Cell Cycle-dependent and DNA Damage-inducible Nuclear Localization of FEN-1 Nuclease Is Consistent with Its Dual Functions in DNA Replication and Repair*

Junzhuan Qiu, Xinwei Li, Geoffrey Frank, and Binghui ShenDagger

From the Department of Cell and Tumor Biology, City of Hope National Medical Center, Duarte, California 91010

Received for publication, August 28, 2000, and in revised form, October 16, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Flap endonuclease-1 (FEN-1), a 43-kDa protein, is a structure-specific and multifunctional nuclease. It plays important roles in RNA primer removal of Okazaki fragments during DNA replication, DNA base excision repair, and maintenance of genome stability. Three functional motifs of the enzyme were proposed to be responsible for its nuclease activities, interaction with proliferating cell nuclear antigen, and nuclear localization. In this study, we demonstrate in HeLa cells that a signal located at the C terminus (the nuclear localization signal (NLS) motif) facilitates nuclear localization of the enzyme during S phase of the cell cycle and in response to DNA damage. Truncation of the NLS motif prevents migration of the protein from the cytoplasm to the nucleus, while having no effect on the nuclease activities and its proliferating cell nuclear antigen interaction capability. Site-directed mutagenesis further revealed that a mutation of the KRK cluster to three alanine residues completely blocked the localization of FEN-1 into the nucleus, whereas mutagenesis of the KKK cluster led to a partial defect of nuclear localization in HeLa cells without observable phenotype in yeast. Therefore, the KRKXXXXXXXXKKK motif may be a bipartite NLS driving the protein into nuclei. Yeast RAD27Delta cells transformed with human mutant Mkrk survived poorly upon methyl methanesulfonate treatment or when they were incubated at an elevated temperature.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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). [alpha -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.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

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.

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.

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.

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.

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.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 delta  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.


    ACKNOWLEDGEMENT

We thank Christina Khadarian, a summer student from California State Polytechnic University, Pomona, for constructing the plasmid pBSK harboring the NLS-truncated FEN-1 cDNA. We are grateful to Drs. Paul Salvaterra and R. J. Lin at the Beckman Research Institute for their kindness and generosity in letting us use their fluorescence microscopes during the course of this study and to David Sadava, Adam Bailis, Qing Chai, and April Armendariz for their critical reading of the manuscript.


    FOOTNOTES

* This work was supported by National Institutes of Health Grant CA73764 (to B. H. S.).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.

Dagger To whom correspondence should be addressed. Tel.: 626-301-8879; Fax: 626-301-8972; E-mail: bshen@coh.org.

