From the Institut für Veterinärbiochemie, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland
Received for publication, September 27, 2000, and in revised form, November 15, 2000
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ABSTRACT |
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Flap endonuclease 1 (Fen1) is a
structure-specific metallonuclease with important functions in DNA
replication and DNA repair. It interacts like many other proteins
involved in DNA metabolic events with proliferating cell nuclear
antigen (PCNA), and its enzymatic activity is stimulated by PCNA
in vitro. The PCNA interaction site is located close to the
C terminus of Fen1 and is flanked by a conserved basic region of 35-38
amino acids in eukaryotic species but not in archaea. We have
constructed two deletion mutants of human Fen1 that lack either the
PCNA interaction motif or a part of its adjacent C-terminal region and
analyzed them in a variety of assays. Remarkably, deletion of the basic
C-terminal region did not affect PCNA interaction but resulted in a
protein with significantly reduced enzymatic activity. Electrophoretic mobility shift analysis revealed that this mutant displayed a severe
defect in substrate binding. Our results suggest that the C terminus of
eukaryotic Fen1 consists of two functionally distinct regions that
together might form an important regulatory domain.
Fen1 (5' exonuclease-1 or flap endonuclease-1) is a
multi-functional structure-specific metallonuclease that is important for DNA metabolic events such as replication and repair. Its main function in replication is proposed to be the removal of the displaced RNA-DNA primers synthesized by DNA polymerase The biochemistry of Fen1 has been studied extensively by several groups
(reviewed in Ref. 11), and the crystal structures of two Fen1
orthologues from archaea have been solved (12, 13). The enzyme seems to
employ a unique cleavage mechanism for substrates containing single
stranded 5' tails or flap structures. It cleaves the flap by
recognizing the 5' end, tracking the length of the tail and cleaving at
the junction between double-stranded and single-stranded DNA (14). The
crystal structures of the archaea orthologues of Fen1 reveal a helical
arch or clamp above the globular domain that contains the active site.
This clamp forms a hole big enough for single-stranded but not for
double-stranded DNA. This structure might be utilized by the nuclease
to recognize and track down the 5' end of a flap structure (13). DNA
substrates containing nicks or small gaps are also cleaved
exonucleolytically by Fen1. Although Fen1 acts less efficiently as an
exonuclease than as a flap endonuclease, it is likely that the enzyme
employs similar mechanisms for both reactions (11).
Eukaryotic Fen1 forms a stable complex with the replication accessory
protein PCNA1 both in
vitro and in vivo, and under physiological salt
conditions PCNA can greatly stimulate the in vitro activity
of Fen1 (15-18). The interaction domain was mapped to a region near
the basic C terminus of Fen1, which contains a consensus motif similar
to that found in several other replication and repair proteins that interact with PCNA (15). Adjacent to the PCNA interaction motif at the
very C terminus, Fen1 from eukaryotic species carries a basic tail of
35-38 amino acids of unknown function. This basic tail is absent in
the solved x-ray structures of both archaea Fen1 orthologues. However,
homologous basic tails are also present at the C termini of the XP-G
structure-specific nucleases known to be involved in nucleotide
excision repair (RAD2 of Saccharomyces cerevisiae and Rad13
of Schizosaccharomyces pombe). Here, this domain seems to be
important for nuclear localization and for dynamic response to UV
radiation (19). Consequently, it was suggested that in the case of
Fen1, this domain might as well contain a nuclear localization signal
that serves to translocate Fen1 into the nucleus where its activity is
required (20). In another report, it was proposed that the entire
C-terminal part of Fen1 might be important for PCNA interaction
(17).
In the present study, we have constructed two deletion mutants of human
Fen1 lacking either the PCNA interaction motif or a part of its
adjacent C-terminal region and analyzed these mutants in a variety of
assays. Consistent with results of a very recent study (21), we found
that the PCNA interaction motif is indispensable for stimulation of
Fen1 activity by PCNA. Moreover, our data reveal that the basic
C-terminal tail is not important for PCNA interaction. However, this
part of the protein is required for substrate binding. Our results
indicate two functionally distinct regions within the C-terminal part
of human Fen1 that might together form an important regulatory domain.
