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INTRODUCTION |
DNA replication and repair are critical for maintaining genome
stability. These processes are in part dependent on the activity of an
emerging family of structure-specific endonucleases. These enzymes,
typified by flap endonuclease-1
(FEN-1),1 are
multifunctional. They possess both flap-specific endo- and nick-specific exo(ribo)nuclease activities and interact with
proliferating cell nuclear antigen (PCNA) (1-8). FEN-1 is required for
the removal of both RNA primers during lagging strand DNA synthesis and
damaged DNA fragments in the long patch DNA base excision repair
pathway (9-14). The fact that FEN-1 is critical for preserving the
integrity of a genome is underscored by recent work showing that
RAD27 (FEN-1 homologue) null mutants in
Saccharomyces cerevisiae have a strong mutator phenotype.
This phenotype is a result of duplication mutations arising from the
inability of the cells to process correctly the RNA primers associated
with Okazaki fragments during lagging strand DNA replication (15).
Duplication mutations are associated with several human disorders such
as recessive retinitis pigmentosa, lethal junctional epidermolysis
bullosa, familial hypertropic cardiomyopathy, and various cancers. In
addition, FEN-1 helps to prevent trinucleotide repeat expansion and
contraction, as a deletion of RAD27 in S. cerevisiae results in length-dependent destabilization of CTG tracts and a marked increase in expansion frequency (16, 17). Trinucleotide expansions are involved in genetic
diseases such as myotonic dystrophy, Huntington's disease, ataxias, and fragile X syndrome.
Nuclear localization is an important component of the ability of the
cell to regulate DNA replication, repair, and other biological functions. Transport occurs through the nuclear pore complex (18, 19).
The nuclear pore allows small molecules to diffuse freely into and out
of the nucleus (18, 19). Targeting of larger proteins to the nucleus
usually requires the presence of a nuclear localization signal (NLS)
(19). A variety of proteins such as heat shock protein factor 2, p53,
transcription factors, DNA repair complex hRad51/BRCA1, and xeroderma
pigmentosum group G (XPG) nuclease have a nuclear localization signal
(20-26). Two major types of NLSs are recognized as follows: 1) a
single cluster of basic amino acids, exemplified by the SV40 T antigen
NLS; and 2) a bipartite NLS composed of one cluster of two basic amino acids, a spacer region of 10-12 amino acids, followed by another basic
cluster, as found in nucleoplasmin (18, 27).
To function, DNA replication and repair proteins have to migrate into
the nucleus after they are translated in the cytoplasm since their
target DNA substrates are located in nucleus. Previously identified
NLSs in DNA repair proteins are bipartite (28). Mutations of the NLS
motif may not only alter regulation of protein movement but also affect
their cellular functions, which in humans may cause diseases. The NLS
of the DNA repair protein XPG resides in the C-terminal region (amino
acid residues 1164-1185) and is essential for the formation of foci
and for response to UV damage (22, 29). The distribution of XPG
proteins could also regulate the rate of DNA repair within
transcriptionally active and inactive compartments of the cell nucleus.
Recently, the NLS of mammalian DNA replication ligase (ligase I) has
been identified at residues 119-131 (30). In addition, the enzyme has
an additional motif of ~20 amino acids at their N terminus, which
functions as a replication factory targeting sequence. This motif,
consisting of two clusters of bulky and positively charged residues, is
necessary and sufficient for the interaction with proliferating cell
nuclear antigen (PCNA). DNA ligase I is recruited to sites of DNA
replication by an interaction with PCNA.
It was proposed that there are three motifs of the FEN-1 nuclease
responsible for three distinct functions as follows: nuclease activities, PCNA interaction, and nuclear localization (1, 4, 31). The
C-terminal 26 amino acids of hFEN-1 are basic and conserved in the
FEN-1/XPG family and may be involved in nuclear localization (31). In
this study, we demonstrate that FEN-1 nuclease localizes into the
nucleus in a cell cycle-dependent manner and in response to
DNA damage in human HeLa cells. Truncational and site-directed
mutagenesis in the C-terminal NLS motif was carried out for in
vitro and in vivo functional analysis in both mammalian
and yeast cells. Truncation of the signal domain effectively blocked
nuclear localization in vivo but had no effect on nuclease activity and PCNA interaction in vitro. Site-directed
mutagenesis revealed that KRKXXXXXXXXKKK is a bipartite NLS
since substitution of KRK with alanines completely eliminate the
nuclear localization, whereas KKK mutagenesis partially abolishes
the function. An NLS-defective FEN-1 transformed into yeast null mutant
cells resulted in an increased sensitivity to methyl methanesulfonate
(MMS) treatment and slower growth at an elevated temperature. These
results indicate that the NLS is essential to the
FEN-1-dependent cellular responses to environmental stresses.
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EXPERIMENTAL PROCEDURES |
Materials
Oligonucleotide primers were synthesized at the City of Hope
Cancer Center core facility. Sources of plasmid vectors used in this
study for subcloning, mutagenesis, and expression are detailed below.
Quick-change site-directed mutagenesis kit, PCR-cloning kit, and
Escherichia coli strain XL1 blue competent cells were purchased from Stratagene (La Jolla, CA). Restriction enzymes were
obtained from New England Biolabs (Beverly, MA).
