From the Department of Biochemistry and Biophysics
and ¶ Cancer Center, University of Rochester School of Medicine
and Dentistry, Rochester, New York 14642
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ABSTRACT |
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Flap endonuclease-1 (FEN1) is proposed to
participate in removal of the initiator RNA of mammalian Okazaki
fragments by two pathways. In one pathway, RNase HI removes most of the
RNA, leaving a single ribonucleotide adjacent to the DNA. FEN1 removes
this ribonucleotide exonucleolytically. In the other pathway, FEN1 removes the entire primer endonucleolytically after displacement of the
5'-end region of the Okazaki fragment. Cleavage would occur beyond the
RNA, a short distance into the DNA. The initiator RNA and an adjacent
short region of DNA are synthesized by DNA polymerase Mammalian DNA replication proceeds by two distinct processes
occurring at replication forks (1). The leading strand is made as a
large continuous segment, whereas the lagging strand is synthesized as
a series of discontinuous segments called Okazaki fragments. Each
segment is individually initiated by a RNA primer. Later, these primers
are removed and replaced with DNA, and the fragments are joined into
one continuous strand. DNA polymerase Initiator RNA removal is accomplished by a eukaryotic 5' to 3'
exonuclease/endonuclease before Okazaki fragment joining (9). Most
frequently called flap endonuclease-1
(FEN1),1 this enzyme is also
known as RAD2 homologue nuclease (10-15). In one proposed pathway,
RNase HI-directed cleavage removes almost the entire RNA primer but
leaves a single RNA residue. FEN1 exonucleolytically removes this last
ribonucleotide (16). However, genetic evidence suggests that
alternative pathways exist. Yeast cells are still viable after deletion
of the gene that accounts for 75% of RNase H activity (17). In a
second proposed pathway, FEN1 alone removes the initiator RNA (18) by
its favored endonucleolytic activity (10, 19). FEN1 endonucleolytic
cleavage requires that the substrate has a downstream primer with an
unannealed 5'-tail, or flap. FEN1 appears to recognize the 5'-end,
slide over the entire length of the tail, and cleave near the junction
of the tail with the template. This releases the tail as an intact
segment (10, 19). Sometimes, the presence of an upstream primer is also
required for FEN1 activity. In fact, for both exonucleolytic and
endonucleolytic substrates, the upstream primer is often stimulatory (10, 20, 21) but can sometimes be inhibitory (18, 22). For
endonucleolytic removal, the RNA must be within a displaced tail. An
upstream primer may or may not be present, as appropriate for the
particular cleavage site. Displacement of the RNA may be accomplished
by a DNA helicase with or without DNA polymerase-directed displacement
synthesis from an upstream fragment (18, 23). Depending on how far the
tail is displaced, FEN1 can cleave within the RNA, at the RNA-DNA
junction, or within the DNA beyond the RNA (18).
FEN1 may also participate in DNA repair. It is a member of the RAD2
family of repair nucleases (24). This family includes XPG in mammals,
RAD2 in Saccharomyces cerevisiae, and RAD13 in Schizosaccharomyces pombe. These homologous nucleases are
important components of the nucleotide excision repair pathway and are
responsible for the incision 3' to the damage site (24). FEN1 is
homologous to the family members called RAD27 or RAD2 homologue in
S. cerevisiae and RAD2 in S. pombe. These family
members are smaller than the XPG nucleases and are believed to function
in other repair processes (24). A third class of members includes
exonuclease I of S. pombe, for which genetic studies suggest
a role in mismatch repair (24). The FEN1 class enzymes are believed to
participate in base excision repair because the null mutant in yeast is
sensitive to methylmethane sulfonate (14, 15). In vitro,
FEN1 can remove abasic sites (25) and can function in reconstitutions
of base excision repair using purified enzymes (26-28). FEN1 can also
remove a more diverse group of flap adducts such as cisplatin
derivatives (29). Additionally, it may also participate in the removal
of monoribonucleotides embedded in chromosomal DNA (30). Finally, FEN1
has been implicated as a part of the mismatch repair pathway (31).
In the current report, we provide evidence that FEN1 participates in
the repair of mismatches produced during the priming and synthesis of
Okazaki fragments. We show that a single mismatch in an otherwise fully
annealed DNA primer changes the pattern of FEN1 cleavage. Typically, on
a fully annealed substrate, FEN1 will exonucleolytically cleave the 5'
most nucleotide. We find that a single nucleotide mismatch up to 15 nucleotides in from the 5'-end promotes endonucleolytic cleavages. This
constitutes a 5' proofreading process in which the mismatch promotes
the nuclease action that leads to its removal.