Nucleic Acid Substrates--
Oligonucleotides for preparing the
substrates for the Fen1 assays were purchased from Microsynth GmbH
(Balgach, Switzerland). Ft2_01 is a 39-mer composed of 19 nucleotides
complementary to nucleotides 698-717 of the plasmid pBluescript
SK+ (pBS) and a 20-nucleotide noncomplementary tail. Ft2_05
is a 19-mer of the same sequence without the noncomplementary tail, and
Ft2_02 is a 30-mer complementary to nucleotides 669-698 of single-stranded pBS. Oligonucleotides Ft2_01 and Ft2_05 were labeled at
the 5' end using [ Enzymes and Proteins--
Human Fen1 cDNA (gift of A. Dutta)
was cloned into the pET23d vector (Novoagene). Deletion mutagenesis was
performed with the QuickChangeTM site-directed mutagenesis
kit from Stratagene according to the instruction manual. The following
primers were used for the Fen1( Native Gradient PAGE--
The electrophoresis was performed in
8-25% polyacrylamide gels using the Phast System (Amersham Pharmacia
Biotech) according to the manufacturer's instructions. The gels were
stained with Coomassie Blue.
Affinity Interaction Binding Assay--
Interaction was measured
as described (25). Briefly, E. coli strain BL21(DE3)pLysS
harboring either wild type or mutant Fen1 and PCNA expression plasmids
was grown at 37 °C, and expression was induced. After harvesting by
centrifugation, the cell pellets were resuspended in buffer A (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride, 10 µM
pepstatin A, 10 µg/ml leupeptin, 10 µg/ml aprotinin) and briefly
sonicated. The cell lysates were clarified by centrifugation. Binding
assay mixtures (500 µl) contained 50 µl of 50%
nickel-nitrilotriacetic acid-Sepharose resin (Invitrogen), 150 µl of
each cell lysate, and 50 mM Tris-HCl, pH 7.5, 150 mM NaCl to increase mixing efficiency. These mixtures were
incubated for 90 min at 4 °C with constant gentle agitation and
washed six times with 0.8 ml of buffer B (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 60 mM imidazole), and
bound protein complexes were subsequently visualized on
Coomassie-stained 12% SDS-polyacrylamide gels.
Fen1 Assay--
The assays were performed in a final volume of
12.5 µl containing 40 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 5 mM dithiothreitol, 200 µg/ml bovine serum albumin, 50 fmol of DNA substrate, 50 ng of RP-A,
and indicated amounts of NaCl, ATP, PCNA, and RF-C. After addition of
wild type or mutant Fen1, the reactions were incubated at 30 °C for
15 min and stopped with 2.5× stop buffer (95% formamid, 20 mM EDTA, 0.05% each bromphenol blue, and Xylene cyanol).
Products were separated on 15% denaturing polyacryamid gels,
visualized by autoradiography and quantified on a PhosphorImager using
the ImageQuant software (Molecular Dynamics).
Mobility Shift Assay for Fen1--
Binding reactions were
carried out as described (26). Briefly, a total volume of 20 µl
contained 50 mM Tris-HCl, pH 8, 10 mM NaCl, 5 mM EDTA, 10% (v/v) glycerol, 50 µg/ml bovine serum albumin, 50 fmol of labeled oligonucleotide template, and indicated amounts of wild type or mutant Fen1. After incubation at 20 °C for
10 min, reactions were loaded on 7% polyacrylamide gels containing 0.5× TBE and run at 50 V for 3 h. The gels were dried and exposed to x-ray films.
A Conserved Basic C-terminal Tail Flanks the PCNA-binding Motif of
Eukaryotic Fen1--
The structures of archaea Fen1 reveal a
saddle-shaped, single-domain The Basic C Terminus of Human Fen1 Is Not Required for PCNA
Binding--
First, we tested whether the C-terminal tail of human
Fen1 is involved in PCNA interaction. We have previously shown that the
complex formed by PCNA and Fen1 is stable enough to survive electrophoresis on native gels (15). The complexes appear as shifted
bands on Coomassie-stained native gels (Fig.