[
32P]dCTP was purchased from PerkinElmer Life
Sciences. Polyclonal antibody against human FEN-1 protein was
custom-produced at Research Genetics, Inc. (Huntsville, AL). The
anti-FEN-1 serum was collected from a rabbit after three boosts and
stored at
80 °C. Monoclonal antibody against human FEN-1 protein
was purchased from Novus (Littleton, CO). Secondary antibody was from
Santa Cruz Biotechnology (Santa Cruz, CA). Nylon membrane was
purchased from Schleicher & Schuell. Fast purification liquid
chromatography accessories and reagents were from Amersham Pharmacia
Biotech. Yeast culture media, including YPD, synthetic complete (SC),
minimal sporulation, and synthetic dextrose minimal (SD), were prepared
according to Sherman et al. (32). Cell cycle inhibitors
(mimosine and nocodazole), amino acids, and all other chemicals were
purchased from Sigma.
cDNA, Plasmids, and Mutagenesis
cDNA for human FEN-1 was the laboratory stock as described
previously (33). Vectors for expression, subcloning, and mutagenesis included pBSK (Stratagene, La Jolla, CA) for subcloning and
mutagenesis, pEGFP-N1 N-terminal protein fusion vector
(CLONTECH, Palo Alto, CA) for the expression of
FEN-1 proteins fused with GFP in human cells, and pET-28b (Novagen,
Madison, WI) for protein overexpression in E. coli. The pBSK
plasmid with the insertion of wild type FEN-1 was used for
site-directed mutagenesis to give rise to the following three mutants.
Mkrk has the mutation of KRK at residues 354-356 to AAA
using the primers 5'GCTCACTCTCTTCAGCTGCGGCTGCTGAGCCAGAACCCAAGG3' and 5'CCTTGGGTTCTGGCTCAGCAGCCGCAGCTGAAGAGAGTGAGC3'. Mkkk
has the mutation of KKK at the residues 365-367 to AAA (KKK/AAA) using the primers 5'ACCCAAGGGATCCACTGCTGCTGCTGCAAAGACTGGGGCAG3' and 5'CTGCCCCAGTCTTTGCAGCAGCAGCAGTGGATCCCTTGGGT3'. The third mutant Mkr has mutation of KR at residues 378 and 379 to AA using
the primers 5'GGCAGCAGGGAAGTTTGCAGCGGGAAAAAGCTTTATTAAA3' and
TTTAATAAAGCTTTTTCCCGCTGCAAACTTCCCTGCTGCC3'. To determine the
localization pattern of the FEN-1 proteins in human cells, we amplified
DNA fragments of the wild type FEN-1 and the three site-directed
mutants Mkkk, Mkrk, and Mkr using PCR based on primers, CK01-
5'CCTCTGTGCTAGCATGGGAATTCAAGGCC3' and
CK02a-5'CCTCTCCAAGCTTTCCCCTTTTAAACTTCC3' (sequences for
desired restriction sites are underlined). These fragments were
then subcloned into the pEGFP-N1 vector, respectively, at the
NheI and HindIII sites. An NLS-truncated FEN-1
cDNA construct (Mnlsd) was also generated using the
primer pair of CK01 and CK03a,
5'CCTCTCCAAGCTTGAAGAAATCATCCAG3', and then cloned into
pEGFP-N1 vector also at the NheI and HindIII sites. To observe the biological functions of the FEN-1 proteins in
yeast, DNA fragments of the wild type FEN-1, Mkkk,
Mkrk, and Mkr cDNA were directly derived
from pBSK-based plasmids using the restriction enzyme NotI.
They were then cloned into the yeast expression vector, pDB20. The
truncation mutant Mnlsd DNA was PCR-amplified using primers
T7 and NLSDNOT (5'ACACACAGCGGCCGCTTACTTGAAGAAATCATCCAG) and
then subcloned into pDB20 at the NotI site. For bacterial overexpression of FEN-1 proteins, the full-length wild type human FEN-1
coding sequence was previously subcloned into pET-28b vector (33). DNA
fragments of mutants Mkrk, Mkkk, and
Mkr from the pBSK-based constructs were excised using
NcoI and XhoI and were subcloned into pET28b. For
the construction of pET28b plasmid of the NLS-truncated mutant, a DNA
fragment of the mutant was amplified using primers T7 and CK03a and was
subcloned into pET28b at the NcoI and XhoI sites.
Overexpression and Purification of FEN-1 NLS Mutants
Truncated and site-directed NLS mutant proteins overexpressed in
bacteria were less soluble than wild type FEN-1 protein, and a majority
of the protein was located in the inclusion bodies. We overcame this
problem by optimizing bacterial expression conditions. Specifically,
the bacterial cells were cultured in 70% Luria broth at 30 °C for
higher solubility of the FEN-1 protein in vivo. Once the
protein was solubilized, they were purified as described previously (34). Briefly, the lysate was sonicated (Branson Sonifier 450, Branson
Ultrasonics) and centrifuged at 15,000 × g for 45 min to remove the cell debris. A 5-ml HiTrap chelating Ni2+
column was equilibrated with buffer A using fast purification liquid
chromatography system (Amersham Pharmacia Biotech). After loading, the
column was washed with 25 ml of buffer A and 25 ml of buffer A plus 60 mM imidazole and was eluted with a 50-ml linear gradient
from 60-500 mM imidazole in buffer A at 2 ml/min. The fractions were run on a 10% SDS-polyacrylamide gel and stained with
Coomassie Brilliant Blue R-250. The buffers of the fractions were
exchanged to 10 mM Tris-HCl, 150 mM NaCl, pH
8.0, by a HiTrap desalting column. Protein concentrations were
determined using the Bio-Rad protein assay.