Published, JBC Papers in Press, October 25, 2000, DOI 10.1074/jbc.M007825200


    ABBREVIATIONS

The abbreviations used are: FEN-1, flap endonuclease-1; GFP, green fluorescence protein, MMS, methyl methanesulfonate; NLS, nuclear localization signal; PCR, polymerase chain reaction; PCNA, proliferating cell nuclear antigen; XPG, xeroderma pigmentosum group G; PI, propidium iodide; FITC, fluorescein isothiocyanate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; Wt, wild type.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Lieber, M. R. (1996) BioEssays 19, 233-240
2. Turchi, J. J., Huang, L., Murante, R. S., Kim, Y., and Bambara, R. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9803-9807[Abstract/Free Full Text]
3. Murante, R. S., Rust, L., and Bambara, R. A. (1995) J. Biol. Chem. 270, 30377-30383[Abstract/Free Full Text]
4. Shen, B., Qiu, J., Hosfield, D. J., and Tainer, J. A. (1998) Trends Biochem. Sci. 23, 171-173[CrossRef][Medline] [Order article via Infotrieve]
5. Li, X., Li, J., Harrington, J. J., Lieber, M. R., and Burgers, P. M. J. (1995) J. Biol. Chem. 270, 22109-22112[Abstract/Free Full Text]
6. Gary, R., Park, M. S., Nolan, J. P., Cornelius, H. L., Kozyreva, O. G., Tran, H. T., Lobachev, K. S., Resnick, M. A., and Gordenin, D. A. (1999) Mol. Cell. Biol. 19, 5373-5382[Abstract/Free Full Text]
7. Tom, S., Henricksen, L. A., and Bambara, R. A. (2000) J. Biol. Chem. 275, 10498-10505[Abstract/Free Full Text]
8. Gomes, X. V., and Burgers, P. M. J. (2000) EMBO J. 19, 3811-3821[Abstract/Free Full Text]
9. Bambara, R. A., Murante, R. S., and Henricksen, L. A. (1997) J. Biol. Chem. 272, 4647-4650[Free Full Text]
10. Johnson, R. E., Kovvali, G. K., Prakash, L., and Prakash, S. (1995) Science 269, 238-240[Medline] [Order article via Infotrieve]
11. Kim, K., Biade, S., and Matsumoto, Y. (1998) J. Biol. Chem. 273, 8842-8848[Abstract/Free Full Text]
12. Klungland, A., and Lindahl, T. (1997) EMBO J. 16, 3341-3348[Abstract/Free Full Text]
13. Waga, S., and Stillman, B. (1994) Nature 369, 207-212[CrossRef][Medline] [Order article via Infotrieve]
14. Waga, S., Bauer, G., and Stillman, B. (1994) J. Biol. Chem. 269, 10923-10934[Abstract/Free Full Text]
15. Tishkoff, D. X., Filosi, N., Gaida, G. M., and Kolodner, R. D. (1997) Cell 88, 263-263
16. Freudenreich, C. H., Kantrow, S. M., and Zakian, V. A. (1998) Science 279, 853-856[Abstract/Free Full Text]
17. Spiro, C., Pelletier, R., Rolfsmeier, M. L., Dixon, M. J., Lahue, R. S., Gupta, G., Park, M. S., Chen, X., Mariappan, S. V., and McMurray, C. T. (1999) Mol. Cell 4, 1079-1085[Medline] [Order article via Infotrieve]
18. Gorlich, D., and Mattaj, I. W. (1996) Science 271, 1513-1518[Abstract]
19. Jans, D. A., and Hubner, S. (1996) Physiol. Rev. 76, 651-685[Abstract/Free Full Text]
20. Baeuerle, P. A., and Baltimore, D. (1988) Cell 53, 211-217[Medline] [Order article via Infotrieve]
21. Baeuerle, P. A., and Baltimore, D. (1988) Science 242, 540-546[Medline] [Order article via Infotrieve]
22. Park, M. S., Knauf, J. A., Pendergrass, S. H., Coulon, C. H., Strniste, G. F., Marrone, B. L., and MacInnes, M. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8368-8373[Abstract/Free Full Text]
23. Scully, R., Chen, J., Ochs, R. L., Keegan, K., Hoekstra, M., Feunteun, J., and Livingston, D. M. (1997) Cell 90, 425-435[Medline] [Order article via Infotrieve]
24. Shaulsky, G., Goldfinger, N., Peled, A., and Rotter, V. (1991) Cell Growth Differ. 2, 661-667[Abstract]