2A). PCNA alone runs at about
67 kDa (lane 1), despite its actual mass of 86.3 kDa,
reflecting the compactness of the trimer and the high overall negative
charge of the protein. Fen1(WT) alone with an estimated pI of 8.78 does
not enter the gel because of its net positive charge at pH 8.44 of the
gel buffer, and the same is true for Fen1(
To confirm these findings, an affinity interaction assay was used to
compare the PCNA binding activities of wild type and mutant Fen1.
Lysates from bacteria expressing polyhistidine
(His6)-tagged wild type and mutant Fen1 and untagged PCNA
were mixed, and nickel-charged metal chelate resin was added to capture
the His6-tagged Fen1 along with any associated PCNA (Fig.
2B). As expected, PCNA bound stably to Fen1(WT) and
Fen1( A Fen1 Mutant Lacking 21 Amino Acids at the C Terminus Is Defective
for PCNA-independent Endo- and Exonuclease Activity--
Fen1 cleaves
endonucleolytically at branched DNA structures that have
single-stranded 5' flaps and exonucleolytically at nicks and, with
lower efficiency, at gaps or recessed 5' ends on double-stranded DNA.
At low salt concentrations, Fen1 displays nuclease activity in the
absence of its accessory protein PCNA. Wild type and mutant Fen1
nuclease activities were first determined in a flap cleavage assay in
the absence of monovalent ions. The circular flap template was created
as described in the experimental section and is documented in Fig.
3A. On this template, Fen1
cleaves at the junction of the flap releasing a labeled 20-mer
fragment, which was quantitated by denaturing urea-polyacrylamide gel
electrophoresis (Fig. 3A). Under low salt conditions, the
activity of Fen1(WT) and Fen1(
Next, we tested whether the exonucleolytic activity of the mutant
Fen1(
In summary, these data are in agreement with a recent study by Gomes
and Burgers (21) showing that a mutation within the PCNA-binding motif
does not affect the nuclease activity of yeast Fen1 at low salt
concentration. In addition, our data reveal that the PCNA independent
endo- and exonucleolytic activities of a Fen1 mutant lacking the basic
C-terminal tail are severely defective when compared with wild type activity.
Both the PCNA-binding Motif and the C-terminal Tail of Human Fen1
Are Important for PCNA-dependent Endo- and Exonuclease
Activity--
Endo- and exonucleolytic activities of Fen1 are
inversely proportional to monovalent salt concentrations in the
physiological range (27). However, it has been shown that PCNA can
stimulate Fen1 activity up to 50-fold under physiological salt
conditions (16, 18). Kinetic analysis revealed that PCNA enhances Fen1 binding stability, thus increasing the cleavage efficiency (28). To
efficiently stimulate Fen1 activity, the PCNA trimer must encircle the
DNA and must be located "below" the flap (15). In our study, PCNA-dependent Fen1 activity was determined at 100 mM NaCl. Under these conditions, Fen1 alone is inactive
(Fig. 4A). For stimulation, the ATP-dependent clamp loader RF-C must be present to load
PCNA at the flap junction and at the position of the nick,
respectively, because PCNA cannot spontaneously load onto circular DNA
molecules in the absence of RF-C. The human single-stranded DNA binding protein RP-A was also present in this assay to prevent nonproductive association of RFC to single-stranded DNA (29). First, wild type and
mutant Fen1 nuclease activities were determined in the presence of 100 mM NaCl (Fig. 4B). Fen1(
In summary, Fen1( The C Terminus of Human Fen1 Is Essential for Substrate
Binding--
Because Fen1( In this study, we have characterized two deletion mutants of human
Fen1 lacking residues at the C terminus that have been proposed to be
critical for interaction with PCNA. These mutants were compared with
wild type Fen1 in a variety of assays. The mutant Fen1( On the other hand, both the endo- and exonucleolytic activities of the
mutant Fen1(
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-primase during discontinuous lagging strand replication (reviewed in Ref. 1). In DNA
repair, Fen1 appears to be required for nonhomologous end joining of
double-strand DNA breaks (2) and for the removal of DNA base damage and
single-strand breaks, likely through participation in the excision and
resynthesis steps of long patch base excision repair and
nucleotide excision repair (reviewed in Ref. 3). The relevance of these
observations is underscored by in vivo data using yeast
Fen1-null mutant strains that display severely impaired phenotypes such
as temperature sensitivity for growth with a terminal phenotype
consistent with a defect in replication, sensitivity for DNA damaging
agents such as UV radiation, and alkylating agents and defects in
telomere maintenance (4-7). Because these deletion strains are strong
mutators with destabilized repetitive sequences, it has been suggested
that Fen1 might also be involved in one of the mechanisms by which
trinucleotide expansions occur (8-10). Such expansions have been
identified as the bases of more than 10 human hereditary diseases. In
sum, Fen1 is a key enzyme for maintaining the genomic stability.