Biochemical Assays
Assays for flap endonuclease and nick-specific exonuclease
activities were carried out as described previously (31). The following
oligonucleotides were used to make the flap and nicked DNA substrates:
a template strand (5'GGACTCTGCCTCAAGACGGTAGTCAACGTG), a 5'-labeled flap
strand (5'GATGTCAAGCAGTCCTAACTTTGAGGCAG AGTCC-3'), and an
adjacent strand (5'CACGTTGACTACCGTC) for the flap DNA substrate; for
the exonuclease DNA substrate, a 5'-labeled downstream strand 5'TTGAGGCAGAGTCC3' was employed instead of the flap strand in the flap
substrate. The reactions were performed in a mixture containing 0.8 pmol of [32P]DNA, 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2, and 30 ng of enzyme in 13 µl at
30 °C for 30 min. The reactions were stopped by adding 3 µl of
stop solution (U. S. Biochemical Corp.), boiled for 3 min, and then
cooled in ice. 4 µl of each reaction product was run on a 15%
denaturing polyacrylamide gel. The gel was exposed to Kodak x-ray film.
For PCNA interaction analysis, E. coli cell crude extracts
of His-tagged proteins of wild type FEN-1 and NLS mutants were
individually mixed with crude extracts of BL21(DE3)/pT7/PCNA expressing
PCNA without a His tag (5, 35). The mixture was loaded onto a Amersham
Pharmacia Biotech nickel column, washed, and eluted with 500 mM imidazole. The eluates were run on a 10% SDS gel to
examine the interaction of PCNA and NLS mutant FEN-1 proteins.
Cell Culture, Transfection with FEN-1/GFP Fusion Constructs, and
MMS Treatment--
HeLa cells (laboratory stock) were cultured in
Dulbecco's modified Eagle's medium plus 10% fetal bovine serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin at 37 °C with 5%
CO2. The cells were passaged until the day before
transfection. When cells achieved 60% of confluency, they were
individually transfected with the plasmid DNAs as follows: wild type
FEN-1, Mnlsd, Mkrk, Mkkk,
Mkr, and pEGFP N-1, with the FuGENE6 reagent (Roche
Molecular Biochemicals). After transfection, the cells were cultured
for 24 h and then washed with PBS and fixed with 4% formaldehyde
for 15 min at room temperature. After a final wash with PBS, the cells were mounted with glycerol. GFP fluorescence signals in transfected cells were detected with an Olympus fluorescence microscope using a
495-nm filter. To assess the nuclear localization response to DNA
damage, cells released from nocodazole inhibition at G2
phase (see below) were treated with 150 µM MMS. Treated
cells harvested at the different time intervals were observed for the
status of nuclear localization under a fluorescent microscope, and the
results were recorded. Nuclear proteins were analyzed by Western
blotting (see below for details).
Cell Cycle Synchronization and Flow Cytometry
HeLa cells were synchronized at the G1/S boundary
using mimosine and at the G2/M boundary using nocodazole as
described previously (36-38). Synchronization in G1 was
achieved by serum starvation to 0.2% fetal bovine serum for 24 h.
The cells were then re-fed in the complete medium supplemented with
mimosine (200 µM, final concentration) for 24 h. To
release cells into S phase, the cells were incubated in mimosine-free
medium. Synchronization in G2 was achieved by culturing
cells with 40 ng/ml nocodazole (final concentration) in complete medium
for 12 h. After removal of drugs, the cells were harvested at
different time points. The cell cycle phase distribution of a cell
population was determined by measuring cell DNA contents in different
phases by flow cytometry. To prepare cell samples for flow cytometry,
cells (1 × 106) were resuspended in complete medium
after trypsinization, pelleted by centrifugation at 500 × g, resuspended in PBS, and then repelleted. The cells
were fixed in 75% cold ethanol and stored at 4 °C. Immediately prior to flow cytometric analysis, the samples were treated with RNase
A for 30 min at room temperature and then stained with propidium iodide
(50 µg/ml, final concentration) overnight. The samples were analyzed
by using a MoFlo FACS (Cytomation, Inc., Fort Collins, CO).
Nuclear Protein Extraction and Western Blotting Analysis
The HeLa cells (2 × 107) treated with MMS (150 µM, final concentration) were harvested by scraping cells
with a rubber policeman and transferred to a 1.5-ml microcentrifuge
tube. The nuclei were first isolated from cells based on the protocol
described by Docherty (39). Briefly, the cells were pelleted, washed,
and resuspended in 400 µl of HEPES buffer (10 mM, pH 7.9)
with 10 mM KCl and 1× protease inhibitor mixture on ice
for 15 min. Then 25 µl of 10% Nonidet P-40 solution was added, the
suspension vortexed vigorously for 15 s, and centrifuged for
30 s at 15,000 × g. To disrupt the nuclear
membrane and extract the nuclear proteins, the pellet was resuspended
in 10 volumes of RIPA buffer (150 mM NaCl, 1% Triton
X-100, 0.5% NaDoC, 0.1% SDS, 50 mM Tris, pH 8.0, 1×
protease inhibitor mixture) at 4 °C for 30 min. After centrifuging
for 20 min at 4 °C, the supernatant was collected and transferred to
a fresh tube. 10 µg of nuclear protein was separated on 10% SDS-PAGE. To visualize uniform loading of nucleic protein extracts taken from different time points, the gel was cut into halves. The
upper part was silver-stained (Bio-Rad), and the proteins on the lower
part of the gel were electrophoretically transferred to a
nitrocellulose membrane for use in Western blotting. The membrane was
blocked with 5% nonfat dry milk in TBST (25 mM Tris, pH
8.0, 150 mM NaCl, 0.5% Tween 20) for 1 h at room
temperature and then probed with anti-FEN-1 polyclonal antibody
(1:4,000) or monoclonal antibody (1:1000) for 2 h at 4 °C.
