present at positions at and near the RNA-DNA
junction.3 This indicates that RNase H1
recognizes the junction and not the intermediate structure between the
A-form helix of the RNA-DNA duplex and the B-form helix of the DNA
duplex. The structure of these regions is likely distorted in the
mismatched substrates.
The remaining ribonucleotide is removed (30, 33) by FEN1/RTH1
(30, 34-37), which contains both an exonucleolytic and endonucleolytic
capability (38, 39). As an exonuclease, it is specific for DNA or
5 Alternative Pathways for Okazaki Fragment Processing Genetic analyses in yeast suggest that there are alternative means
of initiator RNA removal. Null mutants of the primary RNase H in yeast
are not significantly defective in DNA
replication.4 One possibility is that the
yeast RNase H is not equivalent to mammalian RNase H1. Alternatively,
there is an efficient second pathway for RNA removal that does not
require RNase H. Null mutants of FEN1/RTH1 in yeast are
temperature-sensitive for growth and have a hyper-recombination
phenotype (42). This is indicative of long lived regions of ssDNA in
the chromosome and is symptomatic of a defect in Okazaki fragment
processing. This result suggests that a backup pathway which still
involves FEN1/RTH1 could compensate for defective or absent RNase H1.
In the absence of FEN1/RTH1, both pathways would fail, reverting to a
third alternative pathway inefficient enough to produce the
temperature-sensitive phenotype. Recent discovery of at least one
additional gene in yeast that encodes a nuclease with a similar
structure to FEN1/RTH1 (43, 44) suggests the possibility of a third
pathway for RNA primer removal.
Possible Role of the Endonuclease Activity of the FEN1/RTH1
Nuclease in Initiator RNA Removal The mammalian FEN1/RTH1 class nucleases also contain a unique,
structure-specific endonucleolytic activity (38, 39). A favored
substrate consists of two primers on a template having the annealed
portions directly adjacent to each other. However, the 5 Endonucleolytic cleavage by FEN1/RTH1 occurs by a unique mechanism. The
nuclease slides over the 5 Calf FEN1/RTH1 readily recognizes the triphosphorylated 5
The structure of the mammalian 5 Both exo- and endonucleolytic mechanisms of initiator RNA processing
allow for the removal of some of the DNA downstream of the initiator
RNA. This could occur through a nick translation process involving the
exonucleolytic action of FEN1/RTH1. Alternatively, a tail longer than
the initiator RNA would be displaced and then cleaved
endonucleolytically by FEN1/RTH1 near the point of annealing. In either
case it is possible that all of the ribo- and deoxyribonucleotides added by DNA pol How Can the Unannealed Tail Substrate Be Generated? Separation of DNA strands is usually performed by a DNA helicase
and a ssDNA-binding protein (RPA) to stabilize the ssDNA. Human RPA is
a three-subunit protein necessary for SV40 DNA replication (19, 21,
54). Studies indicate that RPA is able to unwind both short oligomers
and long double-stranded regions in the absence of magnesium and ATP
(55, 56). However, the rate of unwinding by RPA is significantly slower
than the unwinding rate of DNA helicases (56). Therefore, it seems
unlikely that RPA alone is responsible for efficient strand separation
in vivo. However, RPA does stimulate several DNA helicases,
suggesting that it aids in the formation of 5 The first is the DNA2 helicase from Saccharomyces cerevisiae
(57). This helicase is a 172-kDa, single subunit protein that copurifies with the yeast FEN1/RTH1. The DNA2 gene is
essential for viability and complements a temperature-sensitive yeast
strain defective in the elongation stage of DNA replication. When an invariant lysine in the nucleotide binding sequence of the helicase domain was altered to glutamic acid, complementation no longer occurred. These results suggest that the helicase activity is at least
part of what makes this protein essential. Its specific substrate is a
primer with an unannealed 5 The second is helicase E from calf thymus (59-61). This helicase also
copurifies with FEN1/RTH1, as well as with DNA pol Stimulation of FEN1/RTH1 Nuclease Action by PCNA Recently Burgers, Lieber, and colleagues (65) demonstrated that
the catalytic action of FEN1/RTH1 is stimulated approximately 10-fold
by PCNA. This was shown on substrates having a primer with a unannealed
5 Why Does FEN1/RTH1 Nuclease Have Such a Unique Sliding
Mechanism? The ssDNA sliding mechanism exhibited by the FEN1/RTH1 nuclease
may have evolved because the nuclease has a dual function in DNA
replication and repair. The null mutant of FEN1/RTH1 in S. cerevisiae has an increased sensitivity to methyl
methanesulfonate, an alkylating agent, implicating FEN1/RTH1 in removal
of adduct-damaged nucleotides (42).
