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Originally published In Press as doi:10.1074/jbc.M103332200 on June 26, 2001

J. Biol. Chem., Vol. 276, Issue 37, 35093-35102, September 14, 2001
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p53 Modulates the Exonuclease Activity of Werner Syndrome Protein*

Robert M. Brosh Jr.Dagger §, Parimal KarmakarDagger §, Joshua A. SommersDagger , Qin Yang, Xin Wei Wang, Elisa A. Spillare, Curtis C. Harris, and Vilhelm A. BohrDagger ||

From the Dagger  Laboratory of Molecular Gerontology, NIA, National Institutes of Health, Baltimore, Maryland 21224, and  Laboratory of Human Carcinogenesis, NCI, National Institutes of Health, Bethesda, Maryland 21892-4255

Received for publication, April 13, 2001, and in revised form, June 11, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Werner syndrome (WS) is characterized by the early onset of symptoms of premature aging, cancer, and genomic instability. The molecular basis of the defects is not understood but presumably relates to the DNA helicase and exonuclease activities of the protein encoded by the WRN gene that is mutated in the disease. The attenuation of p53-mediated apoptosis in WS cells and reported physical interaction between WRN and the tumor suppressor p53 suggest that p53 and WRN functionally interact in a pathway necessary for the normal cellular response. In this study, we have demonstrated that p53 inhibits the exonuclease activity of the purified full-length recombinant WRN protein. p53 did not have an effect on a truncated amino-terminal WRN fragment that retains exonuclease activity but lacks the physical interaction domain for p53 located in the carboxyl terminus. Two naturally occurring p53 mutants found in human cancer displayed a reduced ability to inhibit WRN exonuclease activity. In cells arrested in S phase with hydroxyurea, WRN exits the nucleolus and colocalizes with p53 in the nucleoplasm. The regulation of WRN function by p53 is likely to play an important role in the maintenance of genomic integrity and prevention of cancer and other clinical symptoms associated with WS.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The tumor suppressor gene p53 plays a critical role in the DNA damage response pathway to activate checkpoint control in mammalian cells. Loss of p53 function results in genomic instability, a key feature of carcinogenesis (reviewed in Ref. 1) (2, 3). Key components of checkpoint control mediated by p53 include arrest of cell cycle progression, inhibition of DNA replication, and activation of DNA repair. A DNA damage signaling pathway leads to elevated p53, which up-regulates transcription of a number of genes including the cyclin-dependent kinase inhibitor p21 (4, 5). In addition to growth arrest, p53 mediates damage-induced apoptosis in certain cell types (6). Induction of apoptosis by p53 is thought to be important for the removal of cells that are unable to repair the damage from the population.

Genomic instability is also prevalent in the hereditary disorder Werner syndrome (WS).1 WS is a premature aging disorder characterized by the early onset of age-related symptoms including atherosclerosis, osteoporosis, cataracts, diabetes, and cancer (7-9). WS cells grown in culture display marked chromosomal instability characterized by elevated rates of chromosome translocations, rearrangements, and deletions. Mutations in the WS gene (WRN) are found in patients exhibiting the clinical symptoms of WS (10). The WRN gene encodes a protein that harbors both DNA helicase (11-13) and exonuclease (14-17) activities. The precise molecular roles of the WRN protein are not known but presumably relate to DNA metabolic pathways that influence genome integrity.

A number of recent findings suggest that p53 and WRN proteins function together to maintain genomic stability: (a) p53-mediated apoptosis is attenuated in WS cells (18), (b) overexpression of WRN results in enhanced p53-dependent transcriptional activity (19), (c) Sp1-mediated transcription of the WRN gene is modulated by p53 (20), and (d) WRN knockout mice display accelerated mortality in a p53-null background (21). In support of a molecular interaction between WRN and p53, two groups have reported a physical interaction between the proteins (18, 19). Despite the strong evidence that WRN and p53 associate with each other, there has been no evidence for a functional biochemical interaction between WRN and p53. The p53-WRN physical interaction suggests that the phenotypes of cancer and genomic instability in WS may involve p53 modulation of WRN catalytic function. To investigate the relationship of p53 and WRN protein, we have analyzed whether the ATPase, helicase, and exonuclease activities of WRN are modulated by p53. Our results demonstrate that p53 exerts a strong inhibitory effect on the exonuclease activity of WRN. The regulation of WRN by p53 is relevant to the important roles of these proteins in the maintenance of genome stability.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Proteins-- Baculovirus constructs for a recombinant hexa-histidine-tagged full-length WRN protein or a truncated version of WRN (amino-terminal 368 amino acids, designated N-WRN) were kindly provided by Dr. Matthew Gray (University of Washington, Seattle, WA). Amplified baculovirus was used to infect Sf9 insect cells for overexpression and purification of WRN protein as described elsewhere (22). A recombinant hexa-histidine-tagged carboxyl-terminal fragment of WRN (residues 940-1432, designated C-WRN) was overexpressed in Escherichia coli and purified as described previously (23). Wild-type and mutant p53 proteins (R273H and R249S) used in WRN catalytic assays were purified as described previously (24). Exonuclease III was purchased from Roche Molecular Biochemicals. DNA polymerase I (Klenow) was from New England Biolabs. The rabbit polyclonal antibody against WRN protein and the mouse monoclonal antibody against p53 were from Novus Biologicals. T4 polynucleotide kinase was obtained from New England Biolabs.

Nucleotides and DNA-- M13mp18 ssDNA was from New England Biolabs. The 28-mer oligonucleotide was purchased from Life Technologies, Inc. with the following sequence: 5'-ATGCTGATGCAAATCCAATCGCAAGACA-3'. Polyacrylamide gel electrophoresis-purified 53- and 73-mer oligonucleotides were purchased from Midland Certified Reagent Company. Yeast tRNA was from Roche Molecular Biochemicals. [3H]ATP was from Amersham Pharmacia Biotech , and [gamma -32P]ATP was from PerkinElmer Life Sciences.