After washing with TBST, the membrane was incubated with
peroxidase-conjugated goat anti-rabbit antibody (1:5,000). The Western
blotting image was visualized by the Pierce chemiluminescence detection
system (Pierce).
Immunofluorescent and PI Staining and Microscopy
HeLa cells were cultured in glass chamber slides (Fisher) coated
with 0.01% poly-L-lysine (Sigma). Cells were fixed in 4% formaldehyde for 15 min at room temperature and washed with PBS. They
were then incubated with anti-FEN-1 polyclonal antibody (1:50) for
1 h at room temperature. After washing with PBS, cells were incubated with a fluorescein isothiocyanate (FITC)-conjugated anti-rabbit polyclonal antibody (1:50) for 1 h. The cells were also stained with 0.5% propidium iodide (PI) to visualize nuclei. After washing with PBS, they were then mounted in 90% glycerol/PBS. FEN-1 protein signals and nuclei were observed and recorded with an
Olympus fluorescence microscope (Olympus, Japan).
Yeast Manipulation, Growth Curves, and DNA Damage/Repair
Analysis
Six constructs including pDB20 plasmid with no insert and
pDB20-based plasmids with FEN-1 wild type or different NLS mutants were
transformed into two isogenic yeast strains F3C-15B (wild type) and
IC2-1 (the rad27 null mutant), respectively (40). The null
mutant displayed a distinctive phenotype due to defects in the DNA
replication and repair pathways. Transformants were selected using
SD-URA plates. Nine strains were chosen and grown in SD-URA liquid
medium to saturation at 30 °C. Cell density was measured with a
spectrophotometer at 600 nm and diluted in water if necessary.
Observation of Cell Growth--
The concentration of nine
strains was standardized to OD value of 1.5 (~3 × 107 cells per ml). For growth curves, 100 µl of cells of
each standardized strain was grown in 8 ml of SD-URA liquid medium at
both 30 and 37 °C. OD values were then measured at 5, 10, 15, and
22 h. The growth curves were plotted using the average OD values
of triplicate experiments versus culture time. Different
yeast strains were diluted 1, 5, 25, 125, and 625 times and grown on
SD-URA plates at both 30 and 37 °C. One microliter of each dilutant
was plated on the medium as a dot as shown in the figures.
MMS Treatment--
For each density-standardized strain, 500 µl of cells were used for MMS treatment. Cells were centrifuged and
washed with water and then treated with 500 µl of 0.05 or 0.1% MMS
in 0.1 M potassium phosphate buffer, pH 7.0, for 30 min.
The treated cells were washed with 1.5 ml of water three times and
suspended in water in a final volume of 500 µl. The samples were
further diluted 8,000 times for the control (without MMS treatment),
2,000 times for 0.05% MMS treatment, and 1,000 times for 0.1% MMS
treatment. 100 µl of cells were plated on SD-URA and cultured at
30 °C. After 4 days, colonies were observed and counted. Survival
rate was determined based on the ratio of colony counts with MMS to
colony counts without MMS treatment. Survival histograms represent the mean from at least two independent experiments. In addition, the observation of MMS sensitivity of different strains with various dilutions on plates was made on SD-URA with 0.05% MMS. Five dilutions, 1, 5, 25, 125, and 625 times, were made for each sample. One microliter of each diluent was plated as a dot on a SD-URA plate. Samples were
observed and photographed after 5 days of incubation at 30 °C.
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RESULTS |
FEN-1 Nuclease Is Localized into the Nucleus in G1/S
Phase of the Cell Cycle--
To study the nuclear localization of the
human enzyme, cell cycle phases were first determined by flow cytometry
based on relative fluorescence represented on the x axis and
cell number represented on the y axis of the flow cytometric
profiles (Fig. 1A). After
removal of mimosine (an inhibitor of DNA replication forks), cells
entered the S phase and underwent a period of high DNA synthesis (0-6
h after release corresponding to S phase), followed by G2
phase (8-15 h after release). G2 phase-synchronized cells
were obtained by nocodazole inhibition (Fig. 1A). FEN-1 protein signals were detected at 0, 3, 6, 10, and 13 h after
G1 release as well as at a time immediately after
G2 release (Fig. 1B). Both cytoplasmic and
nuclear immunostainings were observed when cells were immediately
released from the G1/S boundary and G2 phase
(Fig. 1B, 0 h and G2). The FEN-1 protein clearly
moved to and localized in nuclei 3 and 6 h after mimosine release,
which corresponds to S phase (Fig. 1B). The fact that FEN-1
protein was localized into the nuclei in S phase is in agreement with the role of FEN-1 nuclease in DNA replication. When cells entered G2 phase, FEN-1 nuclease migrated back to cytoplasm (Fig.