Materials--
Unlabeled nucleotides were purchased from
Amersham Pharmacia Biotech, and [ Enzyme Purification--
Recombinant human FEN1 was purified as
described previously (18, 29). The final preparation was >95% pure as
determined by silver-stained SDS-polyacrylamide gel electrophoresis.
The final specific activity was 65,000 units/mg, with 1 unit defined as
the amount of nuclease required to exonucleolytically cleave 4,000 fmol
in 30 min at 37 °C of a standard exonucleolytic test substrate consisting of a downstream primer
(5'-TTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTA-3') annealed to a template
(3'-GAGTGATTTCCCTTGTTTTCGAACGTACGGACGTCCAGCTGAGATCTCCTAGGGGCCCATT-5').
Oligonucleotide Substrates--
The sequences of the primers and
the structures of the substrates used in this study are described in
Table I and in the figure legends. Table
I depicts template T1 and the downstream primers used in this study.
The primers are divided into four groups, depending on the location of
the 5' most nucleotide; the 5'-end of all primers in a given group is
located at the same place on the template. The first digit of the
primer name indicates the group: 1, 2, 3, or 4. To generate a mismatch,
each primer has a single base change that forms a single mismatch with
the template, T1. The remaining digits in the name indicate the
distance of this mismatch in nucleotides from the 5'-end of the primer. For all experiments, the substrate name reflects the use of a specific
downstream primer. For example, substrate 2.11 indicates that a primer
from group 2 was annealed to the template to create a mismatch at
position 11. Control substrates 1.0, 2.0, and 4.0 have no mismatch.
Mismatches were made so that nucleotides were paired with themselves
(e.g. G was paired with G). The 5'-end of the template
extends beyond the downstream primer to permit 3'-end labeling. In
substrates 1.T and 1.A, the mismatch is not internal but is, in fact,
the 5' most nucleotide, which is mispaired as T-T or A-A, respectively.
Substrate 3.6 uses a primer that lacks the three 5' most nucleotides of
the primer in substrate 2.9. Thus, both the location of the 5'-end and
the position of mismatch relative to the 5'-end have changed.
Substrates made from primers in group 4 are similar to those made from
primers in group 2, except that 25 nucleotides were added to the 5'-end to form an unannealed 5'-tail, the sequence of which is shown in Table
I.
For 5'-end labeling, downstream primers were incubated with T4
polynucleotide kinase and [ Enzyme Assays--
Assays to monitor cleavage by FEN1 were
performed in FEN1 buffer containing 60 mM BisTris (pH 7.0),
5% glycerol, 0.1 mg/ml bovine serum albumin, 5 mM
A Single Mismatch Alters FEN1 Cleavage Specificity--
We
hypothesized that FEN1 participates in a 5' proofreading reaction
linked to DNA replication. In this reaction, FEN1 would remove
mismatches generated by DNA polymerase
We first compared the cleavage of a fully annealed control substrate
(substrate 1.0) to that of substrates containing a single mismatch
either two or three nucleotides from the 5'-end (substrates 1.2 and
1.3, respectively) (Fig. 1A).
For this and all other experiments in this study, control substrates
were identical to the mismatch substrates, except that they were
without a mismatch. As expected, on these 5'-end-labeled substrates,
FEN1 cleavage in the absence of mismatch resulted in the release of a
single nucleotide (Fig. 1A, lane 2). Cleavage of
fully annealed substrates most often results in exonucleolytic cleavage
(10, 11, 21, 22, 30, 33), although with some sequences, additional
products from cleavage at internal sites are observed. Apparently, FEN1
has the capacity to invade a small proportion of fully annealed
substrates to perform endonucleolytic cleavage. Such minor internal
cleavage products were seen with substrate 1.0. When a mismatch was
present at the second or third nucleotide, the proportion of products resulting from endonucleolytic cleavage (trimers and pentamers) was
greatly enhanced (Fig. 1 A, lanes 3 and
4). The mechanism of FEN1 was shifted from predominantly
exonucleolytic cleavage to predominantly endonucleolytic cleavage
simply by the presence of a single mismatched base pair.