DNA Exonuclease and Helicase Substrates-- The DNA exonuclease substrate consisted of a double-stranded DNA molecule with one blunt end and one recessed 3' end (5' overhang of 20 nucleotides) (see Fig. 1A). To prepare the substrate, single-stranded 53-mer oligonucleotides (7 pmol) were 5'-labeled with [gamma -32P]ATP (60 µCi, 3000 Ci/mmol) and polynucleotide kinase (10 units) using the manufacturer's recommended conditions. The labeled oligomers were mixed with a 2-fold excess of the complementary 73-mer oligonucleotide in 50 mM Tris-HCl (pH 8.0) and 10 mM MgCl2, heated together at 90 °C for 5 min, and then cooled slowly (~3 h) to 25 °C. Annealed double-stranded DNA substrates were separated from unannealed ssDNA oligomers by nondenaturing 12% polyacrylamide gel electrophoresis. Intact double-stranded DNA substrate fragments were recovered using a Qiaex II gel extraction kit (Qiagen) and stored at 4 °C.

The 28-bp M13mp18 partial duplex substrate was constructed with a 28-mer oligonucleotide complementary to positions 3960-3987 in M13mp18. The 28-mer oligonucleotide was labeled at its 5' end as described above and annealed to M13mp18 ssDNA circle. Partial duplex DNA substrates were purified by gel filtration column chromatography using Bio-Gel A-5M resin (Bio-Rad).

Exonuclease Assay-- Exonuclease assay reaction mixtures (10 µl) contained 40 mM Tris (pH 7.4), 5 mM MgCl2, 1 mM dithiothreitol, 0.1 mg/ml BSA, 1 mM ATP, and either WRN full-length recombinant protein (16 nM) or the N-WRN truncated fragment (29 nM). For those reactions containing wild-type p53 or R249S or R273H mutant p53, the concentrations are indicated in the figure legend. The amount of the double-stranded exonuclease substrate in the reaction mixture was ~3 fmol. Reactions were initiated by the addition of WRN protein and incubated at 37 °C for 60 min. Reactions were stopped by the addition of an equal volume of formamide loading buffer (80% formamide, 0.5× Tris-borate EDTA, 0.1% bromphenol blue, and 0.1% xylene cyanol). The digestion products of these reactions were separated on 15% denaturing polyacrylamide gels, visualized using a PhosphorImager (Molecular Dynamics), and quantitated using ImageQuant software (Molecular Dynamics).

Helicase Assay-- Helicase assay reaction mixtures (20 µl) contained 40 mM Tris (pH 7.4), 4 mM MgCl2, 5 mM dithiothreitol, 2 mM ATP, 0.5 mg/ml yeast tRNA, and the indicated concentration of WRN protein. For those reactions containing p53, the concentrations are indicated. The concentration of the 28-bp partial duplex helicase substrate in the reaction mixture was ~2 µM (nucleotide phosphate). Reactions were incubated at 24 °C for 60 min and terminated by the addition of 10 µl of 50 mM EDTA, 40% glycerol, 0.9% SDS, 0.1% bromphenol blue, and 0.1% xylene cyanol. The products of helicase reactions were resolved on 12% nondenaturing polyacrylamide gels. Radiolabeled DNA species in polyacrylamide gels were visualized using a PhosphorImager or film autoradiography and quantitated using ImageQuant software (Molecular Dynamics). The percentage of helicase substrate unwound was calculated by the following formula: % displacement = 100 × P/(S + P). P is the product, and S is the substrate. The values for P and S have been corrected after subtracting the background values in the no enzyme and heat-denatured controls, respectively.

ATPase Assay-- ATPase assay reaction mixtures (30 µl) contained 40 mM Tris (pH 7.4), 4 mM MgCl2, 5 mM dithiothreitol, M13mp18 ssDNA (30 µM nucleotide phosphate), 0.8 mM [3H]ATP (42 cpm/pmol), 138 nM WRN protein (monomer), and the indicated amounts of p53. Reactions were initiated by the addition of WRN protein and incubated at 24 °C. Samples (5 µl) were removed at 2-min intervals and evaluated by thin layer chromatography as described previously (25). Less than 20% of the substrate ATP was consumed in the reaction over the entire time course of the experiment. The kinetic rate constant (kcat) values were expressed as the mean of at least three independent experiments.

DNA Binding Assay-- DNA binding was monitored by gel shift analysis. Solution conditions for DNA binding were essentially the same as those conditions used for the exonuclease assays. Briefly, 3 fmol of the 32P-labeled double-stranded exonuclease substrate were incubated for 10 min on ice with 12.5 ng of wild-type p53, p53-R249S, or p53-R273H proteins in buffer containing 40 mM Tris (pH 7.4), 5 mM MgCl2, 1 mM dithiothreitol, 0.1 mg/ml BSA, and 1 mM ATP. DNA-protein mixtures (10 µl each) were analyzed by native 4% polyacrylamide gel electrophoresis at 4 °C. DNA was visualized using a PhosphorImager and quantitated using ImageQuant software (Molecular Dynamics).

Far Western Blotting-- Far Western blotting analysis was conducted essentially as described previously (26). Briefly, 0.2-1.0 µg of each polypeptide was electrophoresed on 8-16% acrylamide Tris-glycine gels and transferred to polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia Biotech). All subsequent steps were performed at 4 °C. Filters were immersed twice in denaturation buffer (6 M guanidine-HCl in phosphate-buffered saline) for 10 min followed by six 10-min washes in serial dilutions (1:1) of denaturation buffer supplemented with 1 mM dithiothreitol. Filters were blocked in Tris-buffered saline containing 10% powdered milk and 0.3% Tween 20 for 30 min before incubation with p53 (0.5 µg/ml) in Tris-buffered saline supplemented with 0.25% powdered milk, 0.3% Tween 20, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride for 60 min. Filters were washed four times (10 min each) in Tris-buffered saline containing 0.3% Tween 20 and 0.25% powdered milk. The second wash contained 0.0001% glutaraldehyde. Conventional Western analysis was then performed to detect the presence of p53 using mouse monoclonal antibody against p53 (Novus Biologicals; 1:500) as primary antibody. Anti-mouse IgG/horseradish peroxidase conjugate (Vector Laboratories) was used as secondary antibody at a 1:10,000 dilution and detected using ECL (Amersham Pharmacia Biotech) following the manufacturer's instructions.