1B, 10 and 13 h). Fig. 1C is the PI
staining to visualize the nuclei in cell cycle phases corresponding to
the ones in Fig. 1B.

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Fig. 1.
Subcellular localization of FEN-1
nuclease during cell cycle phases. A, determination of
cell cycle phases by flow cytometry. HeLa cells were synchronized in
G1/S phase by serum starvation and addition of mimosine and
in G2 phase by addition of nocodazole. They were then
released by incubating in drug-free complete Dulbecco's modified
Eagle's medium. Cells in the control panel were asynchronous. Samples
for flow cytometric analysis were harvested at the time points
indicated, fixed in 75% ethanol, and stained with propidium iodide.
They were then analyzed using a MoFlo FACS. The cell cycle phases were
determined based on relative fluorescence represented on the
x axis and cell number represented on the y axis.
B, immunofluorescent staining and microscopic imaging of
FEN-1 nuclease in different cell cycle phases. HeLa cells were cultured
in glass chambers and then fixed in 4% formaldehyde at different time
points. They were then incubated with anti-FEN-1 polyclonal antibody
and subsequently with the FITC-conjugated anti-rabbit polyclonal
antibody. FEN-1 protein signals were detected with an Olympus
fluorescence microscope. C, PI staining to visualize nuclei
in the cell cycle phases corresponding to the ones in
B.
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Nuclear Localization of FEN-1 Nuclease Is DNA
Damage-inducible--
A critical function of FEN-1 nuclease is its
structure-specific endonuclease activity in an alternative long patch
DNA base excision repair pathway (13, 14). We hypothesized that the nuclear localization of FEN-1 nuclease is inducible upon DNA damage. Cells in G2 were chosen for this experiment because earlier
experiments (Fig. 1) showed that the FEN-1 protein was distributed in
the cytoplasm and nucleus during this stage. Cells were fixed at
several different time points after MMS treatment. Following initial
treatment with MMS, FEN-1 signals were observed in perinuclei after 10 min (Fig. 2, 10 min). FEN-1
was then localized in nuclei after 20 min and thereafter (Fig. 2,
20 and 40 min, and 1.5, 3, and 5 h). To semi-quantitatively determine nuclear localization of FEN-1 protein upon MMS treatment, Western blotting of nuclear proteins isolated from MMS-treated cells was performed. Cells treated with 150 µM MMS were harvested at different time points as
indicated. Nuclei were isolated, and nuclear proteins were solubilized
and separated on a SDS-PAGE gel. The upper part of the gel was
silver-stained to inspect the normalization of the protein sample
loading (Fig. 3A, lower
panel). The FEN-1 peptide represented in the lower part of the gel
was visualized by Western blotting (Fig. 3A, upper panel).
The relative intensities of FEN-1 from different time points in the
Western blotting were normalized to the marked band in the
silver-staining gel and are shown in Fig. 3B. The nuclear FEN-1 level increased ~10-fold soon after MMS treatment (Fig. 3B).

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Fig. 2.
Effect of DNA damage on nuclear localization
of FEN-1 nuclease. HeLa cells were synchronized in G2
by addition of nocodazole and then incubated in medium containing 0.2%
serum and 150 µM MMS for 10, 20, and 40 min and 1.5, 3, and 5 h. Cells without MMS treatment served as a control. Cells
were fixed in formaldehyde and stained with mono- or polyclonal
anti-FEN-1 antibody. They were then incubated with FITC-conjugated
secondary antibody as described in Fig. 1. 0 min, nuclear
FEN-1 intensity in HeLa cells without MMS treatment; 10 min
to 5 hr, nuclear FEN-1 intensity after MMS
treatment for 10 min to 5 h. PI staining was done to visualize the
nuclei of the cells used in this experiment, but the images were not
shown for clarity of the figure.
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Fig. 3.
Semi-quantitative analysis of nuclear FEN-1
protein upon MMS treatment by Western blotting. A,
cells treated with 150 µM MMS were harvested at different
time points as indicated. Nuclei were isolated and the nuclear membrane
was disrupted using detergents as described under "Experimental
Procedures." 10 µg of nuclear protein was separated on 10%
SDS-PAGE. The upper portion of the gel was silver-stained to
visualize the even loading of nuclear protein extracts (the lower
panel in A). The proteins in the lower portion of the
gel were transferred to a nitrocellulose membrane. The level of FEN-1
in nucleus was detected by monoclonal anti-FEN-1 antibody and
FITC-conjugated secondary antibody. Shown is the Western blotting image
of nuclear FEN-1 protein detected by monoclonal antibody after MMS
treatment for a period as indicated (the upper panel in
A). B, quantitative analysis of Western blotting
of FEN-1 protein localized in nucleus after MMS treatment. The
intensities of FEN-1 protein were normalized using the indicated
control protein band in A. The maximal intensity of the
FEN-1 image was taken as 100% within the time points in experiments
and an average was taken from three independent experiments.
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The NLS of FEN-1 Is Not Required for Nuclease Activity nor Its
Interaction with PCNA in Vitro--
The NLS C-terminal 26-amino acid
motif of the FEN-1 nuclease only exists in eukaryotic enzymes but not
in their prokaryotic homologues. There are three clusters of positively
charged amino acid residues in this
region, which may be important for nuclear localization (31) (Fig.
