The Human dUTPase Gene Encodes both Nuclear and Mitochondrial Isoforms
DIFFERENTIAL EXPRESSION OF THE ISOFORMS AND CHARACTERIZATION OF A cDNA ENCODING THE MITOCHONDRIAL SPECIES*

(Received for publication, March 4, 1997, and in revised form, May 1, 1997)

Robert D. Ladner Dagger and Salvatore J. Caradonna

From the Department of Molecular Biology, The University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, Stratford, New Jersey 08084

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

We have previously identified distinct nuclear and mitochondrial isoforms of dUTPase in human cells, reporting the cDNA sequence of the nuclear isoform (DUT-N). We now report a cDNA corresponding to the mitochondrial isoform (DUT-M). The DUT-M cDNA contains an 252-amino acid open reading frame, encoding a protein with a predicted Mr of 26,704. The amino-terminal region of the protein contains an arginine-rich, 69-residue mitochondrial targeting presequence that is absent in the mature protein. In vitro transcription and translation of the DUT-M cDNA results in the production of a precursor protein with an apparent molecular mass of 31 kDa as judged by SDS-polyacrylamide gel electrophoresis. The DUT-M precursor is enzymatically active and immunoreacts with a dUTPase-specific monoclonal antibody. Mitochondrial import and processing studies demonstrate that the DUT-M precursor is processed into a 23-kDa protein and imported into mitochondria in vitro. Isoelectric focusing experiments demonstrate that the DUT-N has a pI of 6.0, while the processed form of DUT-M has a more basic pI of 8.1, measurements that are in agreement with predicted values. Studies aimed at understanding the expression of these isoforms were performed utilizing quiescent and replicating 34Lu human lung fibroblasts as a model cell culture system. Northern blot analysis, employing an isoform-specific probe, demonstrates that DUT-N and DUT-M are encoded by two distinct mRNA species of 1.1 and 1.4 kilobases, respectively. Western and Northern blot analysis reveal that DUT-M protein and mRNA are expressed in a constitutive fashion, independent of cell cycle phase or proliferation status. In contrast, DUT-N protein and mRNA levels are tightly regulated to coincide with nuclear DNA replication status. Because DUT-N and DUT-M have identical amino acid and cDNA sequences in their overlapping regions, we set out to determine if they were encoded by the same gene. The 5' region of the gene encoding dUTPase was isolated and characterized by a combination of Southern hybridization and DNA sequencing. These analyses demonstrate that the dUTPase isoforms are encoded by the same gene with isoform-specific transcripts arising through the use of alternative 5' exons. This finding represents the first example in humans of alternative 5' exon usage to generate differentially expressed nuclear and mitochondrial specific protein isoforms.


INTRODUCTION

dUTPase (EC 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, simultaneously removing dUTP from the DNA biosynthetic pathway and providing substrate (dUMP) for the de novo synthesis of thymidylate (1). The importance of the dUTPase function in prokaryotic, eukaryotic, and viral systems has been firmly established in recent years. dUTPase is essential for viability in Escherichia coli and Saccharomyces cerevisiae and was shown to be required for efficient DNA replication in several viral systems (2-5). The importance of the dUTPase function in the human system has been further distinguished by its importance in anti-thymidylate chemotherapy.

Thymidylate metabolism has been an important target for the development of widely utilized chemotherapeutic agents such as 5-fluorouracil, fluorodeoxyuridine, and ZD1694 that are used in the treatment of breast and gastrointestinal tumors. The enzymatic target of these compounds, thymidylate synthase (TS),1 generates dTMP from dUMP and a folate cofactor. Investigations by several laboratories suggest that the basis of cytotoxicity caused by inhibition of de novo thymidylate metabolism may be the accumulation of excessive dUTP pools (6-9). TS inhibition induces a dramatic elevation of dUTP pools resulting in chronic dUMP misincorporation into DNA during replication and repair, leading to DNA fragmentation and cell death (7). Work published by Curtin et al. (7) demonstrates a significant correlation between intracellular dUTP levels and the magnitude of DNA damage resulting from TS inhibition. dUTPase (the major regulator of dUTP pools in humans) plays a protective role by limiting dUTP accumulation in the cell and countering the cytotoxic effect of TS inhibition. Recently, direct evidence supporting this role was demonstrated. Overexpression of the E. coli dUTPase in HT29 human colorectal tumor cells resulted in the induction of resistance to the TS inhibitor fluorodeoxyuridine (10). These studies provide substantial evidence suggesting that levels of the dUTPase enzyme may be a critical factor in determining fluorodeoxyuridine toxicity in certain cancer types. The essential nature of dUTPase during DNA replication and its role in anti-thymidylate chemotherapy has led many authors to speculate on the use of human dUTPase as a target for future chemotherapeutic design (6, 11, 12). Indeed, the recent determination of the human dUTPase crystal structure represents a clear step toward the development of structure-based dUTPase inhibitors (13).