For consistency, mismatched substrates were produced by pairing
nucleotides with themselves (i.e. G with G). However, we
have noticed that the sequence of the mismatch affects the distribution of internal cleavages, as shown in Fig. 1B. Lane
1 shows the cleavage of control substrate 1.0, as in Fig.
1A. For lane 2, the sequence of the primer was
modified to generate a T-T mispair with the template at the 5'-end of
the primer (substrate 1.T). For lane 3, the template
sequence was modified so that the 5'-end nucleotide of the original
primer formed an A-A mismatch with the new template (substrate 1.A). In
comparing lanes 2 and 3, the identity of the mismatch clearly alters the ratio of the three major products. There is
a higher percentage of pentamer released with the A-A mismatch.
These results suggest that the specific nucleotides of the mismatch
affect the internal cleavage capacity of FEN1, perhaps by altering the
exact amount of primer destabilization or the shape of helical
distortion. On the other hand, the pattern of products is similar to
those generated when the mismatch was at position 2 or 3 from the
5'-end (Fig. 1, compare A with B). This suggests
that the mismatch alters the structure of the substrate around it to
favor endonucleolytic cleavage but does not define the position of
cleavage. Instead, the nuclease appears to cleave at sites that it
favors, irrespective of the exact location of the mismatch.
Internal Cleavages Allow Efficient Endonucleolytic Mismatch
Removal--
As the mismatch was moved further into the strand, the
effect on the distribution of 5' products decreased. Mismatches at 7, 9, 11, 13, and 15 nucleotides from the 5'-end produced a product distribution that differed little from the distribution seen with no
mismatch (data not shown). A primer template in which the primer has an
unannealed 5'-tail is the preferred substrate of FEN1 (10, 18, 19). We
wanted to determine whether a mismatch in the annealed region
downstream of an unannealed 5'-tail would alter the pattern of
cleavage. We thought the mismatch might effectively lengthen the tail,
enabling FEN1 to cleave further internally than it otherwise would.
FEN1 can remove initiator RNA endonucleolytically when it has been
displaced into a tail (18). The helical disruption caused by a mismatch
might allow FEN1 to remove the initiator RNA and a mismatch
simultaneously, with an appropriately placed endonucleolytic cut. Fig.
2 clearly shows no difference in cleavage on endonucleolytic substrates with no mismatch (lane 1), a
mismatch seven nucleotides into the annealed region (lane
2), and a mismatch nine nucleotides into the annealed region
(lane 3). Cleavage of each substrate results in the
production of two major products of ~26 and 10 nucleotides. The
expected product is ~26 nucleotides, given that the unannealed
5'-tail is 25 nucleotides. The smaller product is the result of
cleavage within the tail. This might occur from either transient
annealing between the tail and the upstream portion of the template or
the secondary structure within the unannealed tail. The presence of the
tail might have facilitated the binding of FEN1 and movement of the
nuclease to the site of the mismatch to make an initial cleavage.
However, these data provide no evidence that the two features
collaborate to favor an altered position of cleavage. Presumably, after
the tail has been removed, the mismatch will promote further internal
cleavages as suggested by the results shown below.
To perform effective 5' proofreading, FEN1 must cleave beyond the
mismatch. It would appear that if the mismatch is sufficiently far from
the 5'-end, it cannot be removed by a single cut. However, because the
primers examined to this point had been 5'-end-labeled, only the first
cleavage event could be observed. A mismatch that is initially deep
within a primer becomes increasingly close to the 5'-end as FEN1
cleaves the substrate. As this occurs, the mismatch would be expected
to have an increasingly greater influence on the cleavage events.
Although the mismatch is not removed in a single cut, FEN1 may do so
through a series of cleavage events. In Fig.
3, substrate 2.9 has a mismatched base
pair at position 9. Lane 1 demonstrates that FEN1 can
release a monomer, trimer, or hexamer on this substrate. In each event,
the mismatch is not removed in a single cut. After FEN1 removes the
first three nucleotides, the mismatched base pair at position 9 is now
at position 6. We used a second substrate, 3.6, to model this
intermediate and examine FEN1 activity. Using substrate 3.6, we can
observe the results of subsequent cuts.