4A). These amino acids are KRK (residues 354-356),
KKK (residues 365-367), and KR (residues 377-378), and all were
converted to alanines in this study to test their role in nuclear
localization. In addition, a truncated version of FEN-1 without the
last 26 amino acids was tested. The four mutant versions and wild type
FEN-1 proteins were overexpressed in bacteria and purified (Fig.
4B). By using purified proteins, flap endonuclease and
exonuclease assays were performed using different DNA substrates as
described under "Experimental Procedures." No significant
difference in enzyme activities was observed between the NLS-truncated
mutant enzyme and wild type (Fig. 5,
A and B). The experiments were repeated independently with the point mutant enzymes (Mkkk,
Mkrk, and Mkr, data not shown). FEN-1/PCNA
interaction experiments were also performed and showed that both wild
type and NLS mutant proteins could bind to PCNA without any observable
defect (Fig. 5C).

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Fig. 4.
Design of mutations and purification of NLS
mutant FEN-1 proteins. A, illustration of three
functional motifs of human flap endonuclease-1 based on previous
studies (1, 4, 31). These motifs are proposed to be responsible for
nuclease activities, PCNA interaction, and nuclear localization. The
amino acids shown above the bar are key residues determined
by our mutagenesis experiments (51). Residues in the nuclease regions
are involved in Mg2+ coordination and are critical for
various activities. The region from residues 337 to 345 is critical for
PCNA interaction (52, 53; G. Frank, J. Qiu, and B. Shen, unpublished
observations). The last 26 amino acid residues enlarged below the
bar represent the motif responsible for nuclear localization. All
26 residues were deleted in mutant Mnlsd. Three clusters of
positively charged amino acid residues labeled with residue numbers
have been converted to alanines. The resulting mutants were named as
Mkrk, Mkkk, and Mkr. B,
purification of wild type human FEN-1 nuclease (lane 2),
Mnlsd (lane 3), Mkrk (lane
4), Mkkk (lane 5), and Mkr
(lane 6). 70 ng of proteins were loaded and were visualized
by Coomassie Brilliant Blue R staining on a 10% SDS gel. Lane
1 is the molecular weight marker.
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Fig. 5.
The NLS mutant FEN-1s possess wild type flap
endo- and exonuclease activity and interact with PCNA.
A, flap endonuclease activity of the wild type and NLS
mutant Mnlsd. Substrate and products of the flap
endonuclease activity were labeled as 34, 21, and
19nt, respectively. Experiments were carried out as
described under "Experimental Procedures." Lane 1, negative control without enzyme; lane 2, wild type FEN-1;
lane 3, NLS-deleted mutant. Approximately 10 ng of proteins
were used in the assay reactions. B, exonuclease activity.
Substrate and products were labeled as 15, 3, 2, and
1nt, respectively. Lane 1, negative control
without enzyme; lane 2, wild type FEN-1; lane 3, NLS-deleted mutant. The same amount of proteins were used as in
A. C, SDS gel image of FEN-1/PCNA interactions.
The interaction assays were carried out as described previously (5).
The E. coli crude extract with His-tagged FEN-1 nuclease was
mixed with the crude extract of non-His-tagged human PCNA. The
mixture was loaded onto a Amersham Pharmacia Biotech nickel chelating
column, washed, and then eluted with 500 mM imidazole. The
elutes were separated on SDS-PAGE and visualized with Coomassie
Brilliant Blue R staining. Lane 1, molecular weight markers;
lane 2, elute from the nickel column loaded with PCNA only;
lane 3, elute from the nickel column loaded with wild type
human FEN-1 only; lane 4, elute from the nickel column
loaded with wild type hFEN-1and hPCNA; and lane 5, elute
from the nickel column loaded with C-terminal truncated mutant FEN-1
protein and hPCNA.
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Deletion or Mutations of the NLS Motif Abolishes the Nuclear
Localization of FEN-1--
The role of the NLS in nuclear targeting
was tested by using wild type and NLS mutant FEN-1 coding regions fused
to GFP. The expression of the FEN-1/GFP fusion protein was under
control of the PCMV lE promoter. Fig.
6 shows that wild type (Fig. 6b) and Mkr (Fig. 6c) human FEN-1
fused with GFP were able to localize into nuclei. However, conversion
of amino acids KRK to alanines or deletion of the whole NLS motif (Fig.
6, e and f) completely abolished nuclear
localization. The mutant Mkkk was still able to drive
inefficiently the fusion protein into the nucleus (Fig. 6d).
From these data, we conclude that the bipartite sequence
KRKXXXXXXXXKKK is an NLS for FEN-1 nuclease.

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Fig. 6.
Mutations in the NLS affect nuclear
localization of FEN-1 in HeLa cells. HeLa cells were transfected
with wild type and mutant FEN-1 fused with GFP in the plasmid vector
pEGFP N-1. Transfected cells were cultured for 24 h and fixed in
4% formaldehyde for 15 min. After washing with PBS, the cells were
mounted with glycerol, and green fluorescence signals were detected
under an Olympus fluorescence microscope. a, cells
transfected with vector pEGFP harboring GFP only. b, cells
transfected with wild type FEN-1in the vector pEGFP. c,
cells transfected with FEN-1 mutant Mkr in the vector
pEGFP. d, cells transfected with FEN-1 mutant
Mkkk in the vector pEGFP. e, cells transfected
with FEN-1 mutant Mkrk in the vector pEGFP. f,
cells transfected with FEN-1 mutant Mnlsd in the vector
pEGFP.