In recent years, there have been major advances in our understanding of the protein biochemistry of human dUTPase. Several laboratories have described the cloning of dUTPase cDNAs (11, 12, 14), and the crystal structure of a recombinant form of human dUTPase has recently been solved (13). The resulting structural data demonstrate that human dUTPase exists as a homotrimer with active site residues contributed by adjacent subunits (13). Our laboratory has recently discovered that distinct mitochondrial (DUT-M) and nuclear (DUT-N) isoforms of this enzyme exist in human cells (11). The isoforms are separable by SDS-PAGE, with DUT-N having an apparent molecular mass of 22 kDa and the processed form of DUT-M having an apparent molecular mass of 23 kDa. To determine the structural differences between these isoforms, a combination of NH2-terminal protein sequencing and mass spectrometry was utilized to characterize each form in detail, demonstrating that the two isoforms are largely identical, differing only in a short region of their amino termini. Analysis of dUTPase phosphorylation demonstrated that DUT-N is serine-phosphorylated at a consensus cyclin-dependent protein kinase phosphorylation site (Ser11 of DUT-N), suggesting a link to cell cycle regulation (15). While DUT-M contains the identical site, it does not undergo phosphorylation in HeLa cells. Site-directed mutagenesis analysis demonstrates that serine phosphorylation of DUT-N does not regulate enzymatic activity, and the biological significance of DUT-N phosphorylation remains unclear (15). Kinetic analysis of each isoform demonstrates that they retain identical affinities for dUTP despite their structural differences.

Our current knowledge of dUTPase expression in higher eukaryotes is limited. Studies of dUTPase in plants suggest that expression is linked to the rate of proliferation, as demonstrated by a close association of dUTPase activity and replicative vegetative and floral meristem regions (16, 17). Recently, the dUTPase gene was isolated from the human fungal pathogen, Candida albicans, the expression of which is governed in part by an MluI cell cycle box element located in the promoter region (18). This element was shown to be critical for the proliferation-dependent transcriptional regulation of the dUTPase gene. In the human system, studies utilizing peripheral blood lymphocytes suggest that dUTPase expression is also associated with proliferative status (14). However, this previous study is unclear with respect to the expression of distinct nuclear and mitochondrial isoforms. The present investigation was undertaken to extend our previous work characterizing human dUTPase isoforms and elucidate the mechanisms governing isoform expression.


EXPERIMENTAL PROCEDURES

Cell Culture and Metabolic Labeling

34Lu human lung fibroblasts (CRL 1491) were obtained from the American Type Culture Collection and maintained in Eagle's minimal essential medium supplemented with 10% fetal calf serum purchased from Life Technologies, Inc. Cells were synchronized by incubation in serum-free minimal essential medium for 72 h. Cells were then released from G0 by the addition of minimal essential medium containing 10% fetal bovine serum. Progression through the cell cycle was monitored in serum-stimulated and -starved cells by [3H]thymidine incorporation. At 3- or 6-h intervals after serum stimulation/starvation, cells were labeled for 20 min with 10 µl of [3H]thymidine (1 mCi/ml). The remainder of the procedures were performed as described previously (19).

Isolation and Sequencing of DUT-M cDNA Clones

A lambda gt10 cDNA library was constructed from mRNA derived from serum-starved 34Lu human lung fibroblasts. Total RNA was isolated using the reagents and protocols of TRIZOL reagent (Life Technologies, Inc.). Poly(A)+ RNA was purified with the Poly(A)Ttract mRNA isolation system (Promega). cDNA construction was performed using the Superscript Choice system for cDNA synthesis (Life Technologies). The library was screened using a 935-bp SphI/EcoRI fragment of the DUT-N cDNA cloned by this laboratory (11). This DNA fragment contains sequence common to both the DUT-N and DUT-M isoforms. The library screening protocol was based on standard procedures as described by Sambrook et al. (20). Several cDNA isolates were subcloned into the EcoRI site of pGEM-3Z (Promega). These clones were sequenced using the Sequenase dideoxy chain termination kit (U.S. Biochemical Corp.) according to the manufacturer's recommendations. A series of subclones were generated using internal restriction sites to facilitate sequencing of the cDNA isolates in their entirety. The sequences were determined from both strands.