The sliding mechanism of FEN1/RTH1 suggests a means by which it could
participate in repair of adduct-damaged DNA. It could slide past damage
on an unannealed 5
As discussed earlier, biotin adducts can be traversed but not if they
are conjugated with streptavidin (46). This suggests an ultimate limit
on adduct size, possibly imposed by the size and flexibility of the
anticipated arch structure in the nuclease. It appears that the
nuclease is designed to tolerate a variety of adduct structures.
However, damage could inhibit catalysis if it is too close to the point
of cleavage (66). This problem could be remedied in vivo by
further strand displacement.
DNA having abasic lesions is cleaved in a similar fashion. Abasic sites
on chromosomal DNA are sensitive to type II abasic endonucleases. These
cleave upstream of the damage to create a strand with a phosphorylated
abasic sugar at its 5 Why Are There Three Nuclear DNA Polymerases? Genetic studies in yeast demonstrate that DNA polymerases
Recent years have seen a major expansion of our knowledge of the
eukaryotic replication fork. As summarized here, nearly all of the
necessary reactions and enzymes involved have been identified, characterized genetically, and reconstituted in vitro.
Nevertheless, the exact complement of components, their contacts and
interactions, and structure of the complex remain to be determined.
-5
-helicase activity to separate
the strands creating two replication forks. Unwinding of the origin by
large T antigen is stimulated by the ssDNA-binding protein replication protein A (RPA) (18-21). After unwinding, each leading strand is initiated by an RNA primer generated by the primase subunits of DNA pol
(22). The polymerase subunit of DNA pol
then adds a stretch of
deoxyribonucleotides to the RNA primer. Next, replication factor C
(RFC) initiates a reaction called polymerase switching (23, 24). In an
ATP-dependent process, RFC dissociates DNA pol
and
assembles proliferating cell nuclear antigen (PCNA) in the region of
the primer terminus. PCNA is a homotrimer of 36-kDa subunits that form
a toroid structure. The current model suggests that RFC transiently
opens the toroid of PCNA and then allows PCNA to reclose, encircling
the double helix adjacent to the primer terminus (25, 26). Structural
analysis indicates that the central opening of PCNA contains sufficient
clearance that the toroid can slide freely (27). Then, DNA pol
interacts with PCNA, which functions as a sliding clamp holding the
polymerase on the primer terminus. The clamped DNA polymerase is highly
processive, adding thousands of nucleotides without dissociating.
), the clamp-loading subunit (
), and the
polymerizing subunit (
) form a complex at the primer terminus.