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-32P]ATP and T4 polynucleotide
kinase. Free ATP was removed on MicroSpinTM G-25 columns.
To generate the substrates for the endo- and exonuclease assays, the
appropriate primers were mixed with single-stranded pBS DNA in 1:1
molar ratio in 20 mM Tris-HCl, pH 7.4, 150 mM
NaCl, heated to 75 °C, and slowly cooled to room temperature. The
templates for the mobility shift assay were created by hybridizing
5'-labeled Ft2_01 or Ft2_05 and unlabeled Ft2_02 to a complementary
49mer oligonucleotide, corresponding to nucleotides 669-717 of pBS.
P) mutant (amino acids 337-344):
5'-AAGAGCCGCCAAGGCAGCACCAAGGTGACCGGCTCACTCT-3' and
5'-AGAGTGAGCCGGTCACCTTGGTGCTGCCTTGGCGGCTCTT-3'. For the Fen1(
C) mutant (aa 360-380), oligos
5'-GCGCAAGGAGCCAGAAGGAAAGCTTGCGGCCG-3' and
5'-CGGCCGCAAGCTTTCCTTCTGGCTCCTTGCGC-3' were used, respectively. Wild type and mutant Fen1 were overexpressed in Escherichia
coli strain BL21(DE3)pLysS as histidine-tagged proteins and
purified to homogeneity via Nickel charged metal chelating resin
(HiTrap; Amersham Pharmacia Biotech) and fast protein liquid
chromatography MonoS chromatography. Human PCNA was produced in
E. coli using the plasmid pT7/hPCNA (gift from B. Stillman)
and purified to homogeneity as described (22). Replication factor C
(RF-C) was purified from nuclear extract of 60 g of harvested HeLa
cells as described (23). Human replication protein A (RP-A) was
overexpressed in E. coli strain BL21(DE3) harboring the
expression plasmid p11d-tRP-A and purified according to Ref. 24.
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/
protein with a deep positively
charged cleft along one face and an unusual "helical clamp" through
which a single-stranded flap strand is likely threaded. The C-terminal
nine residues extend away from the body of the enzyme (Fig.
1A and Refs. 12 and 13). These
residues are homologous to critical residues of eukaryotic proteins
known to interact with PCNA (15), and, based on these data, a model of
how Fen1 could be hooked to PCNA during DNA replication was proposed
(12). However, Fen1 from eukaryotic species have additional 35-38
residues at the C terminus that are not present in the archaea enzymes
(Fig. 1B). Chen and colleagues (17) have deleted 17 residues
at the very C terminus of human Fen1 and tested this mutant for its
ability to interact with PCNA. Surprisingly, their mutant Fen1 failed
to interact with PCNA in several assays, although it still contained an
intact PCNA interaction motif. Thus, these C-terminal residues could be
involved in PCNA interaction, or they could play another as yet unknown
function. In a quest for Fen1 mutants that would differ from wild type
Fen1 in their affinities for PCNA, we selectively deleted two regions
at the C terminus of human Fen1. The first deletion comprised the
entire PCNA interaction motif (amino acids 337-343) and was called
Fen1(
P). The second deletion mutation, called Fen1(
C), enclosed
the last 21 C-terminal amino acids (amino acids 360-380). Wild type
and mutant Fen1 were overexpressed in E. coli as
polyhistidine (His6)-tagged proteins and purified to
homogeneity (Fig. 1C).