Isolation of the Human dUTPase Gene

An EMBL-3 human leukocyte genomic library (CLONTECH) was screened with the DUT-N cDNA (11). The library screening protocol was based on standard procedures as described by Sambrook et al. (20). Several clones were isolated, and the three largest (9-15 kb) were further characterized by a combination of restriction digestion, Southern hybridization, and sequence analysis. An oligonucleotide probe (5'-GGTGTCTCTTCAGAGCAGG-3') corresponding to the extreme 5' end of the DUT-N cDNA was synthesized and used to identify a 2.7-kb EcoRI/BamHI genomic fragment. This fragment was subsequently sequenced using a series of nested sequencing primers designed from newly obtained sequence information.

In Vitro Transcription/Translation of DUT-N and DUT-M

Coupled in vitro transcription and translation was performed using the TNT system purchased from Promega. The open reading frames of both DUT-M and DUT-N were cloned into pGEM-3Z in the T7 and SP6 orientations, respectively. The reaction mixture (50 µl) contained 25 µl of rabbit reticulocyte lysate; 2 µl of TNT reaction buffer; 20 µM concentration of each amino acid minus methionine; 45 µCi of [35S]methionine (Amersham, SJ235, 15 mCi/ml, >1000 Ci/mmol); 2 units of either SP6 or T7 RNA polymerase; and 2 µg of plasmid (pGEM-3Z) containing either DUT-N or DUT-M. Reactions were incubated at 30 °C for 90 min.

Cellular Fractionation: Isolation of Functionally Intact Mitochondria from HeLa Cells for Mitochondrial Import Studies

Mitochondria were isolated by the procedure of Rickwood et al. (21) using differential centrifugation after cells were disrupted by homogenization. For complete details of this procedure, see Caradonna et al. (23). The purified mitochondria were washed twice in MESH buffer (20 mM Hepes-NaOH, pH 7.4; 220 mM mannitol; 70 mM sucrose; and 0.1 mM EDTA) and used directly for import studies.

In Vitro Mitochondrial Import of DUT-M

Mitochondrial import of in vitro expressed proteins was performed as described previously (22). Briefly, 25 µl of radiolabeled translation reaction was mixed with 25 µl of the prepared mitochondria (at 5 mg/ml total protein). Reactions were allowed to proceed for the indicated times at 30 °C and then chilled on ice. The sample was either disrupted by sonication followed by dUTPase protein immunoprecipitation or further processed as described next. The mitochondria were pelleted by centrifugation and washed twice in 500 µl of MESH buffer. The mitochondria were subsequently resuspended in 20 µl of MESH buffer and incubated with 20 µg/ml trypsin (L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated and affinity-purified; Promega) for 30 min on ice. The mitochondria were washed twice more with 500 µl each of MESH buffer and sonicated, and dUTPase protein was immunoprecipitated. dUTPase protein was subsequently fractionated by 15% PAGE, and the gel was dried and exposed to x-ray film to visualize the radiolabeled protein.

Antibodies, Immunoprecipitation, SDS-PAGE, Two-dimensional PAGE, Western Blot Analysis, and Enzyme Assays

dUTPase-specific monoclonal antibodies (mAb 415) were generated and prepared as described previously (23). For immunoprecipitation, 5 µl of mAb (1 mg/ml) was used. Immunoprecipitation was performed as described previously (23). dUTPase-specific polyclonal antibodies were raised against recombinant DUT-N protein (expressed in the baculovirus system) as described previously (11). The polyclonal antibodies were purified by dUTPase protein affinity chromatography and used for immunoblot analysis at a dilution of 1:1000. Enzyme assays were performed as described previously (10). Two-dimensional PAGE was performed using a Mini-PROTEAN II cell obtained from Bio-Rad. Procedures were performed according to the manufacturer's recommendations and as described in Strahler et al. (14). Western blot analysis was performed as described previously (11).