Indirect Immunolabeling and Confocal Microscopy-- U2OS cells (obtained from American Type Culture Collection) derived from a human osteogenic sarcoma that contains wild-type p53 and p53-null SAOS-2 cells (obtained from American Type Culture Collection) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. SV40-transformed normal human skin cell line GM00637D fibroblasts and WRN-/--transformed human AG11395 fibroblasts were grown in minimum essential medium with 10% fetal bovine serum, 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, 1% vitamin, and 1% amino acids (Life Technologies, Inc.). The cells were grown on coverslips to a density of 105 cells/ml. After 24 h, HU (Sigma) was added to the cells to a final concentration of 10 mM, and the cells were incubated for 5 h. The cells were fixed with 2% freshly prepared paraformaldehye (Sigma) for 10 min at room temperature, followed by two washes with phosphate-buffered saline containing 100 mM glycine (Aldrich). The cells were subsequently permeabilized with methanol at -20 °C for 20 min, followed by three washes with cold phosphate-buffered saline. Finally, the coverslips were incubated with primary antibodies for 16 h at 4 °C. WRN, p53, and nucleolin (C23) proteins were detected immunologically by incubating the coverslips with the appropriate antibodies for 16 h at 4 °C. For simultaneous detection of WRN and p53, rabbit polyclonal antibody against WRN (1:1000; Novus Biologicals) and mouse monoclonal antibody against p53 (1:500; Novus Biologicals) were used. For simultaneous detection of WRN and nucleolin (C23), the mouse monoclonal antibody against C23 (1:200; Santa Cruz Biotechnology) and rabbit polyclonal antibody against WRN (as described above) were used. After washing three times (10 min each) with phosphate-buffered saline containing 0.05% Tween and 0.1% BSA, the coverslips were incubated simultaneously with donkey anti-rabbit Texas Red (1:200; Jackson Laboratories) and donkey anti-mouse CY2 (1:500; Jackson Laboratories) for 1 h at room temperature. After washing three times (10 min each), the coverslips were mounted on Vectashield (Vector Laboratories) and viewed under a laser scan confocal microscope (Zeiss 410) in separate channels (green, 488 nm; red, 568 nm). The images were then overlaid and analyzed with Metamorp imaging system 4.1 (Universal Imaging Corp.). Immunofluorescence experiments were repeated at least three times. Approximately 30 cells were analyzed for each treatment, and representative photographs are shown.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we investigated the hypothesis that WRN protein functionally interacts with p53. It was previously shown that the carboxyl terminus of p53 (18, 19) interacts with the carboxyl terminus of WRN. However, a functional biochemical interaction between the proteins has not been reported. The effect of p53 protein on WRN exonuclease, helicase, and ATPase activities was tested.

Effect of p53 on WRN Exonuclease Activity-- We first tested the effect of p53 on WRN exonuclease activity using a duplex DNA substrate containing a recessed 3' end (Fig. 1A). This substrate is efficiently degraded by WRN protein in a 3' to 5' direction (Fig. 1B). The exonuclease activity of WRN protein (16 nM monomer) was effectively inhibited by p53 at concentrations as low as 12 nM p53 monomer (Fig. 1, B and C). The inhibition of WRN 3' to 5' exonuclease activity was p53 dose-dependent, with maximal inhibition achieved at the highest p53 concentration tested (71 nM monomer), an ~4:1 molar ratio of p53 monomer to WRN monomer (Fig. 1, B and C). From multiple experiments using different preparations of recombinant wild-type WRN protein and p53, we consistently observed a 2-fold or greater increase in labeled oligonucleotides ranging in size from 49-53 nucleotides for those WRN exonuclease reactions conducted in the presence of p53 (71 nM) compared with reactions in which p53 was omitted. In these experiments, the effect of p53 on WRN exonuclease activity resulted in attenuated degradation, yielding a population of products of greater size compared with those obtained from WRN exonuclease reactions lacking p53 (Fig. 1C). Maximal p53 inhibition resulted in an increase in full-length oligonucleotide (53-mer) or oligonucleotides slightly shorter (by 1-5 nucleotides) than the full-length 53-mer, depending on p53 concentration and exonuclease activity of WRN protein preparations. The enrichment of larger-sized products was accompanied by a corresponding decrease in the faster-migrating species (shorter labeled fragments) (Fig. 1, B and C). Quantitative analysis of the products from WRN exonuclease experiments containing only WRN (16 nM monomer) indicated that 29% of the DNA substrate was >= 52-mer (Fig. 1C). In the presence of WRN (16 nM monomer) and increasing p53 concentrations (12, 24, 35, and 71 nM monomer), the percentage of oligonucleotides >=  52-mer increased progressively to 35%, 43%, 53%, and 66%, respectively.



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  		Fig. 1.   Inhibition of WRN exonuclease activity by p53. A, 3'-recessed DNA substrate used for WRN exonuclease studies. B, WRN protein (16 nM monomer) was incubated with the exonuclease substrate in the presence of the indicated concentrations of p53 monomer under the standard exonuclease reaction conditions as described under "Materials and Methods." DNA products were resolved by denaturing polyacrylamide gel electrophoresis. Results of a typical experiment are shown. Similar results were obtained from independent experiments that were repeated at least four times. C, scans of lanes from resolved products of WRN exonuclease reactions conducted in the presence of the indicated concentrations of p53.

p53 preferentially binds to ssDNA ends (90% of total binding) (27). The inhibitory effect of p53 on WRN exonuclease activity may be due to a direct protein interaction with WRN or binding to the termini of the DNA substrate itself, preventing the loading of WRN protein onto the DNA substrate. Alternatively, p53 may prevent the initiation of the WRN exonuclease reaction. In contrast to its effect on WRN exonuclease, p53 was unable to affect the 3' to 5' exonuclease activity of either exonuclease III (Fig. 2A) or DNA polymerase I (Klenow) (Fig. 2B). These data suggest that the ability of p53 to inhibit digestion is specific to WRN exonuclease.


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  		Fig. 2.   Exonuclease activity of bacterial DNA exonucleases is not inhibited by p53. WRN protein (16 nM monomer), exonuclease III (0.1 unit; A), or DNA polymerase I (Klenow; 2 units; B) was incubated with the exonuclease substrate in the presence of p53 (71 nM monomer) as indicated under the standard exonuclease reaction conditions for either 10 min (exonuclease III) or 60 min (WRN protein and Klenow) as described under "Materials and Methods." DNA products were resolved by denaturing polyacrylamide gel electrophoresis.