25. Sheldon, L. A., and Kingston, R. E. (1993) Genes Dev. 7, 1549-1558[Abstract]
26. Whiteside, S. T., and Goodbourn, S. (1993) J. Cell Sci. 104, 949-955[Free Full Text]
27. Dingwall, C., and Laskey, R. A. (1991) Trends Biochem. Sci. 16, 478-481[CrossRef][Medline] [Order article via Infotrieve]
28. Friedberg, E. C. (1992) Trends Biochem. Sci. 17, 347[CrossRef][Medline] [Order article via Infotrieve]
29. Knauf, J. A., Pendergrass, S. H., Marrone, B. L., Strniste, G. F., MacInnes, M. A., and Park, M. S. (1996) Mutat. Res. 363, 67-75[Medline] [Order article via Infotrieve]
30. Montecucco, A., Rossi, R., Levin, D. S., Gary, R., Park, M. S., Motycka, T. A., Ciarrocchi, G., Villa, A., Biamonti, G., and Tomkinson, A. E. (1998) EMBO J. 17, 3786-3795[Abstract/Free Full Text]
31. Harrington, J. J., and Lieber, M. R. (1994) Genes Dev. 8, 1344-1355[Abstract]
32. Sherman, F., Fink, G. R., and Hicks, J. B. (1986) Methods in Yeast Genetics , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
33. Nolan, J. P., Shen, B., Park, M. S., and Sklar, L A. (1996) Biochemistry 35, 11668-11676[CrossRef][Medline] [Order article via Infotrieve]
34. Shen, B., Nolan, J. P., Sklar, L. A., and Park, M. S. (1996) J. Biol. Chem. 271, 9173-9176[Abstract/Free Full Text]
35. Fien, K., and Stillman, B. (1992) Mol. Cell. Biol. 12, 155-162[Abstract]
36. Lee, S. E., Mitchell, R. A., Cheng, A., and Hendrickson, E. A. (1997) Mol. Cell. Biol. 17, 1425-1433[Abstract]
37. Petersen, L. N., Orren, D. K., and Bohr, V. A. (1995) Mol. Cell. Biol. 15, 3731-3737[Abstract]
38. Taylor, R. M., Moore, D. J., Whitehouse, J., Johnson, P., and Caldecott, K. W. (2000) Mol. Cell. Biol. 20, 735-740[Abstract/Free Full Text]
39. Allan, J. (1996) in Gene Transcription: DNA Binding Proteins (Docherty, K., ed) , pp. 140-141, John Wiley & Sons, Inc., New York
40. Frank, G., Qiu, J., Somsouk, M., Weng, Y., Somsouk, L., Nolan, J. P., and Shen, B. (1998) J. Biol. Chem. 273, 33064-33072[Abstract/Free Full Text]
41. Liang, S. H., and Clarke, M. F. (1999) J. Biol. Chem. 274, 32699-32703[Abstract/Free Full Text]
42. Boulikas, T. (1997) Anticancer Res. 17, 843-864[Medline] [Order article via Infotrieve]
43. Featherstone, C., Darby, M. K., and Gerace, L. (1988) J. Cell Biol. 107, 1289-1297[Abstract]
44. Qiu, J., Qian, Y., Frank, P., Wintersberger, U., and Shen, B. (1999) Mol. Cell. Biol. 19, 8361-8371[Abstract/Free Full Text]
45. Murray, J. M., Tavassoli, M., Al-Harithy, R., Sheldrick, K. S., Lehmann, A. R., Carr, A. R., and Watts, F. Z. (1994) Mol. Cell. Biol. 14, 4878-4888[Abstract]
46. Reagan, M. S., Pittenberger, C., Siede, W., and Friedberg, E. C. (1995) J. Bacteriol. 177, 364-371[Abstract]
47. Templeton, D. J., Park, S. H., Lanier, L., and Weinberg, R. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3033-3037[Abstract]
48. McVey, D., Brizuela, L., Mohr, I., Marshak, D. R., Gluzman, Y., and Beach, D. (1989) Nature 341, 503-507[CrossRef][Medline] [Order article via Infotrieve]
49. Rihs, H. P., Jans, D. A., Fan, H., and Peters, R. (1991) EMBO J. 10, 633-639[Abstract]
50. Yoneda, Y. (1997) J. Biochem. (Tokyo) 121, 811-817[Abstract]
51. Shen, B., Nolan, J. P., Sklar, L. A., and Park, M. S. (1997) Nucleic Acids Res. 25, 3332-3338[Abstract/Free Full Text]
52. Jonsson, Z. O., Hindges, R., and Hubscher, U. (1998) EMBO J. 17, 2412-2425[Abstract/Free Full Text]
53. Warbrick, E., Lane, D. P., Glover, D. M., and Cox, L. S. (1997) Oncogene 14, 2313-2321[CrossRef][Medline] [Order article via Infotrieve]


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