Cleavage of substrate 3.6 released monomers, dimers, and tetramers
(Fig. 3, lane 2). The mobilities of products in lanes
2 and 3 differ from those in lane 1 because
of the sequence difference between substrates 2.9 and 3.6. In
particular, released monomer dC migrates farther in lane 2 than does monomer dT in lane 1. To verify the exact lengths
of the products from both substrates, we made molecular weight ladders
of each primer by digestion with snake venom phosphodiesterase. Because
substrate 3.6 already has three nucleotides removed compared with
substrate 2.9, the release of a monomer, dimer, etc. in lane
2 is equivalent to cleavage beyond the fourth, fifth, etc.
nucleotide in lane 1. Removal of the original mismatch nine
nucleotides into the substrate requires the release of a hexamer in
lanes 2 and 3. The monomers, dimers, and
tetramers released in lane 2 correspond to a second cut 3' of the fourth, fifth, and seventh nucleotides of the original substrate
2.9. Even this second cut falls short of the goal of mismatch removal.
However, the process has now placed the mismatch as little as two
nucleotides from the 5'-end.
The fact that cleavage did not reach the mismatch was surprising. In
substrate 3.6, the mismatch is only six nucleotides from the 5'-end, a
distance reached on substrate 2.9 (lane 1). One reason we
failed to observe cleavage beyond the mismatch could be because of its
distance from the 5'-end. Alternatively, failure to cleave beyond the
mismatch may reflect the influence of specific sequences. In many
sequence contexts, FEN1 requires an upstream primer for stimulation of
cleavage (10, 20, 21). To allow for such stimulation, we added an
upstream primer (UP) to substrate 3.6. This upstream primer
produces a nick with the nucleotide of the downstream primer that is
located immediately 3' to the mismatch. The presence of this upstream
primer did indeed stimulate cleavage after the original ninth
nucleotide mismatch, releasing a hexamer from substrate 3.6 (lane
3). Results here show how a combination of several cuts and
synthesis from the upstream fragment could collaborate to achieve
removal of the mismatched nucleotide.
Internal Mismatches Relieve Internal Pause Sites--
We were
concerned that measuring subsequent cuts, as seen in Fig. 3, on a
substrate in which the first cut has been artificially simulated might
produce different results than when FEN1 must actually make the first
cut itself. By labeling at the 3'-end of the downstream primer (Fig.
4), we were able to examine all of the
products generated by FEN1 cleavage and verify the results of Fig. 3.
Fig. 4 shows a time course of cleavage by FEN1 of 3'-labeled substrates
containing no mismatch (substrate 2.0) or a single mismatch at position
9, 11, or 15 (substrate 2.9, 2.11, or 2.15). Progressive cleavage
events can be seen over time. Because a particular cleavage may be
upstream primer-independent, bands at some positions are more dense
than others. The densest bands represent pauses in the progression of
cleavage. On the control with no mismatch, multiple FEN1 cleavages over
time result in products up to 14 nucleotides into the downstream primer
(lanes 2-7). The presence of a mismatch has a number of
effects. In all cases, FEN1 is able to remove the mismatch. Pauses in
the region of the mismatch disappear. For example, on substrate 2.15, a
group of pause sites is mostly eliminated from nucleotides 12, 13, and
14 just before the mismatch at nucleotide 15 (lane 7 versus lane
28). Similar results are seen around mismatches at positions 9 and
11 for substrates 2.9 and 2.11, respectively (lane 7 versus lanes
14 and 21). The mismatches promote their own removal by
relieving the sequence constraints of the substrate. This increased
capacity for cleavage is also demonstrated by an increase in the amount
of cleavage beyond the mismatch. In fact, the mismatched substrates are
more extensively digested throughout the substrate, not just near the
mismatch. Presumably, a mismatch allows a short flap to be created.
Sites near the mismatch that might normally cause pausing could be
displaced into the flap. Efficient cleavage at the base of the flap
would bypass these pause sites, allowing the nuclease to progress more rapidly. The presence of a mismatch clearly alters sequence effects, permitting more efficient FEN1 action and promoting removal of the
mismatch itself.