To address the mechanism of p53 inhibition of WRN exonuclease activity, we measured the effect of p53 on a truncated recombinant WRN protein (residues 1-368) designated N-WRN that lacks the ATPase/helicase domain and the carboxyl terminus but retains the exonuclease motif (Fig. 3A). This N-WRN recombinant fragment is an active exonuclease that degrades with a 3' to 5' directionality (Fig. 3B ) (17, 28). The presence of p53 did not have an effect on the exonuclease activity of the truncated WRN protein (Fig. 3B). These results suggest that p53 inhibits the exonuclease activity of the full-length recombinant WRN protein by a direct protein interaction with a region other than the amino-terminal 368 amino acids of WRN.


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  		Fig. 3.   Exonuclease activity of the truncated N-WRN recombinant fragment is not inhibited by p53. A, schematic of WRN protein and WRN protein fragments used in this study. B, the N-WRN protein fragment (29 nM monomer) was incubated with the exonuclease substrate in the presence of the indicated concentrations of p53 monomer under the standard exonuclease reaction conditions described under "Materials and Methods."

To further address the mapping of the WRN-p53 interaction, far Western analysis was performed. For this, full-length WRN protein, N-WRN (residues 1-368), and C-WRN (residues 940-1432) (Fig. 3A) were immobilized on a PVDF membrane, which was then incubated with purified recombinant p53 protein. The membrane was then washed to remove unbound protein, and the presence of p53 was detected by conventional Western blotting. As controls, the membrane also contained BSA and p53 itself. In addition, a second membrane containing the same proteins was prepared and incubated in buffer alone. The anti-p53 antibody detected a band at the position of the carboxyl-terminal WRN fragment as well as at the position of the full-length WRN protein (Fig. 4). However, the amino-terminal WRN fragment was not detected by this far Western analysis. These results are consistent with a previous report that the p53-binding domain of WRN protein resides within the carboxyl-terminal 419 residues of WRN (19). These physical interaction data, together with our results from the exonuclease studies, suggest (but do not prove) that p53 inhibits the exonuclease activity of the full-length WRN protein by a direct protein interaction with a carboxyl-terminal region that resides after the amino-terminal domain of WRN that harbors the exonuclease activity.


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  		Fig. 4.   p53 binds to the carboxyl-terminal region of WRN, but not to the amino-terminal fragment. Purified recombinant WRN proteins (full-length WRN, N-WRN, and C-WRN), BSA, and p53 (as indicated above the lanes) were subjected to SDS-polyacrylamide gel electrophoresis. The proteins were transferred to PVDV membrane and then incubated with either purified p53 (A) or buffer alone (B). Western blotting using anti-p53 antibody was then used to detect the presence of p53 on each membrane. The positions of the molecular mass standards running parallel are shown on the right.

The ability of p53 to modulate WRN exonuclease activity is the first demonstration of a direct biochemical functional interaction between the proteins. The critical role of p53 in maintaining genome integrity in mammalian cells may be related to its biochemical interaction with the WRN protein. The mutational status of p53 may influence its ability to affect WRN exonuclease function. To explore this issue further, we tested two naturally occurring p53 mutants (R273H and R249S) found in human cancer for their ability to modulate the exonuclease activity of WRN. R273H and R249S are p53 hot spot missense mutations that reside in the core domain for sequence-specific DNA binding (residues102-290) (29-32). It was consistently observed that the 3'-recessed duplex substrate used for exonuclease studies was degraded more extensively by WRN in the presence of either the R273H or R249S p53 mutant proteins compared with wild-type p53 protein (Fig. 5, A and B). Scan analysis of products resolved on denaturing polyacrylamide gels demonstrated that the maximal product peak of the WRN exonuclease reaction was shifted farther from the origin for the R249S and R273H mutant proteins compared with wild-type p53 (Fig. 5B). Quantitative analysis of the products from WRN exonuclease experiments containing only WRN (16 nM monomer) indicated that 24% of the DNA substrate was >= 49-mer (Fig. 5B). In the presence of WRN (16 nM monomer) and wild-type R249 and R273 p53 proteins (24 nM monomer), the percentage of oligonucleotides >=  49-mer was 39%, 25%, and 28%, respectively. Thus, both p53 mutant proteins were less effective as inhibitors of WRN exonuclease activity than wild-type p53. These results suggest that the region of p53 containing the R249S and R273H mutations, which were previously shown to be important in DNA binding (33), plays a role in the modulation of WRN exonuclease activity. To explore the DNA binding status of the p53 proteins, we tested wild-type and mutant p53 proteins for binding to the DNA substrate used for the exonuclease studies by gel shift experiments. Wild-type p53 protein was able to bind the exonuclease DNA substrate as detected by a shift of the free substrate to a specific retarded species on native polyacrylamide gels; however, both the R273H and R249S p53 mutant proteins failed to bind the DNA substrate (Fig. 6). The faint intermediate species in the lane corresponding to the R249S p53 mutant was not reproducibly observed in any of the p53-substrate DNA binding experiments. These data suggest that DNA binding by p53 contributes to the inhibition of WRN exonuclease activity on the 3'-recessed duplex DNA substrate.


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  		Fig. 5.   p53 mutants show a reduced ability to inhibit WRN exonuclease activity. WRN protein (16 nM monomer) was incubated with the exonuclease substrate in the presence of wild-type p53 (WT), R249S p53 (249), or R273H p53 (273) (24 nM monomer) as indicated under the standard exonuclease reaction conditions described under "Materials and Methods." Results of a typical experiment are shown in A. Experiments were repeated at least three times. Scan analysis of WRN exonuclease reactions conducted in the presence of wild-type or mutant (R249S and R273H) p53 proteins is shown in B.


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  		Fig. 6.   Wild-type p53, but not mutant p53, binds the exonuclease substrate. The exonuclease substrate was incubated with wild-type (WT) or mutant (249 and 273) p53 protein (35 nM monomer) as indicated under the standard binding conditions described under "Materials and Methods."