Northern Hybridization Analysis

Total RNA was isolated from 34Lu cells using the Trizol Reagent method (Life Technologies), and poly(A)+ RNA was isolated with the Poly(A)Ttract mRNA isolation system (Promega) using the protocols supplied by the manufacturer. In each case, 1 µg of mRNA was electrophoresed in a 1.5% agarose formaldehyde gel and transferred to nitrocellulose. Blots were probed with 32P-labeled random-primed cDNA probe (SphI/EcoRI DUT-N fragment or GAPDH cDNA) or with a DUT-M-specific probe (290-bp AluI/EcoRI fragment). Blots probed with random-primed cDNAs were hybridized at 42 °C overnight in 50% formamide, 5 × SSPE (1 × SSPE: 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA (pH 7.7)), 5 × Denhardt's solution, 1% SDS, 100 µg/ml denatured salmon sperm DNA. The filters were washed twice at room temperature in 2 × SSC, 0.1% SDS (5 min); twice in 0.2 × SSC, 0.1% SDS at 50 °C (15 min); and twice in 0.2 × SSC at 65 °C (15 min). The blots were subsequently exposed to Kodak X-Omat film. Blots were stripped by boiling for 10 min in 0.5% SDS prior to reprobing.


RESULTS

Isolation of the cDNA Encoding the Mitochondrial Isoform of Human dUTPase

Previous studies by this laboratory have demonstrated that DUT-N and DUT-M proteins are nearly identical, differing only in a short region of their amino termini (11). Based on this evidence, we speculated that their respective mRNAs would also be identical in their overlapping regions. We therefore utilized the 3' end of the DUT-N cDNA (SphI/EcoRI, 935-bp restriction fragment) to screen for a cDNA isolate corresponding to the DUT-M isoform. This restriction fragment removes the 5' end of the DUT-N cDNA coding for DUT-N-specific amino acids, leaving the 3' end containing sequence shared between the isoforms. A cDNA library was constructed from mRNA derived from 34Lu human lung fibroblasts that were serum-starved. Quiescent 34Lu cells were shown to express only the mitochondrial isoform, thus providing a greater likelihood of identifying a DUT-M-specific cDNA (described hereafter).

Screening the 34Lu library resulted in the identification of 10 positive clones, four of which were chosen for plaque purification and subsequent characterization. The isolates were subcloned into the EcoRI site of pGEM-3Z, and subsequent sequence analysis indicated that all four isolates encode the DUT-M isoform based on comparison of the deduced amino acid sequence with the experimentally determined protein sequence of DUT-M (11). The nucleotide sequence and open reading frame of the largest DUT-M cDNA (960 bp) is presented in Fig. 1. The DUT-M open reading frame (starting at nucleotide 63) corresponds to a 252-amino acid polypeptide with a predicted molecular weight of 26,704. The first 342 nucleotides of the DUT-M cDNA are unique. Nucleotides 343-960 are identical to residues 46-664 of the DUT-N cDNA previously reported by this lab (Fig. 2A). The deduced amino acid sequence indicates that the DUT-M protein possesses an arginine-rich, 69-amino acid leader sequence, which is removed in the mature protein. Amino acid residues 1-93 are unique to DUT-M, while residues 94-252 are common to both nuclear and mitochondrial forms (Fig. 2B). The cDNA does not contain a consensus polyadenylation signal and appears to be incomplete at the 3' end as evidenced by a lack of a poly(A) tail. During cDNA library construction, random hexamers were used to prime during first strand cDNA synthesis instead of oligo-dT; this may account for the lack of a complete 3' end.


Fig. 1. Nucleotide and amino acid sequence of the mitochondrial isoform of human dUTPase, DUT-M. A 252-amino acid open reading frame is encoded beginning at position 63. A 69-residue, NH2-terminal mitochondrial targeting presequence is proteolytically removed upon import into the mitochondria. The amino termini of the mature, processed DUT-M protein begins at Ala70.
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Fig. 2. Comparison of the cDNA and amino acid sequences of DUT-N and DUT-M. A, schematic illustration comparing the cDNA sequences of DUT-N and DUT-M isoforms. Translation initiation and termination codons are indicated. B, schematic illustration comparing DUT-N and DUT-M protein sequences. Isoform-specific and common sequences are indicated.
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Isoelectric Points of DUT-N and DUT-M

Current models of mitochondrial protein import suggest that a positively charged targeting presequence is required for protein import into mitochondria (24). In agreement with this model, an arginine-rich, 69-residue presequence is predicted by the DUT-M cDNA. The 69-residue presequence has a calculated pI of 12.9, while the full-length DUT-M sequence has a calculated pI of 9.97. Reports of homologous nuclear encoded mitochondrial and cytosolic/nuclear isoproteins confirm that mitochondrial proteins have a more basic overall charge than their cytosolic/nuclear counterparts (25). DUT-N has a predicted pI of 6.16, while the processed form of DUT-M has a predicted pI of 8.23. To experimentally determine the pI values of the dUTPase isoforms, two-dimensional SDS-PAGE was employed. Total HeLa cell extract and extract from purified mitochondria were fractionated separately by two-dimensional PAGE and transferred to nitrocellulose, and dUTPase isoforms were detected by Western blotting (Fig. 3, A and B). Detection of dUTPase from total cell extract (Fig. 3A) reveals two proteins with isoelectric points of 6.0 and 8.1. These pI values correlate with the predicted values for DUT-N and DUT-M, respectively. Fractionation of protein derived from purified mitochondria confirms that the protein with the pI of 8.1 is in fact DUT-M (Fig. 3B).