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Biological Deficiency of NLS Mutants in DNA Replication and
Repair--
We performed two experiments to investigate the effects of
NLS mutants of FEN-1 on cellular DNA replication and repair. The human
wild type FEN-1 and three site-directed NLS mutant
FEN-1 genes were used to transform into yeast. We
expressed the human proteins in yeast wild type and rad27
null mutant strains, the latter showing conditional lethality and MMS
sensitivity due to defective RNA primer removal and long patch DNA base
excision repair. Our previous studies demonstrated that functional
FEN-1 can complement a rad27 null (40). All of
the strains were individually transformed with the vector alone (pDB),
and wild type and mutant FEN-1 genes Mkrk,
Mkkk, and Mkr can grow at 30 °C in both wild type and rad27 null backgrounds. However, the growth of a
rad27 null mutant transformed with pDB and
pDB/Mkrk was significantly retarded at 30 °C (Fig.
7A, lanes 6 and 9).
When the duplicated plate was incubated at 37 °C, the
rad27 null strains could survive when they were transformed
with wild type, Mkkk, and Mkr FEN-1 expression
plasmids (Fig. 7B, lanes 5, 7, and 8). Null
mutant cells proliferated much slower when they were transformed with NLS-defective FEN-1s (Fig. 7B, lanes 6 and
9). The growth curves displayed in Fig. 7C showed
quantitatively similar results to Fig. 7B when cells were
grown in liquid medium at 37 °C.

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Fig. 7.
NLS mutant FEN-1s were not able to compensate
temperature sensitivity of yeast null mutant. Plasmids of pDB20
with and without wild type and mutant FEN-1 genes were
transformed into wild type (F3C-15B) and RAD27-disrupted
mutant (IC2-1) S. cerevisiae strains, respectively.
Duplicate plates were incubated at 30 and 37 °C. A,
30 °C; B, 37 °C; and C, cell growth curves
at 37 °C. Lanes 1, Wt + pDB20; 2, Wt
+Mkkk-pDB; 3, Wt + Mkrk-pDB;
4, Wt + Mnlsd-pDB; 5, rad27 + Mkkk-pDB; 6, rad27 + Mkrk-pDB; 7, rad27 + Mkr-pDB; 8, rad27 + FEN1-pDB;
9, rad27 + pDB20. The numbers on the
right sides of the A and B are
dilutions of yeast cells.
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To test the complementation of MMS hypersensitivity in the
rad27 null strain transformed with a human NLS mutant
FEN-1s, we measured the cell survival rates after MMS treatment. Five
dilutions of each strain were plated onto YPD medium containing 0.05 or 0.1% of MMS. Fig. 8A shows
that complementation using Mkrk and pDB constructs in the
null mutant could not reverse the phenotype of MMS hypersensitivity
(Fig. 8, lanes 6 and 9), whereas the other two
mutants, Mkkk and Mkr, were able to reverse the
null yeast phenotypes. The survival of these four different strains at
MMS concentrations of 0.05 and 0.1% is displayed in Fig.
8B, which quantitatively reflects the results of Fig.
8A. Although Mkkk showed a partial defect in the
nuclear localization of the protein in HeLa cells, it reversed the
defective phenotype of the yeast null mutant. This is probably due to
the high level exogenous expression of FEN-1 nuclease and because
partial localization is sufficient to support this particular
biological activity.

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Fig. 8.
MMS sensitivity of S. cerevisiae
cells transformed with FEN-1s with or without
mutations in NLS region. A, samples of five different
dilutions of each strain were plated on SD-URA medium with 0.05% MMS.
For each dot, 1 µl of cells with 1.5 OD unit was added and cultured
for 5 days. B, survival histogram was prepared based on the
average from three independent experiments. Numbers on the
top of A and the bottom of
B (lanes 1-9) represent Wt + pDB20, Wt + Mkkk-pDB, Wt + Mkrk-pDB, Wt + Mnlsd-pDB, rad27 + Mkkk-pDB,
rad27 + Mkrk-pDB, rad27 + Mkr-pDB, rad27 + FEN1-pDB, and rad27 + pDB20, respectively. The numbers on the right
side of the A are dilutions of yeast cells.
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DISCUSSION |
Although biochemical and genetic analysis has provided convincing
evidence for the uniqueness and importance of FEN-1 in DNA replication
and repair (for recent reviews see Refs. 1 and 9 and references
therein), the functional significance of nuclear localization of FEN-1
was unclear. Our studies provide evidence regarding the NLS of FEN-1
and its importance in response to the cell cycle and DNA damage.