The DNA polymerase The FEN1 nuclease has a unique substrate specificity for flap
structures. Although FEN1 cleaves exonucleolytically, it prefers a
substrate with an unannnealed 5'-tail and presumably removes a portion
of initiator RNA by an endonucleolytic mechanism (18). If cleavage
occurs within the DNA, either as a single cut or as a series of cuts,
mismatches incorporated in the DNA by DNA polymerase One advantage of this FEN1 reaction, which we term 5' proofreading, is
that it can be performed as part of the replication process. In this
way, it is similar to the 3' proofreading function associated with DNA
polymerases To test the hypothesis that FEN1 could perform 5' proofreading, we have
designed a series of substrates to model potential mismatch
intermediates during Okazaki fragment processing. A single mismatch was
used to represent errors made by DNA polymerase We initially hypothesized that mismatches near the 5'-end of a primer
would encourage endonucleolytic cleavage by FEN1. However, we were
surprised to find that the influence of the mismatch extended for 15 or
more residues. This is particularly significant considering that the
experimental conditions used here lack factors present in the cell that
would favor flap formation. As discussed above, the presence of a
growing upstream primer would encourage the displacement of the
mismatched region, promoting cleavage. Furthermore, FEN1 in S. cerevisiae has been found to associate with the Dna2 helicase,
which moves in the same direction on the template as synthesis (36).
This helicase is expected to promote dissociation of the 5'-end region
of the downstream primer. The action of a homologous mammalian helicase
and the presence of other helix-destabilizing proteins such as RPA (37,
38) should contribute to even more efficient internal cleavages.
Current technology does not allow us to exactly recreate the conditions
present within the cell where 5' proofreading normally occurs.
Nevertheless, our results emphasize that the intrinsic structural
features of a substrate with a mismatch are sufficient for FEN1 to
distinguish it from a fully base-paired substrate. Overall, these
considerations are consistent with the proposal that the presence of a
mismatch throughout the 10-20 nucleotides of DNA expected to be added
by DNA polymerase For FEN1 to be effective at 5' proofreading, it must not just respond
to the mismatched substrate. It must be able to cut beyond the mismatch
and remove it. FEN1 can remove distant mismatches by making several
cuts in a progression. Using 3'-end labeling, Fig. 4 demonstrates that
given enough time and utilizing multiple cleavages, FEN1 can easily cut
beyond a mismatch even 15 residues deep. These results present 5'
proofreading as a multistep process. As the mismatch is moved further
internally, an increasing number of cuts are required to reach the
mismatch, and the process becomes less and less efficient. Clearly, at
some point, if a mismatch were positioned far enough internally, it
would not be reached by FEN1 before the region was ligated into a
continuous strand of chromosomal DNA. In such a case, the mismatch
would be repaired as usual by post-replication repair processes.
Fig. 5 presents a model illustrating the
selective form of replication-coupled repair proposed to be mediated by
FEN1 5' proofreading. In the absence of a mismatch, FEN1 activity is
minimal after the removal of initiator RNA, whereas the presence of a
mismatch encourages the 5' proofreading mechanism. Because of the
results shown in Fig. 2, we depict removal of the initiator RNA as a
separate step from removal of the mismatch. The results suggest that
the creation of a flap does not particularly activate nearby mismatches
for cleavage.
/primase.
Because the fidelity of DNA polymerase
is lower than that of the
DNA polymerases that complete DNA extension, mismatches occur
relatively frequently near the 5'-ends of Okazaki fragments. We have
examined the ability of FEN1 to repair such errors. Results show that
mismatched bases up to 15 nucleotides from the 5'-end of an annealed
DNA strand change the pattern of FEN1 cleavage. Instead of removing
terminal nucleotides sequentially, FEN1 appears to cleave a portion of
the mismatched strand endonucleolytically. We propose that a mismatch
destabilizes the helical structure over a nearby area. This allows FEN1
to cleave more efficiently, facilitating removal of the mismatch. If
mismatches were not introduced during synthesis of the Okazaki
fragment, helical disruption would not occur, nor would unnecessary
degradation of the 5'-end of the fragment.
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ABSTRACT
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/primase synthesizes every RNA
primer (2, 3) and the adjacent 10-20 nucleotides of DNA (4). It is
then replaced by either DNA polymerase
or
via a process called
polymerase switching (5-7). DNA polymerase
lacks the proofreading
3'-5' exonuclease activity present in polymerases
and
(2).
Because DNA polymerase
has no mechanism to remove erroneously
inserted nucleotides, it can continue synthesis only by mismatch
extension. DNA polymerase
incorporates an incorrect nucleotide at a
rate of approximately 1 in every 10,000 nucleotides, whereas the error
rate of polymerases
and
is at most 1 in 50,000-200,000
nucleotides (8).