is at the 3
-end and immediately behind are the other subunits
contacting the double-stranded region of the primer-template (28). By
both analogy and preliminary experimental evidence, RFC is retained in
the complex of PCNA and DNA pol
.2
and switching to DNA pol
occur in
essentially the same way on both the lagging and leading strand of SV40
(29, 30) as illustrated in Fig. 1A. In fact,
polymerase switching during synthesis of Okazaki fragments was found to
be a necessary prerequisite for complete gap filling (30). Analysis of
SV40 replication intermediates indicates that priming on the lagging
strand is very frequent, with initial placement of primers ~ 50 nucleotides apart (31). Okazaki fragment intermediates consist of
initiator RNAs averaging about 10 nucleotides in length, extended with
10-20 additional deoxyribonucleotides (31). Because Okazaki fragment
intermediates are made in the absence of ATP, and RFC requires ATP for
polymerase switching, the deoxyribonucleotides of the intermediates
appear to be added by DNA pol
prior to the switch. After loading of
PCNA and DNA pol
, an additional 10-20 nucleotides are added prior
to the position of the next downstream initiator RNA primer. There must
be further extension of the upstream primer during or after removal of
the initiator RNA and possibly nick translation synthesis through the
first deoxyribonucleotides of the downstream primer. DNA pol
should then dissociate. Results from the homologous E. coli system
for DNA pol III (32) indicate that encountering a downstream primer induces the dissociation of the processive complex. If eukaryotes operate similarly, the necessary frequent dissociations of DNA pol
would be promoted by contact with the downstream primer.
Fig. 1.
DNA polymerase switching and processing of an
Okazaki fragment on the lagging strand. A, as the DNA
helicase promotes unwinding at the replication fork, DNA pol with
RFC and PCNA synthesizes DNA on the leading strand. DNA pol
initiates synthesis on the lagging strand by generating an RNA primer
(red segment) followed by a short segment of DNA. Then, RFC
and PCNA load a second DNA polymerase (
or
) to continue
synthesis of the Okazaki fragment. B, as DNA pol
approaches the downstream Okazaki fragment, cleavage by RNase H1
removes the initiator RNA primer leaving a single 5
-ribonucleotide.
Then, FEN1/RTH1 removes the 5
-ribonucleotide. The resulting nick is
sealed by DNA ligase.
[View Larger Version of this Image (28K GIF file)]
-RNA terminated DNA primers annealed to templates (40). Depending on
the sequence of the region, it may be stimulated, unaffected, or
inhibited by the presence of a primer bound immediately upstream of the
cleavage site (40). Removal of the junctional ribonucleotide is
generally inhibited by an upstream primer. This suggests that the
initiator RNA removed by RNase H1 dissociates before FEN1/RTH1-directed
cleavage of the junctional ribonucleotide. During synthesis, the
extension of the upstream primer by polymerization could stimulate nick translation. When a sequence is encountered such that the upstream primer is inhibitory, nick translation would stop, allowing ligation of
the Okazaki fragments. We found that the simultaneous action of a DNA
polymerase, RNase H1, FEN1/RTH1, and DNA ligase I results in correct
Okazaki fragment processing in vitro (41) as depicted in
Fig. 1B.
-end of the
downstream primer forms an unannealed 5
-tail. Then, FEN1/RTH1 could
cleave the tail endonucleolytically in the DNA region downstream of the
initiator RNA. After the gap between the remaining DNA portions of the
two primers is filled, ligation would complete Okazaki fragment
processing.
-end of the unannealed tail and traverses
the entire length of the tail before arriving at the point of cleavage
near the annealing point of the tail (39, 45). One of the most
definitive experiments demonstrating an obligatory sliding mechanism
involved use of a tail 73 nucleotides long. Annealing of a
20-nucleotide-long primer anywhere on the tail inhibited cleavage (46).
Additionally, modification of the most 5
-nucleotide with a
biotin-containing side chain, followed by binding of streptavidin,
prevented entry of the nuclease and cleavage (46).
-end region
of a displaced initiator RNA and cleaves in the downstream DNA (47).
Surprisingly, several of the substrates used in this study were cleaved
in the absence of a nick-like structure. Influences of nucleotide
sequence on nick-dependent stimulation and specificity of
cleavage were examined; however, no specific criteria could be
discerned (47). Overall, results demonstrate that the endonucleolytic activity of the nuclease is capable of bypassing the need for RNase H1
in Okazaki fragment processing. The proposed cleavage mechanism of a
displaced Okazaki fragment by FEN1/RTH1 nuclease is shown in Fig.