Effect of p53 on WRN Helicase Activity-- It has been demonstrated previously that p53 inhibits the unwinding activity of a number of helicases (24, 35, 36). To determine whether p53 exerts a direct effect on WRN helicase activity, we preincubated WRN helicase (92 nM monomer) with increasing amounts of p53 (0, 47, 94, and 184 nM monomer) before incubation with an M13 28-bp partial duplex DNA substrate. As shown in Fig. 7, p53 did not stimulate or inhibit WRN-catalyzed unwinding of the M13 partial duplex. Moreover, p53 (47-184 nM) did not affect WRN helicase activity on the 28-bp partial duplex substrate with an amount of WRN protein (26 nM monomer) that unwinds only 10% of the duplex DNA substrate (data not shown).


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  		Fig. 7.   p53 does not affect WRN helicase activity on a 28-bp M13 partial duplex substrate. WRN protein (92 nM monomer) was incubated with the M13 partial duplex in the presence of the indicated concentrations of p53 monomer under the standard helicase reaction conditions described under "Materials and Methods." Reaction products were analyzed by nondenaturing gel electrophoresis. Data presented are typical of at least four independent experiments. black-triangle, heat-denatured control.

Effect of p53 on WRN ATPase Activity-- Although the helicase activity of WRN was not affected by p53 on the short 28-bp M13 partial duplex substrate, it was possible that the ATPase activity of the enzyme might be modulated by p53. We have reported that the specific ATPase activity of WRN protein is significantly increased by increasing the length of the DNA effector used as a cofactor in the ATPase reaction (22). These data suggest that WRN protein translocates processively along ssDNA. We have found that the exonuclease activity of WRN protein on a 3'-recessed DNA duplex is strongly stimulated by ATP.2 The inhibitory effect of p53 on WRN exonuclease activity raised the possibility that WRN ATPase activity might also be inhibited by p53. As shown in Table I, kcat values for WRN ATPase activity were not significantly altered by the presence of p53. There was a small reduction (~8%) in the kcat values at the higher p53 concentrations (94 and 188 nM). The effect of p53 on the kinetic parameter Km for ATP or DNA binding by WRN was not determined in this study. These data would suggest that the modulation of WRN exonuclease activity by p53 is not simply due to a decreased efficiency in hydrolyzing ATP by WRN.

                              
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Table I
The effect of p53 on the kcat for ATP hydrolysis catalyzed by WRN
ATPase assay reaction mixtures (30 µl) contained 40 mM Tris (pH 7.4), 4 mM MgCl2, 5 mM dithiothreitol, M13mp18 ssDNA (2 µM nucleotide phosphate), 0.8 mM [3H]ATP (42 cpm/pmol), 138 nM WRN protein, and the indicated concentrations of p53. Reactions were initiated by the addition of WRN protein and incubated at 24 °C. Samples (5 µl) were removed at 2-min intervals and evaluated by thin layer chromatography.

Colocalization of WRN and p53-- Human WRN has been reported to localize to nucleoli as demonstrated by a diffuse staining of the nucleolar chromatin using anti-WRN antibodies (38). Upon treatment of cells with HU, an agent that depletes the cell of its deoxynucleotide triphosphate supply and results in S-phase arrest, WRN changes its localization from the nucleoli to nuclear staining characteristic of replication foci (39). The functional and physical interaction between WRN and p53 suggests that the two proteins may be associated in vivo. The previous observation that WRN is recruited from the nucleoli and repositioned at sites of stalled replication (39) suggested to us that p53 may also be recruited to these sites to modulate WRN function. To directly test this possibility, we examined the localization of WRN and p53 in cells exposed to HU. The results from these studies are shown in Fig. 8. In untreated U2OS cells, p53 was distributed throughout the nucleoplasm, whereas WRN displayed a diffuse nucleolar localization and nucleoplasmic staining to a lesser degree. Specificity of anti-WRN and anti-p53 antibodies was verified by the absence of detectable staining using WRN-/- AG11395 skin fibroblasts and p53-null SAOS-2 cells, respectively (data not shown). Nucleolar localization of WRN was verified by dual labeling with an anti-nucleolin antibody (data not shown). The merged picture demonstrated that nucleolar WRN did not colocalize with p53, which resides primarily outside the nucleoli. After HU treatment, WRN is redistributed to the nucleoplasm. Relocalization of WRN from nucleoli to nucleoplasm was verified by colabeling with anti-nucleolin antibody (data not shown). After treatment with HU, 70-80% of the cells displayed redistribution of WRN from the nucleoli to the nucleoplasm (data not shown). Staining with an anti-p53 antibody demonstrated that the WRN foci coincided with p53 foci (Fig. 8). Confocal analysis of the pixels from WRN and p53 labeling in U2OS cells indicated a 3.6-fold increase in the percentage of WRN protein colocalized with p53 and a 2.2-fold increase in the percentage of p53 colocalized with WRN in response to HU treatment (Table II). Similarly, WRN and p53 were found to colocalize after S-phase arrest in the SV40-transformed skin fibroblast cell line GM00637D (Fig. 8). Confocal analysis of the pixels from WRN and p53 labeling in GM00637D cells indicated a 2.1-fold increase in the percentage of WRN protein colocalized with p53 and a 2.1-fold increase in the percentage of p53 colocalized with WRN in response to HU treatment (Table II). These colocalization patterns provide evidence that WRN is recruited from nucleoli to sites in the nucleoplasm where p53 is also found in cells that have been arrested in S phase.


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  		Fig. 8.   Colocalization of WRN and p53 in the nuclei of human cells arrested in S phase by HU treatment. Human U2OS cells or GM00637D cells were arrested in S phase by HU treatment as described under "Materials and Methods." Cell cycle analysis demonstrated that the percentage of U2OS cells in G2 decreased from 18 + 3% to 6 + 2% after treatment with 10 mM HU (panel 1). After fixation and permeabilization, cells were incubated with anti-p53 (green) and anti-WRN (red) antibodies. After treatment with HU, WRN localizes in nuclear foci that coincide with p53 foci as demonstrated in the overlapped images. The yellow color results from the overlapping of the red and green foci in the merged images. In control cells not treated with HU, WRN is localized largely to the nucleoli, where p53 is not found.