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-32P]ATP (3,000 or
6,000 mCi/mmol) and [
-32P]dATP (3,000 mCi/mmol) were
from NEN Life Science Products. Oligonucleotides were synthesized by
Genosys Biotechnologies (The Woodlands, TX). T4 polynucleotide kinase
and SequenaseTM version 2.0 were obtained from U. S.
Biochemical Corp. RNase inhibitor and snake venom phosphodiesterase
were from Roche Molecular Biochemicals. All other reagents were from
Sigma Chemical Co.
Oligonucleotide sequences
-32P]ATP according to the
manufacturer's protocol and then annealed to the template in annealing
buffer (50 mM Tris, pH 8, 10 mM magnesium acetate, 50 mM NaCl, and 1 mM dithiothreitol).
For 3'-end-labeled substrates, downstream primers were first
phosphorylated using T4 polynucleotide kinase and ATP. Then, after
annealing to template T1, they were extended by addition to the 3'
terminus using [
-32P]dATP and SequenaseTM
version 2.0. Substrates were isolated via 12% native gel
electrophoresis (32), eluted from the gel using elution buffer (0.5 M ammonium acetate, 0.1% SDS, and 0.1 mM
EDTA), precipitated in ethanol, and resuspended in annealing buffer or
TE, pH 8.0.
-mercaptoethanol, 10 mM MgCl2, and 10 fmol
of substrate/reaction in a volume of 25 µl/reaction. Reactions were
initiated by the addition of 15 ng (340 fmol) of FEN1/reaction and
incubated at 37 °C for 30 min. Reactions were stopped with 25 µl
of 2× formamide loading dye (98% formamide, 10 mM EDTA
(pH 8.0), 0.01% (w/v) each of xylene cyanole and bromphenol blue) and
heated at 95 °C for 5 min. Controls lacking enzyme were also assayed. Products were separated on either 12% or 18% polyacrylamide, 7 M urea denaturing gel electrophoresis (32) and visualized by autoradiography. DNA size markers were generated by digesting 5'-end-labeled primers with snake venom phosphodiesterase. Any adjustments to the above are noted in the figure legends.
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in the region of DNA just
beyond the initiator RNA. On a fully annealed DNA primer, the action of
FEN1 is exonucleolytic, whereas on a flap, it is endonucleolytic. Our
initial experiments were designed to determine whether the presence of
a mismatch would destabilize a primer so that it would appear as a flap
to the nuclease. If so, we wanted to measure the distance over which
the mismatch could influence cleavage activity.
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Fig. 1.
Mismatch location and sequence affect FEN1
cleavage specificity. Assays measuring the internal cleavage of
substrates with a single mismatch are depicted. Each substrate was
5'-end-radiolabeled (*) and incubated with FEN1 as described under
"Experimental Procedures." Schematic diagrams of the substrates are
depicted at the top of the figure. A,
substrate 1.0 (lanes 1 and 2) has no mismatch.
Substrate 1.2 (lane 3) and substrate 1.3 (lane 4)
contain a mismatch at either position 2 or 3, respectively. Lane
1 contains no FEN1. B, lane 1 contains the same control substrate as in A. Substrate 1.T
(lane 2) contains a T-T mismatch, and substrate 1.A
(lane 3) contains an A-A mismatch, both of which are located
at the 5'-end of the primer. The sizes of the starting material and
cleavage products are indicated to the right. In
B, the starting material bands have been omitted for
simplicity, but they were similar to that in A.
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Fig. 2.
A mismatch in an endonucleolytic substrate
does not affect FEN1 cleavage. Assays measuring the
endonucleolytic cleavage of substrates with unannealed 5'-tails
combined with a single mismatch within the annealed region are
depicted. Each substrate has a 25-nucleotide unannealed 5'-tail with
either no mismatch (substrate 4.0) or a mismatch at position 7 or 9 (substrates 4.7 and 4.9) within the annealed region. Each substrate was
5'-end-radiolabeled (*) and incubated in the presence (lanes
1-3) or absence (lanes 4-6) of FEN1 as described
under "Experimental Procedures." Sizes are indicated to the
left. A schematic diagram of the substrates is depicted
above the figure.
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Fig. 3.