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Fig. 1.
Mutations at the C terminus of human
Fen1. A, structure of Methnococcus
jannaschii Fen1 (13). The region of homology to the PCNA-binding
motif in human Fen1 is indicated (P). The basic C-terminal
tail is not present in the archaea orthologue. B, sequence
alignment of the Fen1 C termini from various eukaryotic species. The
PCNA-binding motif and the basic C-terminal tail are
shaded and the conserved residues are printed in
bold. C, SDS-PAGE analysis of purified wild type
and mutant Fen1. 1.3 µg of each sample were separated on a 12% SDS
polyacrylamid gel and stained with Coomassie Blue.
P) (lanes 2 and
3). However, Fen1(
C) lacking several basic residues at
the C terminus slowly enters the gel and appears between 170-180 kDa
(lane 4). By separating a mixture of equimolar amounts of
purified PCNA (as trimer) and Fen1(WT), a complex with a molar mass of
263 kDa was apparent (lane 5). Most likely, the high pI of
Fen1 is responsible for the extent of the observed band shift. A
similar shift appeared when equimolar amounts of PCNA and Fen1(
C)
were separated, although in this case the complex ran at around 190 kDa
(lane 6). However, when equimolar amounts of PCNA and
Fen1(
P) were separated, no such band shift could be observed
(lane 7). This suggests that Fen1(WT) and Fen1(
C) but not
Fen1(
P) form complexes with PCNA that are stable enough to survive
native PAGE.
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Fig. 2.
The mutant Fen1( C)
but not the mutant Fen1(
P) is able to interact
with PCNA. A, samples containing 4 µg of PCNA (40 pmol of trimer), 5 µg (120 pmol) of wild type, or mutant Fen1 and
wild type or mutant Fen1 in combination with PCNA, respectively, were
incubated at 37 °C for 15 min, separated on a 8-25% native
gradient gel using the Phast System (Amersham Pharmacia Biotech), and
stained with Coomassie Blue. Markers are ovalbumin (43 kDa), bovine
serum albumin (67 kDa), aldolase (158 kDa), catalase (232 kDa), and
ferritin (440 kDa). B, nickel-nitrilotriacetic acid resin
was incubated with cleared bacterial lysates from
isopropyl-1-thio-
-D-galactopyranoside-induced cells
containing human PCNA expression plasmid (lane 1) and
His6-tagged wild type and mutant human Fen1 expression
plasmids (lanes 2-4). Cleared lysates from cells expressing
His6-tagged wild type (WT, lane 5)
and mutant (
P, lane 6;
C, lane 7) Fen1 were mixed with
nickel-nitrilotriacetic acid resin and equal aliquots of lysates from
bacteria expressing untagged PCNA. After washing, protein complexes
were denatured, separated by SDS-PAGE, and analyzed by Coomassie Blue
staining. Arrows indicate the mobility of individual
proteins.
C) (lane 5 and 6) but not at all to
Fen1(
P) (lane 7). The same results were obtained when
His6-tagged PCNA was used to pull down untagged wild type
and mutant Fen1, thus ruling out the possibility of a binding
interference with the polyhistidine tag (data not shown). Taken
together, these data suggested that the PCNA binding activity does not
depend on the presence of the basic C-terminal tail of human Fen1,
although a slight reduction in affinity cannot be ruled out.
P) was almost identical (Fig.
3A, lanes 2-4 and 5-7), whereas the activity of Fen1(
C) was clearly impaired (lane 8-10). Although 2.5 ng (60 fmol) of Fen1(WT) and Fen1(
P) cleaved 80 and 90% of the
input template (50 fmol) in 15 min, respectively, the same amount of
Fen1(
C) cleaved only about 8% of the template. Thus, at
nonsaturating enzyme concentrations, Fen1(
C) is about 10 times less
active than Fen1(WT) and Fen1(
P). Fen1(
C) displayed a reduced enzymatic activity even at highly saturating enzyme concentrations (25 ng; 0.6 pmol).