2.
Fig. 2.
Removal of a displaced Okazaki initiator RNA
by FEN1/RTH1 nuclease. As synthesis of the upstream Okazaki
fragment is completed, DNA polymerase with a DNA helicase displaces
both RNA and DNA of the downstream fragment, generating an unannealed
5-tail. FEN1/RTH1 binds and tracks over the 5
-tail. Then, FEN1/RTH1
endonucleolytically cleaves the 5
-tail removing the entire RNA
primer.
[View Larger Version of this Image (46K GIF file)]
-3
-exo/endonuclease has not yet been
determined. However, eukaryotic and prokaryotic nucleases of this type
have homologous sequences (48). Recently, the x-ray crystal structures
of three of the prokaryotic nucleases, Taq 5
-exonuclease
(49), the bacteriophage T5 5
-3
-exo/endonuclease (50), and the
bacteriophage T4 5
-nuclease (also called RNase H) (51), were
determined. While the overall structures are similar, Ceska et
al. (50) specifically pointed out an arch shape in the T5
nuclease, rising above the globular main section of the protein. Two
-helical structures make up the supports of the arch, and the
keystone region is an area of random coil. Working from known
mechanistic information about this class of nucleases, Ceska et
al. (50) proposed a way for the nuclease to interact with its
substrate. Their model shows how the double-stranded region of a
5
-tailed primer could bind a wide cleft in the globular portion of the
protein and the unannealed tail could fit through the arch.
Significantly, the opening in the arch is sufficiently wide to allow
passage of single but not dsDNA. The single strand then threads through
the archway until the region around the annealing point of the tail can
bind the cleft for catalysis. The free unannealed strand can then drift
through the archway, and the rest of the substrate would dissociate
from the cleft.
would be removed. This would explain why the high
fidelity of chromosomal DNA replication could be maintained, even
though the fidelity of synthesis of both RNA and DNA by DNA pol
is
relatively low (52). It also could explain why DNA pol
(53) had no
need for 3
-5
-exonuclease activity.
-tails on Okazaki
fragments. There are at least three current candidates for a helicase
that would create the 5
-tail. All track 3
to 5
on the template
strand.
-tail. This originally suggested a role in
separating the replication fork. It would appear that DNA2 helicase
could propagate, but not initiate, formation of a 5
-tail of an Okazaki
fragment. However, such initiation could be performed by polymerases,
RPA, another helicase, or other replication proteins. High expression
of FEN1/RTH1 complements a temperature-sensitive dna2
mutation (58) supporting the likelihood of a functional interaction
in vivo. Together these results argue that the DNA2 helicase
is responsible for the creation of unannealed tails on Okazaki
fragments.
. It is
apparently a monomer of approximately 100 kDa. It can act on a fully
annealed primer and is capable of displacing DNA up to several hundred
nucleotides in length (59-61). The third is the Ku helicase (62). This
enzyme associates with human DNA pol
(63) and has two subunits of
72 and 80 kDa (62). It can also displace fully annealed primers. Ku has
recently been identified as part of the complex of proteins that binds
the yeast origin of DNA replication ARS121 (64). It is the protein
previously designated OBF2. This observation supports a significant
role for the Ku protein in DNA replication and possibly the separation of the replication fork. Either of these helicases could have a role in
the creation of an unannealed 5
-tail on Okazaki fragments.