A Bipartite Sequence in C Terminus of FEN-1 Nuclease Is a Nuclear
Localization Signal--
Three functional motifs of FEN-1 were
identified in previous reports (1, 31). The putative NLS was proposed
to be located in the C terminus of the protein. NLS sequences only
occur in eukaryotic FEN-1. Our mutagenesis data indicate that removal
of, or mutations in, the C-terminal last 26 amino acids did not affect flap endonuclease or nick-specific exonuclease activities or PCNA interaction (Fig. 4). However, specific mutations abolished the nuclear
localization of the nuclease (Fig. 5). There are three clusters of
positively charged amino acids in this region. Mutation of the KKK
cluster partially diminished the nuclear localization in HeLa cells,
whereas mutation of KRK completely abolished this activity. However,
conversion of KR in the third cluster did not show any effect on
nuclear localization, indicating that these two residues are not
critical. KRK and KKK are separated by eight other amino acids, a
distance similar to most identified bipartite NLSs, e.g. as
in XPG and p53 (19, 22, 41). Previously, Friedberg (28) and then
Boulikas (42) computer-searched and aligned the NLS regions of DNA
repair proteins and found that typical NLS sequences are bipartite. The
bipartite NLS in XPG nuclease efficiently drives the 185-kDa protein
into nuclei in response to UV irradiation and retains the protein on
the repair sites as demonstrated by Park et al. (22). Our
results have shown that the bipartite NLS of FEN-1 nuclease plays a
critical role in localizing this DNA replication/repair nuclease into
nuclei, possibly resembling the transportation mechanism demonstrated in other systems.
Nuclear Localization of FEN-1 Nuclease Is Cell
Cycle-dependent and DNA Damage-inducible--
Although the
size of FEN-1 nuclease (43 kDa) is such that it could diffuse passively
through the nuclear pores, the rate of the diffusion, based on
comparison with ovalbumin, which is of similar size (43), is likely to
be too low to be practicable. Both in vitro biochemical and
in vivo yeast genetic data indicated that the enzyme is
involved in RNA primer removal during lagging strand DNA synthesis of
DNA replication (44) and alternative PCNA-dependent long
patch base excision repair (13, 14). FEN-1, as a riboexonuclease,
removes the last ribonucleotide of RNA primers after RNase H
endonucleotically digests the major portion of the RNA primer during
the Okazaki fragment processing. As a structure-specific flap
endonuclease, it removes the DNA fragment displaced by a DNA polymerase
during DNA base excision repair. Both of these processes may
require a rapid transport of the nuclease from cytoplasm to nucleus in
response to cellular DNA synthesis and damage.
Immunoflourescent staining of the endogenous FEN-1 proteins in
different phases of the synchronized HeLa cells showed different distribution patterns (Fig. 1). The pattern of migration correlated well with DNA synthesis. Cells in G1 phase had little FEN
nuclease localized into nuclei corresponding to the low level of DNA
synthesis, whereas cells in S phase had FEN-1 exclusively localized
into the nucleus. FEN-1 remained in the nuclei until cells entered the
G2 phase (Fig. 1). In addition, upon treatment with the
DNA-damaging reagent MMS, the FEN-1 protein immediately moved into the
nucleus (Fig. 2 and 3). This presumably allowed it to function in DNA repair. The localization of FEN-1 nuclease was also observed in yeast
cells transformed by FEN-1-GFP fusion constructs (data not shown). The
ratio of protein in the nucleus to that in the cytoplasm has been
significantly increased to a similar degree that is observed in
mammalian cells. It is noteworthy that the yeast
Rad27 gene was preferentially expressed at the
G1/S transition and transcription was also damage-inducible
(45, 46).
How signals of DNA damage and DNA synthesis activities in nuclei travel
from the nucleus to the cytoplasm and activate the NLS to initiate the
localization process is largely unknown. However, recent results
indicate that NLS-dependent nuclear import is precisely regulated, and phosphorylation is involved. For example,
phosphorylation of human retinoblastoma protein results in its cell
cycle-dependent nuclear localization (47). For the SV40
antigen, the presence of phosphorylatable sequences in the N-terminal
region of the NLS increases nuclear localization efficiency. Removal of
these residues decreases the nuclear uptake rate of this protein (48, 49). In addition, the transcription factor Stat 1 is phosphorylated by
the Jak family of tyrosine kinases, which results in dimer formation followed by translocation into the nucleus to activate directly target genes (50). The phosphorylation sites, together with
the NLS, constitute phosphorylation-mediated regulatory modules for
nuclear protein localization. The detailed mechanism relating the
signaling of DNA damage to FEN-1 migration into nuclei needs further investigation.
Defects in the NLS of FEN-1 Protein Abolish Specific in Vivo
Biological Functions--
Our biochemical experiments showed that
deletion or alterations of the C-terminal amino acids of FEN-1 did not
affect its in vitro flap endonuclease, nick-specific
exonuclease, or PCNA-interaction activities (Fig. 5). However,
abolishing the nuclear localization of this protein both in mammalian
and yeast cells resulted in in vivo defects of cellular DNA
synthesis activities and repair of DNA damage. The wild type human
enzyme could support the survival of the yeast rad27 null
mutant at 37 °C, whereas the Mkrk mutant cannot (Fig.
7). In the latter case, cells accumulated in the S phase apparently due
to failure of the nuclear localization of FEN-1 proteins. In addition,
expression of wild type FEN-1 enzyme reversed the MMS sensitivity of
the yeast rad27 null mutants, while localization-deficient
NLS mutants had dramatically reduced recovery ability similar to the
null mutants (Fig. 7 and 8). Although Mkkk showed a partial
defect in the nuclear localization of the protein, it reversed the
defective phenotype of the yeast null mutant in a similar degree to
that by Mkr and wild type FEN-1s. This is probably due to
the high level exogenous expression of FEN-1 nuclease and because
partial localization is sufficient to support the biological
activities. All of these data indicate that nuclear localization is a
crucial process for a biochemically active enzyme to perform its
replication and repair functions in nucleus.