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Fig. 3.
Fen1( C) but not
Fen1(
P) activity is reduced in
PCNA-independent nuclease assays. Activity of buffer alone
(B) wild type (WT), and mutant
(
P and
C) Fen1 was
measured in the absence of ATP, PCNA and RF-C. Proteins were titrated
to 50 fmol of RP-A-coated circular substrate in reaction buffer
containing no salt. Products were separated on 15% urea polyacrylamid
gels and visualized by autoradiography. Quantification was done with a
PhosphorImager. A, flap endonucleolytic activity. The
amounts of Fen1 were 0.25 ng (6 fmol), 2.5 ng (60 fmol), and 25 ng (0.6 pmol), respectively. B, exonucleolytic activity. The amounts
of Fen1 were 0.5 ng (12 fmol), 5 ng (120 fmol), and 50 ng (1.2 pmol),
respectively. Right panel, schematic view of the circular
flap and nicked DNA substrates.
C) was impaired as well. Purified wild type and mutant Fen1
were examined on a circular partial duplex DNA molecule containing a
nick (Fig. 3B). Fen1 digests the upstream oligonucleotide
exonucleolytically in the 5' to 3' direction, thus releasing a labeled
5' mononucleotide, which could be resolved and quantitated on 15% urea
polyacrylamid gels. As expected, Fen1(
C) exonucleolytic activity was
also severely reduced (lanes 18-20). Again, the activities
of Fen1(WT) and Fen1(
P) were almost identical (Fig. 3B,
lanes 12-14 and 15-17). Although 5 ng of (120 fmol) of Fen1(WT) and Fen1(
P) cleaved 84 and 81% of the input
template (50 fmol) in 15 min, respectively, no detectable cleavage
product could be observed when 50 fmol of the template was incubated
with 5 ng of Fen1(
C). When the same amount of template was incubated
together with 50 ng of Fen1(
C), only 24% were cleaved in 15 min. At
these concentrations, Fen1(WT) and Fen1(
P) cleaved almost 100%.
P) was inactive, even at the highest concentration tested (lanes 5-7). This
result is again in agreement with a recently characterized similar
yeast Fen1 mutant (21), although this mutant carrying two point
mutations within the PCNA-binding motif shows weak activity at long
incubation times. Fen1(
C) displayed some activity at higher
concentrations, but this activity was also severely impaired compared
with Fen1(WT) activity; in the presence of PCNA, as little as 0.05 ng
(1.2 fmol) Fen1(WT) cleaved 85% of the input template in 15 min
(lane 2), whereas the same amount of Fen1(
C) cleaved only
8.5% (lane 8). Remarkably, the difference between Fen1(WT)
and Fen1(
C) activity seems to be the same as in the absence of PCNA,
although in the PCNA independent assay, the enzyme concentration was
50-fold higher. When the PCNA-dependent exonucleolytic
activity of wild type and mutant Fen1 was tested, Fen1(
P) was, as
expected, completely inactive (lanes 15-17), and Fen1(
C)
was about 10 times less active than Fen1(WT) (lanes 12-14
and 18-20).
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Fig. 4.
Fen1( P) is inactive
and Fen1(
C) activity is severely reduced in
PCNA-dependent Fen1 assays. Activity of buffer alone
(B), wild type (WT), and mutant
(
P and
C) Fen1 was
measured in the presence of 1 mM ATP, 0.5 pmol of PCNA, and
2.5 pmol of RF-C. Proteins were titrated to 50 fmol of RP-A-coated
circular substrate in reaction buffer containing 100 mM
NaCl. Products were separated on 15% urea polyacrylamid gels and
visualized by autoradiography. Quantification was done with a
PhosphorImager. A, flap endonucleolytic activity. The
amounts of Fen1 were 0.05 ng (1.2 fmol), 0.5 ng (12 fmol), and 5 ng
(120 fmol), respectively. B, exonucleolytic activity. The
amounts of Fen1 were 0.1 ng (2.4 fmol), 1 ng (24 fmol), and 10 ng (240 fmol), respectively. C, depletion of the assay reveals that
Fen1 activity is dependent on the presence of both PCNA and RF-C.