FEN1 can efficiently remove single, internal
mismatches. Assays measuring the internal cleavage of substrates
with a single mismatch are depicted. Substrate 2.9 contains a mismatch
nine nucleotides from the 5'-end of the primer. Substrate 3.6 is
similar to substrate 2.9, except that the three 5' most residues of
primer 2.9 are lacking in 3.6, making the mismatch only six nucleotides
from the 5'-end. Lanes 3 and 6 contain substrate
3.6, except that an upstream primer (UP) has been added to
form a nick with the first nucleotide after the mismatch in the
downstream primer. Each substrate was 5'-end-radiolabeled (*) and
incubated in the presence (lanes 1-3) or absence
(lanes 4-6) of FEN1 as described under "Experimental
Procedures." The sizes of the starting material and cleavage products
are indicated to the left. Schematic diagrams of the
substrates are depicted above the figure.
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Fig. 4.
Removal of a mismatch requires multiple
cleavage events. Oligonucleotide substrates containing either no
mismatch (lanes 1-7) or a single mismatch at position 9 (lanes 8-14), position 11 (lanes 15-21), or
position 15 (lanes 22-28) were radiolabeled at the 3'-end
(*). For each substrate, a single reaction (180 µl) containing 90 fmol of substrate was assembled at 4 °C. After the addition of 900 fmol of FEN1, the reaction was started by placing the sample at
37 °C. At 1, 3, 5, 10, 20, and 30 min, 20-µl aliquots were
removed, and the reaction was stopped by the addition of 10 µl of
formamide dye. Aliquots were heated to 70 °C for 10 min and
separated on a 12% polyacrylamide, 7 M urea denaturating
gel. For the no enzyme control ( ), an aliquot of the reaction was
removed before the addition of FEN1. The number of nucleotides removed
by FEN1 are noted to the left and right.
Schematic diagrams of each substrate are depicted above the
figure.
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/primase complex lacks a 3' to 5'
proofreading exonuclease; thus, it is relatively error prone (2). It
would seem that the 5'-ends of Okazaki fragments would have an
increased frequency of mismatch incorporation during DNA replication. Such errors would pose a threat to the fidelity of DNA replication if
they were not removed. If ligation of Okazaki fragments occurred before
removal, such errors could be corrected through the mismatch DNA repair
pathway. However, it would be more efficient to remove the mismatches
immediately, as part of the replication process. In fact, the overall
error rate of synthesis on the lagging strand is similar to that on the
leading strand (34). This result is consistent with the removal and
replacement of the entire polymerase
-synthesized region of every
Okazaki fragment by nick translation (34, 35). Recent estimates place
the average length of mammalian Okazaki fragments at 50-100
nucleotides (4). If so, extensive degradation of the 5'-end of every
fragment would be very inefficient. Alternatively, Okazaki fragment
processing could have a different proofreading mechanism, whereby only
regions containing a mismatch are degraded.
could be
removed. The mismatch could destabilize the helical structure of the
DNA and promote its own removal by relieving any sequence constraints.
In fact, the results shown here suggest that the presence of a mismatch
promotes extensive endonucleolytic cleavage. The absence of a mismatch
would encourage the minimum cleavage before ligation. Apparently,
mismatches create a helical disruption, allowing FEN1 to recognize the
presence of the mismatch and efficiently remove it. Because the
structure-specific requirements of FEN1 cleavage make the template
inert to FEN1 degradation (11), FEN1 would be able to distinguish the
nascent strand from the template strand and promote the repair of only
the incorrectly inserted nucleotide.
and
in that errors can be corrected as part of the
process in which they were created. Another similarity to 3'
proofreading is that the mismatch creates the substrate for the
proofreading nuclease. Because the polymerase-associated 3' exonuclease
is single strand-specific, only mismatched nucleotides are recognized
for degradation. The presence of a mismatched base pair promotes the
formation of 5'-flaps, the favored substrate of FEN1. This makes FEN1 a
logical choice to perform the proposed 5' proofreading reaction. We
predict that this process, as a form of replication-coupled mismatch
repair, is used in vivo to proofread and remove polymerase
errors from the chromosome.