-tail. PCNA and FEN1/RTH1 were the only two proteins in the reaction,
proving that their interaction created the stimulation. These two
proteins can interact in the absence of substrate, as indicated by the
binding of FEN1/RTH1 to PCNA-containing affinity resin. Stimulation
requires a large excess of PCNA over substrate molecules, presumably
because PCNA freely slides on and off of the linear template. When PCNA
was loaded using RFC and ATP onto M13 DNA having two adjacent primers,
PCNA stimulated FEN1/RTH1 nuclease-directed cleavage of the terminal
5
-nucleotide of the downstream primer (65). A PCNA mutant protein
(pcna-52), incapable of trimerization, was inactive (65). The results
show that PCNA only stimulates FEN1/RTH1 after encircling the
substrate. Blocking the unannealed strand with a primer prevents
FEN1/RTH1 nuclease-directed cleavage even in the presence of PCNA (45). This shows that PCNA does not forgive FEN1/RTH1 of its obligation to
slide down the unannealed tail. Overall these results suggest that PCNA
acts downstream of the displaced tail and possibly stabilizes FEN1/RTH1
at the position of cleavage. If PCNA is always loaded directionally by
RFC, one face of PCNA may be designed to interact with polymerases
while the other binds FEN1/RTH1. This implies that there are two PCNAs
at the replication fork, one serving as the sliding clamp for DNA pol
and the other interacting with FEN1/RTH1. These interactions are
part of the model of the proposed pathways for Okazaki fragment
processing as shown in Figs. 1 and 2.
-tail and then cleave the tail removing the damaged
nucleotides (Fig. 3). This approach allows cleavage of a
variety of types of damage, without needing to specifically recognize
the structure of the damaged nucleotide. To assess the mechanism of
nuclease tracking, and its ability to cleave modified DNA, adducts were
placed at various locations on the tails of substrates (66). Footprint
analysis using micrococcal nuclease indicates that after tracking, but
before cleavage, FEN1/RTH1 protects a region of the tail 25 nucleotides
long, adjacent to the cleavage site. When
cis-diamminedichloroplatinum(II) (CDDP) adducts were placed
within or beyond the region protected by FEN1/RTH1, the 5
-tail was
cleaved. A CDDP adduct bound to the last two nucleotides at the very
5
-end of an eight-nucleotide-long tail was also cleaved by FEN1/RTH1.
The nuclease also removes tails containing adducts on the 2
-position
of the ribose. In contrast, a CDDP adduct just adjacent to the expected
cleavage point was inhibitory to the nuclease.
Fig. 3.
Repair of adduct-damaged DNA by the FEN1/RTH1
nuclease. FEN1/RTH1 could participate in the repair of damaged DNA
by the mechanism shown here. A DNA helicase displaces the damaged strand. If the damaged or adducted nucleotide can pass through the
archway, FEN1/RTH1 tracks over the damage and cleaves an oligomer containing the damaged site.
[View Larger Version of this Image (50K GIF file)]
-end, annealed to a template just downstream of a
second primer. FEN1/RTH1 cannot remove the abasic sugar
exonucleolytically. However, strand displacement synthesis from the
upstream primer can create a tail, over which the nuclease can slide,
and then remove the abasic deoxyribose as part of an
endonucleolytically cleaved oligomer (67).
,
, and
are all essential (68-70). These determinations
prompted suggestions that all three performed specific functions at the replication fork (4, 71, 72). DNA pol
, although capable of highly
processive DNA synthesis, was stimulated at high salt concentrations by
interaction with PCNA (72). This suggests that it also participates in
polymerase switching, having a similar role to that of DNA pol
.
Consistent with these observations, DNA pol
was proposed to perform
the majority of elongation of either the leading or lagging strand.
However, reconstitution of SV40 DNA replication with purified proteins
showed that both leading and lagging strand DNA replication is
performed efficiently with only DNA pol
and
. Recent
cross-linking experiments were done to determine the DNA polymerases
bound to nascent DNA during replication in cell extracts (73). For SV40
replication, DNA pol
but not
was found to cross-link. For
cellular DNA, both were found to cross-link, but mitogenic stimulation
induced pol
to a considerably lesser extent than DNA polymerases
and
. The authors concluded that only DNA pol
and
participate in SV40 DNA replication. Measurements with chromosomal DNA
suggest that DNA pol
is not participating as a sole elongating
enzyme for either the leading or lagging strand. Instead it is likely to be performing an essential process that occurs during DNA
replication.