P) is completely defective under
PCNA-dependent conditions. Because this mutant is unable to
form a complex with PCNA (Fig. 2), we conclude that the physical
interaction between Fen1 and PCNA is absolutely required for Fen1
stimulation. Moreover, PCNA-dependent endo- and
exonucleolytic activity of Fen1(
C) is as much reduced as the
PCNA-independent activity, which suggests that the C-terminal tail has
no influence on PCNA interaction and stimulation.
C) activity was 10-fold reduced both in
PCNA-independent and in PCNA-dependent nuclease assays, we
next tested whether this mutant had a defect in substrate binding. To
investigate the substrate binding characteristics of wild type and
mutant Fen1, we employed the mobility shift assay developed by
Harrington and Lieber (26). Because Fen1 is a potent nuclease in the
presence of divalent metal ions, EDTA was included in the binding
reaction. The labeled binding substrates were created as described
under "Experimental Procedures." Fen1(WT) and Fen1(
P) were found
to bind to both flap and nick substrates in a
dose-dependent manner. At concentrations between 60 and 240 nM of Fen1, a single shifted species was observed (Fig.
5, lanes 2-7 and
12-17, respectively). However, when equal amounts of
Fen1(
C) were incubated with the binding substrates, no detectable
shift could be observed, indicating that this C-terminal mutant
displayed a severe defect in substrate binding, suggesting that the
reduced nuclease activity of this mutant is caused by a defect in the
binding portion of the nucleolytic reaction.
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Fig. 5.
The Fen1( C) mutant
has a defect in substrate binding. Substrate binding activity of
buffer alone (B), wild type (WT), and mutant
(
P and
C) Fen1 was
measured in the presence of 50 fmol of labeled oligonucleotide template
and EDTA to prevent nucleolytic degradation of the substrate.
Protein-DNA complexes (**) were separated from free DNA probe (*) on
7% nondenaturing polyacrylamide gels, and the band shifts were
visualized by autoradiography. The Fen1 concentrations were 60, 120, and 240 nM, respectively. Left panel, binding
activity to an oligo flap template. Right panel, binding
activity to nicked double-stranded DNA. Bottom panels,
schematic views of the linear flap and nicked DNA substrates.
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P) lacking 7 amino acids (amino acids 337-343) near the C terminus behaved very
similarly to mutants recently characterized by other groups (21, 25).
These studies and the data presented here provide a consistent body of
evidence that the bimolecular interaction between these two proteins is
mediated by a consensus PCNA-binding motif
(QXX(M/I/L)XXF(F/Y)), which is present in several other proteins known to interact with PCNA
including p21, XP-G and DNA ligase I. On the other hand, at least two
regions within PCNA seem to mediate the interaction with Fen1, and the
protein-protein contacts between Fen1 and PCNA are slightly different
in solution from the contacts that occur when the proteins are
complexed with DNA (15, 21). The relevance of the PCNA/Fen1 interaction
via the interaction motif is underscored by in vivo data
using yeast strains with mutations within this conserved region (21,
30). In another report, it was suggested that the basic C-terminal tail
of Fen1 was also important for the interaction with PCNA, because a
deletion mutant lacking 17 amino acids at the C terminus failed to
interact with PCNA, although this mutant still contained an intact PCNA
interaction motif (17). We have constructed a similar mutant,
Fen1(
C), lacking 21 amino acids at the very C terminus and tested it
for PCNA interaction in a native PAGE-based assay and in an affinity
interaction binding assay. In our hands, no difference in the ability
to interact with PCNA could be detected between this mutant and wild
type Fen1, whereas the Fen1(
P) mutant was completely unable to
interact with PCNA. This discrepancy may partially result from
differences in the experimental design or alternatively the additional
C-terminal residues on the Fen1 mutant of Chen et al. (17)
may affect the folding of the PCNA-binding motif so that it no longer
can interact with PCNA. Our data, however, show that the short
PCNA-binding motif of Fen1 is responsible for the bulk of the PCNA
interaction. Consistent with this, we showed in this study that wild
type Fen1 and the Fen1(
C) mutant were stimulated by PCNA to a
relatively equal extent under physiological salt conditions.