                              
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Table II
Colocalization of WRN and p53 proteins in cells arrested in S phase with HU


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we have provided the first evidence of a direct biochemical functional interaction between the WRN protein and p53. p53 is able to effectively inhibit the 3' to 5' exonuclease activity of WRN. p53 fails to perturb the exonuclease activity of two bacterial exonucleases (exonuclease III and DNA polymerase I), suggesting a specific interaction between WRN and p53. In support of this notion, p53 does not inhibit the exonuclease activity of N-WRN, a recombinant truncated WRN fragment that lacks p53 binding. This finding suggests that a protein interaction between p53 and WRN is important in the mechanism for p53 inhibition of WRN exonuclease activity. The 3' to 5' exonuclease activity of WRN protein is effectively inhibited by stoichiometrically equivalent levels of p53 monomer compared with WRN monomer. These results indicate that the inhibition of WRN exonuclease activity does not require a vast excess of p53 and suggest an important biological mechanism to regulate the degradative capacity of WRN at recessed 3' ends.

Two prevalent p53 mutations (R249S and R273H) in human cancer were tested for their abilities to block WRN exonuclease activity. Both mutants displayed a reduced ability to inhibit WRN exonuclease activity, suggesting that this region may mediate the functional interaction with WRN; however, these p53 mutations reside outside the reported physical interaction domain for WRN, residues 318-393 of p53 (19). The p53 mutant R249S was found to display a partially reduced affinity for its interaction with WRN in vivo, suggesting that other regions of p53 may contribute to WRN binding (18). The R273H p53 mutant protein was reported to interact strongly with WRN in vivo (18); however, this mutant also displayed a reduced effect on WRN exonuclease activity. The R273H mutant was also found to bind the TFIIH particle helicases XPB and XPD similarly to wild-type p53, yet unlike wild-type p53, the R273H mutant did not inhibit the helicase activity of these proteins (24). Our results demonstrate that the R249S and R273H p53 mutant proteins both failed to bind the DNA substrate used for the WRN exonuclease studies, suggesting that an abnormal interaction with DNA might influence the ability of the p53 mutants to inhibit WRN exonuclease activity. However, other factors may contribute to the reduced effect of the p53 mutants on WRN exonuclease activity including the affinity of the p53 for WRN in vitro and the effects of p53 on WRN binding to DNA. The purified R273H mutant protein was previously shown to display a 77% reduction in binding to calf thymus double-stranded DNA cellulose compared with wild-type p53 (40), a loss of DNA binding that was comparable to but not quite as great as that found in our study with the 3' recessed oligonucleotide duplex substrate. In summary, we conclude that modulation of WRN exonuclease activity by p53 is dependent on protein interaction and likely to be influenced by the DNA binding properties of p53.

The inhibition of WRN exonuclease activity by p53 is likely to have biological consequences. The immunofluorescence data presented here indicate that a significant fraction of p53 molecules colocalize with WRN when a replication block is imposed. This finding suggests that WRN and p53 may be associated with the DNA replication complex. p53 is able to inhibit SV40 viral replication (36) and nuclear DNA replication in a transcription-free DNA replication extract from Xenopus eggs (41), suggesting that p53 may bind directly to proteins of the replication complex and interfere with DNA replication. WRN copurifies with a DNA replication complex (42) and may function during replication as suggested by the defect in DNA synthesis in WS cells. Specifically, some WS cells have an extended S phase (43) and a reduced frequency of initiation sites (44, 45). Genomic instability in WS cells, as characterized by extensive deletions and chromosomal rearrangements, may arise due to basic defects in some aspect of replication that involves p53.

The interaction of p53 with WRN protein may be critical to direct S-phase cells into apoptosis. The attenuation of p53-mediated apoptosis in WS cells might possibly be explained by the absence of a p53-WRN direct interaction that could serve as a signal for programmed cell death (18). Previous studies (37, 46-50) demonstrating that protein synthesis is not required for p53-induced apoptosis suggest that p53 directly targets downstream members, such as WRN, in the apoptotic pathway. Additional studies that explore the in vivo associations of WRN and p53 during DNA replication, repair, or recombination should help to elucidate the functional importance of the observed biochemical interactions.

The intrinsic ability of 3' to 5' exonucleases to sequentially remove nucleotides from the 3' terminus of DNA strands and thus produce a substrate receptive to polymerization renders this class of enzymes essential in DNA replication, repair, and recombination. The observation that certain types of DNA lesions uniquely inhibited or blocked WRN exonuclease activity suggests that WRN protein could be involved in sensing (but not removing) these types of DNA damage (28). An alternative hypothesis is that the degradative function of WRN protein should be down-regulated at certain sites of DNA damage or abnormal bases to allow appropriate processing to take place. In this study, we provide the first evidence that the exonuclease activity of WRN may be blocked by a cellular protein. The ability of p53 to inhibit WRN degradation of DNA may provide a regulatory mechanism for DNA surveillance.

p53 regulation of WRN exonuclease activity may be important in a specific pathway of DNA damage processing that involves other proteins. We have recently found that Ku functionally interacts with WRN exonuclease to facilitate digestion of damaged DNA (23, 34). Conversely, p53 may serve to inhibit WRN exonuclease activity on certain damaged DNA substrates. The exonuclease activity of WRN protein may be tightly regulated by the inhibitory effect of p53 as well as by the stimulatory effect of Ku. Ku stimulates WRN exonuclease activity on the same DNA substrate that p53 inhibits (23).2 We have tested WRN exonuclease activity in the presence of both Ku and p53 and found that Ku is still able to stimulate WRN exonuclease activity (data not shown). Thus, the functional interactions of p53 or Ku with WRN may operate in different pathways. Alternatively, both p53 and Ku may function together to regulate WRN exonuclease activity on some other type of DNA substrate. Additional in vitro and in vivo studies are necessary to address these issues. The modulation of WRN exonuclease activity by p53 may play a critical role in the maintenance of genomic stability by preventing deleterious degradation of DNA at inappropriate loci or during specific periods of the cell cycle. For example, after exposure to DNA damage, one cellular response would be to prevent inadvertent WRN nuclease attack at sites of strand breaks that arise as a consequence of DNA damage. p53 inhibition of WRN exonuclease activity would allow the DNA repair machinery to correct the damage without losing genetic information.