in the DNA near the
5'-end of an Okazaki fragment. In vivo, an upstream primer
might also be present because FEN1 often requires a primer adjacent to
the site of cleavage (10, 20, 21). On the other hand, in some sequence
contexts, the upstream primer is inhibitory (18, 22). A delay in FEN1
activity would allow synthesis from the upstream Okazaki fragment to
approach the sites of cleavage, providing an upstream primer when
needed. In most experiments, we have not used an upstream primer. The
presence of an upstream primer that is designed to bind competitively
with the 5'-region of the downstream primer would create a transient flap structure, allowing for normal endonucleolytic cleavage as opposed
to mismatch-directed internal cleavage. This would complicate the
interpretation of our results. However, not using upstream primers has
at least two consequences: (a) it biases against detecting internal cleavages because synthesis from an upstream fragment would
normally collaborate with the mismatch to destabilize the helix
structure of the downstream fragment; and (b) it creates the
observed uneven cleavage patterns. These arise because some sites can
be cleaved readily without an upstream primer, whereas others are
upstream primer-dependent. When we used an upstream primer
designed to bind adjacent to an internal site on the downstream primer
(Fig. 3, lane 3), it had the expected effect of stimulating cleavage and allowing mismatch removal.
will promote FEN1 cleavage. Such an ability would
allow FEN1 to preferentially cleave those fragments that require
mismatch correction.
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Fig. 5.
Model for repair of polymerase
-incorporated mismatches and completion of Okazaki
fragment processing. First, the DNA polymerase
/primase complex
makes a RNA primer (hatched line) and begins DNA synthesis,
and then FEN1 removes the initiator RNA, perhaps including some DNA as
well, with or without extension from an upstream fragment. Meanwhile,
DNA polymerase
continues DNA synthesis and inserts a mismatch that
slightly disrupts the DNA helix. This disruption promotes the removal
of the mismatch by FEN1 in one cut or a series of endonucleolytic cuts,
depending on the location of the mismatch. After synthesizing 10-20
nucleotides, DNA polymerase
is replaced by DNA polymerase
or
for processive synthesis. The upstream fragment is extended up to
the downstream fragment, and ligation occurs. If DNA polymerase
does not incorporate a mismatch, unnecessary cleavage by FEN1 is
minimized, and ligation ensues immediately after RNA removal.
The crystal structures of several FEN1 homologues have been solved, and they suggest that FEN1 has an arch or loop structure enabling the nuclease to bind a flap and position the active site for cleavage (39-41). FEN1 may have evolved a strong preference for flap structures to carry out more efficient removal of initiator RNA and to participate in long patch base excision repair, a process that occurs by a flap mechanism (42, 43). Strong flap specificity explains why mismatches are so effective at stimulating FEN1 activity, as seen in our 3'-end-labeled time course experiment. A region around the mismatch is converted from an exonucleolytic substrate to the preferred flap substrate. The unannealed region can be removed quickly, allowing the nuclease to progress further on the substrate than if it were completely annealed.
Deletion of the yeast FEN1 caused an increase in the expansion frequency of trinucleotide repeats (44). This suggests that a mutation of FEN1 may be involved in human diseases such as myotonic dystrophy, Huntington's disease, several ataxias, and fragile X syndrome, all of which involve the expansion of repeat sequences (44). The role that FEN1 may play in DNA replication and repair and in preventing human diseases indicates the value of examining the exact mechanisms of this enzyme and defining the complete range of its substrate specificity.
In this report, we describe the specificity of FEN1 for DNA substrates
with mismatches near the 5'-end of a primer. We show that the presence
of a mismatch alters FEN1 specificity, promoting the nuclease to remove
the mismatch. We propose that this activity has evolved to remove
mismatches introduced by DNA polymerase during the initiation of
Okazaki fragments. In effect, FEN1 would then be the proofreading
nuclease for DNA polymerase
.
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ACKNOWLEDGEMENTS |
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We gratefully thank Dr. Min S. Park of Los Alamos National Laboratories for the generous gift of a human FEN1 expression vector. We thank E. V. Sechman for assistance in purifying recombinant human FEN1.
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FOOTNOTES |
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* This research was supported in part by National Institutes of Health Grant GM24441 (to R. A. B.) and Fellowship Grant GM18961 (to L. A. H.) and by Cancer Center Core Grant CA11198.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ A student in the Medical Scientist Training Program, funded by NIH Grant T32GM07356, and supported in part by an E. H. Hooker Fellowship.
To whom correspondence should be addressed: University of
Rochester Medical Center, Box 712, 601 Elmwood Ave., Rochester, NY
14642. Tel.: 716-275-3269; Fax: 716-271-2683.
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ABBREVIATIONS |
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The abbreviation used is: FEN1, flap endonuclease-1.
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REFERENCES |
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