C) were significantly reduced compared with the wild
type enzyme. This defect could be observed in a PCNA-dependent background, as well as under conditions
where Fen1 displays activity without PCNA. We therefore conclude that
this defect is not due to the inability of Fen1(
C) to interact with PCNA. Moreover, the ability of this mutant to associate with an oligo
flap template and a short nicked double-stranded DNA was severely
reduced. Our results clearly demonstrate the importance of the
C-terminal basic tail for the binding portion of the nucleolytic reaction. Because the active site of the enzyme is composed of residues
of the N-terminal and intermediate conserved regions (31, 32), it is
rather unlikely that the C terminus is also involved in the cleavage
portion of the enzymatic reaction. The simplest explanation for the
defect in substrate binding of Fen1(
C) would be the lack of several
positively charged residues (8 lysines and 1 arginine) that may
specifically or unspecifically contact the negatively charged phosphate
groups of the DNA backbone and contribute to the stability of the
enzyme substrate complex. If this is true, then the question arises why
such an "anchor" is not present in the archaea Fen1 orthologues,
which have structure-specific mechanisms for DNA substrate binding and
catalysis resembling the human enzyme (33). Another even more
interesting possibility would be that the C-terminal tail loops back
toward the enzymatic center, thus providing structural elements that
might modulate the stability of the enzyme substrate complex. We could
recently show that Fen1 is an acetylated protein in
vivo.2 This Fen1
acetylation is significantly increased after UV treatment of the cells
and is most probably mediated by the histoneacetyl transferase domain
of the transcriptional coactivator p300. There is a detectable
interaction between these two proteins both in vitro and
in vivo, which is mediated by the C-terminal tail of Fen1.
Moreover, we showed that the acetylated Fen1 possessed a reduced
enzymatic activity, thus resembling our Fen1(
C) mutant. Possibly,
one or several lysine residues at the C-terminal tail are
"neutralized" upon acetylation, which could lead to a destabilized enzyme-substrate complex, as shown here for the Fen1(
C) mutant. This
model suggests that the C-terminal tail of eukaryotic Fen1 contains
regulatory regions that are modified upon treatment of the cells with
damaging agents. This modification might regulate the enzymatic
activity itself or the subnuclear localization by modulating the DNA
binding stability and/or specificity. In summary, our results suggest
that the C terminus of eukaryotic Fen1 consists of two functionally
distinct regions: one that mediates PCNA interaction, which is
important for stimulation of enzymatic activity and/or targeting Fen1
to sites where its action is required, and a second region that
functions in recognition of the DNA substrate. This second region is
not present in archaea orthologues of Fen1. It therefore has been
suggested that it might be required for nuclear localization of the
enzyme in eukaryotes (20, 34), although the importance of such a
putative nuclear targeting signal for Fen1 has not been shown. Our data
suggest that this basic C-terminal tail of eukaryotic Fen1 most likely
has an additional function.
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ACKNOWLEDGEMENTS |
---|
We thank Robert Hindges for the purification of human RP-A and Samez Hasan for suggestions and critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by Swiss National Science Foundation Grants 31 43.138 95/2 and 31 61361.00 and by the Kanton of Zürich.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.
Present address: Dept. of Pathology, Brigham and Women's
Hospital, Harvard Medical School, 75 Francis St., Boston, MA 02115.
§ To whom correspondence should be addressed. Tel.: 411-635-54-72; Fax: 411-635-68-40; e-mail: hubscher@vetbio.unizh.ch.
Published, JBC Papers in Press, November 16, 2000, DOI 10.1074/jbc.M008829200
2 S. Hasan, M. Stucki, P. O. Hassa, R. Imhof, U. Hübscher, and M. O. Hottiger, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: PCNA, proliferating cell nuclear antigen; RF-C, replication factor C; RP-A, replication protein A; PAGE, polyacrylamide gel electrophoresis.
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