Our observation that WRN exonuclease activity is modulated by p53 has significant biological implications. The down-regulation of WRN catalytic activity by p53 may deter spontaneous mutation and genomic instability associated with malignant progression. This does not imply that the absence of WRN protein in WS enables cells to avoid neoplastic transformation. On the contrary, genomic instability and a high incidence of certain cancers (sarcomas) characterize WS. Thus, WRN protein may serve multiple functions in cellular DNA metabolic pathways that are regulated by proteins such as p53, replication protein A (RPA) (13), or Ku (23).

It was recently reported that overexpression of WRN enhances p53-mediated transcription (19). However, a carboxyl-terminal fragment of WRN lacking the helicase and exonuclease domains of WRN but responsible for the physical interaction with p53 does not elevate transcription activity of p53 (19). Blander et al. (19) concluded that binding of p53 to WRN alone is not likely to potentiate the transcription activity of p53. Our results demonstrating that p53 directly modulates the exonuclease activity of WRN would be consistent with the hypothesis that catalytic domains of WRN may be required to activate a p53-mediated pathway in vivo. At this point, the pathway for activation of p53-mediated transcription by WRN protein is not understood, and a role for the catalytic activity of WRN protein remains to be elucidated.

p53 modulation of WRN catalytic function may be an important feature of p53-mediated apoptosis. Defects in this pathway may contribute to the prevalent cancer disposition in WS patients. Spillare et al. (18) demonstrated that p53-mediated apoptosis in WS fibroblasts could be rescued by expression of wild-type WRN protein. It would be insightful to determine whether expression of the carboxyl-terminal domain of WRN responsible for physical interaction with p53 is sufficient for rescue of the attenuated p53-mediated apoptosis in WS-/- cells. Perhaps other domains of WRN are also involved in p53-mediated apoptosis because they are required for enhancement of p53-dependent transcriptional activity (19). Although we did not detect a direct inhibitory effect of p53 on WRN helicase activity using the M13 28-bp partial duplex helicase substrate, it is quite possible that p53 would exert an effect on WRN helicase activity on certain DNA substrates. In fact, we have detected a direct effect of p53 on WRN helicase unwinding of Holliday junctions.3 Thus, p53 may directly affect the helicase activity of WRN on specific structures. The genomic instability and cancer predisposition observed in WS may be a consequence of a defect in p53-mediated apoptosis due to the absence of a p53-WRN functional interaction.

    ACKNOWLEDGEMENTS

We thank Dr. Jan Nehlin and members of the Danish Center for Molecular Gerontology for helpful comments. We thank Dr. Larry Loeb and members of his laboratory for critical reading of the manuscript. We acknowledge Dr. Ian Hickson for useful comments and suggestions. We are grateful to Drs. Elizabeth Mambo and Patricia Opresko for critical reading of the manuscript.

    FOOTNOTES

* 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.

§ Both authors contributed equally to this work.

|| To whom correspondence should be addressed: Laboratory of Molecular Genetics, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Dr., Baltimore, MD 21224. Tel.: 410-558-8562; Fax: 410-558-8157; E-mail:vbohr@nih.gov.

Published, JBC Papers in Press, June 26, 2001, DOI 10.1074/jbc.M103332200

2 R. M. Brosh, Jr., P. Karmakar, J. A. Sommers, Q. Yang, X. W. Wang, E. A. Spillare, C. C. Harris, and V. A. Bohr, unpublished data.

3 Q. Yang, R. Zhang, X. W. Wang, E. Spillare, D. Subramanian, J. D. Griffith, J.-L. Li, I. D. Hickson, J.-C. Shen, L. A. Loeb, R. M. Brosh, Jr., P. Karmakar, V. A. Bohr, and C. C. Harris, submitted for publication.

    ABBREVIATIONS

The abbreviations used are: WS, Werner syndrome; HU, hydroxyurea; ssDNA, single-stranded DNA; bp, base pair(s); BSA, bovine serum albumin; PVDF, polyvinylidene difluoride.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Lane, D. P. (1992) Nature 358, 15-16
2. Bennett, M. R. (1999) Biochem. Pharmacol. 58, 1089-1095
3. El-Deiry, W. S. (1998) Curr. Top. Microbiol. Immunol. 227, 121-137
4. Kastan, M. B., Zhan, Q., el-Deiry, W. S., Carrier, F., Jacks, T., Walsh, W. V., Plunkett, B. S., Vogelstein, B., and Fornace, A. J. J. (1992) Cell 71, 587-597
5. El-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J. M., Lin, D., Mercer, W. E., Kinzler, K. W., and Vogelstein, B. (1993) Cell 75, 817-825
6. Levine, A. J. (1997) Cell 88, 323-331
7. Epstein, C. J., Martin, G. M., Schultz, A. L., and Motulsky, A. G. (1966) Medicine (Baltimore) 45, 177-221
8. Salk, D. (1982) Hum. Genet. 62, 1-5
9. Goto, M., Miller, R. W., Ishikawa, Y., and Sugano, H. (1996) Cancer Epidemiol. Biomark. Prev. 5, 239-246
10. Yu, C. E., Oshima, J., Fu, Y. H., Wijsman, E. M., Hisama, F., Alisch, R., Matthews, S., Nakura, J., Miki, T., Ouais, S., Martin, G. M., Mulligan, J., and Schellenberg, G. D. (1996) Science 272, 258-262
11. Gray, M. D., Shen, J. C., Kamath-Loeb, A. S., Blank, A., Sopher, B. L., Martin, G. M., Oshima, J., and Loeb, L. A. (1997) Nat. Genet. 17, 100-103
12. Shen, J. C., Gray, M. D., Oshima, J., and Loeb, L. A. (1998) Nucleic Acids Res. 26, 2879-2885
13. Brosh, R. M. J., Orren, D. K., Nehlin, J. O., Ravn, P. H., Kenny, M. K., Machwe, A., and Bohr, V. A. (1999) J. Biol. Chem. 274, 18341-18350
14. Huang, S., Li, B., Gray, M. D., Oshima, J., Mian, I. S., and Campisi, J. (1998) Nat. Genet. 20, 114-116
15. Kamath-Loeb, A. S., Shen, J. C., Loeb, L. A., and Fry, M. (1998) J. Biol. Chem. 273, 34145-34150
16. Shen, J. C., Gray, M. D., Oshima, J., Kamath-Loeb, A. S., Fry, M., and Loeb, L. A. (1998) J. Biol. Chem. 273, 34139-34144
17. Huang, S., Beresten, S., Li, B., Oshima, J., Ellis, N. A., and Campisi, J. (2000) Nucleic Acids Res. 28, 2396-2405
18. Spillare, E. A., Robles, A. I., Wang, X. W., Shen, J. C., Yu, C. E., Schellenberg, G. D., and Harris, C. C. (1999) Genes Dev. 13, 1355-1360
19. Blander, G., Kipnis, J., Leal, J. F., Yu, C. E., Schellenberg, G. D., and Oren, M. (1999) J. Biol. Chem. 274, 29463-29469
20. Yamabe, Y., Shimamoto, A., Goto, M., Yokota, J., Sugawara, M., and Furuichi, Y. (1998) Mol. Cell. Biol. 18, 6191-6200
21. Lombard, D. B., Beard, C., Johnson, B., Marciniak, R. A., Dausman, J., Bronson, R., Buhlmann, J. E., Lipman, R., Curry, R., Sharpe, A., Jaenisch, R., and Guarente, L. (1999) Mol. Cell. Biol. 20, 3286-3291
22. Orren, D. K., Brosh, R. M., Jr., Nehlin, J. O., Machwe, A., Gray, M. D., and Bohr, V. A. (1999) Nucleic Acids Res. 27, 3557-3566
23. Cooper, M. P., Machwe, A., Orren, D. K., Brosh, R. M., Ramsden, D., and Bohr, V. A. (2000) Genes Dev. 14, 907-912
24. Wang, X. W., Yeh, H., Schaeffer, L., Roy, R., Moncollin, V., Egly, J. M., Wang, Z., Freidberg, E. C., Evans, M. K., Taffe, B. G., Bohr, V. A., Weeda, G., Hoeijmakers, J. H. J., Forrester, K., and Harris, C. C. (1995) Nat. Genet. 10, 188-195
25. Matson, S. W., and Richardson, C. C. (1983) J. Biol. Chem. 258, 14009-14016
26. Brosh, R. M. J., Li, J. L., Kenny, M. K., Karow, J. K., Cooper, M. P., Kureekattil, R. P., Hickson, I. D., and Bohr, V. A. (2000) J. Biol. Chem. 275, 23500-23508
27. Bakalkin, G., Yakovleva, T., Selivanova, G., Magnusson, K. P., Szekely, L., Kiseleva, E., Klein, G., Terenius, L., and Wiman, K. G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 413-417
28. Machwe, A., Ganunis, R., Bohr, V. A., and Orren, D. K. (2000) Nucleic Acids Res. 28, 2762-2770
29. Bargonetti, J., Manfredi, J. J., Chen, X., Marshak, D. R., and Prives, C. (1993) Genes Dev. 7, 2565-2574
30. Halazonetis, T. D., and Kandil, A. N. (1993) EMBO J. 12, 5057-5064
31. Pavletich, N. P., Chambers, K. A., and Pabo, C. O. (1993) Genes Dev. 7, 2556-2564
32. Wang, Y., Reed, M., Wang, P., Stenger, J. E., Mayr, G., Anderson, M. E., Schwedes, J. F., and Tegtmeyer, P. (1993) Genes Dev. 7, 2575-2586
33. Kern, S. E., Kinzler, K. W., Bruskin, A., Jarosz, D., Friedman, P., Prives, C., and Vogelstein, B. (1991) Science 252, 1708-1711
34. Orren, D. K., Machwe, A., Karmakar, P., Piotrowski, J., Cooper, M. P., and Bohr, V. A. (2001) Nucleic Acids Res. 29, 1926-1934
35. Kienzle, H., Baack, M., and Knippers, R. (1989) Eur. J. Biochem. 184, 181-186
36. Friedman, P. N., Kern, S. E., Vogelstein, B., and Prives, C. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9275-9279
37. Zhou, X., Wang, X. W., Xu, L., Hagiwara, K., Nagashima, M., Wolkowicz, R., Zurer, I., Rotter, V., and Harris, C. C. (1999) Cancer Res. 59, 843-848
38. Marciniak, R. A., Lombard, D. B., Johnson, F. B., and Guarente, L. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 6887-6892
39. Constantinou, A., Tarsounas, M., Karow, J. K., Brosh, R. M. J., Bohr, V. A., Hickson, I. D., and West, S. C. (2000) EMBO Reports 1, 80-84
40. Kern, S. E., Kinzler, K. W., Baker, S. J., Nigro, J. M., Rotter, V., Levine, A. J., Friedman, P., Prives, C., and Vogelstein, B. (1991) Oncogene 6, 131-136
41. Cox, L. S., Hupp, T., Midgley, C. A., and Lane, D. P. (1995) EMBO J. 14, 2099-2105
42. Lebel, M., Spillare, E. A., Harris, C. C., and Leder, P. (1999) J. Biol. Chem. 274, 37795-37799
43. Poot, M., Hoehn, H., Runger, T. M., and Martin, G. M. (1992) Exp. Cell Res. 202, 267-273
44. Takeuchi, F., Hanaoka, F., Goto, M., Akaoka, I., Hori, T., Yamada, M., and Miyamoto, T. (1982) Hum. Genet. 60, 365-368
45. Hanaoka, F., Yamada, M., Takeuchi, F., Goto, M., Miyamoto, T., and Hori, T. (1985) Adv. Exp. Med. Biol. 190, 439-457
46. Wagner, A. J., Kokontis, J. M., and Hay, N. (1994) Genes Dev. 8, 2817-2830
47. Caelles, C., Helmberg, A., and Karin, M. (1994) Nature 370, 220-223
48. Friedlander, P., Haupt, Y., Prives, C., and Oren, M. (1996) Mol. Cell. Biol. 16, 4961-4971
49. Wang, X. W., Vermeulen, W., Coursen, J. D., Gibson, M., Lupold, S. E., Forrester, K., Xu, G., Elmore, L., Yeh, H., Hoeijmakers, J. H., and Harris, C. C. (1996) Genes Dev. 10, 1219-1232
50. Robles, A. I., Wang, X. W., and Harris, C. C. (1999) Oncogene 18, 4681-4688.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


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