Fig. 3. Western blot analysis of dUTPase isoforms separated by two-dimensional PAGE. HeLa-S3 cell protein (10 µg) from either whole cell extract (A) or mitochondrial extracts (B) were separated by two-dimensional PAGE and transferred to nitrocellulose. dUTPase protein was detected by Western blot analysis utilizing dUTPase-specific polyclonal antibodies.
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In Vitro Characterization of DUT-M

To confirm the identity of the DUT-M cDNA, a plasmid construct containing the DUT-M open reading frame was subjected to in vitro transcription and translation in the presence of [35S]methionine. dUTPase protein was immunoprecipitated from the lysate with dUTPase-specific monoclonal antibody, fractionated by SDS-PAGE, and exposed to x-ray film. Alternatively, the immunoprecipitated protein was assayed for dUTPase activity. The DUT-N open reading frame was used as a positive control in these experiments (Fig. 4, lane 1). As demonstrated in Fig. 4, lane 2, in vitro transcription/translation of the DUT-M cDNA results in the production of a precursor protein with an apparent molecular mass of 31 kDa that immunoreacts with dUTPase monoclonal antibody. A second minor protein species is also evident of slightly lower molecular weight. We believe this is an artifact of the in vitro transcription/translation system, possibly due to the use of a second in-frame methionine codon at position 129 of the DUT-M nucleotide sequence. In vitro dUTPase assays demonstrate that the DUT-M precursor protein retains dUTPase activity despite an extended amino terminus contributed by the mitochondrial targeting presequence (data not shown).


Fig. 4. In vitro transcription and translation of the DUT-M cDNA. The DUT-M and DUT-N open reading frames were cloned into pGEM-3Z in SP6 and T7 orientations, respectively. These plasmids were used for in vitro transcription/translation in the presence of [35S]methionine. dUTPase protein was immunoprecipitated from the lysate, fractionated by 15% SDS-PAGE, and exposed to x-ray film. Lane 1, DUT-N; lane 2, DUT-M; lane 3, plasmid with no insert.
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In Vitro Mitochondrial Processing of DUT-M

To further establish the mitochondrial localization of the DUT-M protein, in vitro import experiments were performed to demonstrate that the DUT-M precursor is taken up by mitochondria and proteolytically cleaved into a lower molecular weight, mature form. DUT-M was transcribed and translated in the presence of [35S]methionine in vitro and incubated for various times with mitochondria isolated from HeLa cells. DUT-N was used as a negative control in these experiments. The results of these experiments are seen in Fig. 5A. Lane 1 shows DUT-M protein without treatment, and lanes 2-5 show DUT-M protein that was incubated with purified mitochondria for 10, 20, 30, and 40 min, respectively. These data clearly demonstrate that, upon incubation with mitochondria, the DUT-M protein is proteolytically cleaved into a 23-kDa form. This processed molecular weight is in agreement with observations of the mature DUT-M protein in vivo (11). Lane 6 was left blank. Lane 7 shows DUT-N protein untreated. Lane 8 shows DUT-N protein that was incubated with mitochondria for 60 min; no proteolytic processing is evident.


Fig. 5. Mitochondrial Import of DUT-M. The DUT-M and DUT-N open reading frames were transcribed/translated in vitro in the presence of [35S]methionine, and 25-µl aliquots of the lysates were incubated with purified mitochondria at 30 °C for the indicated times. A, after incubation, the samples were sonicated, and dUTPase protein was immunoprecipitated and fractionated by 15% SDS-PAGE. The gel was dried and exposed to x-ray film for 48 h. Lane 1, DUT-M left untreated; lanes 2-5, DUT-M protein incubated with mitochondria for 10, 20, 30, and 40 min, respectively; lane 6, left blank; lane 7, DUT-N protein left untreated; lane 8, DUT-N protein incubated with mitochondria for 60 min. B, after incubation, the mitochondria were pelleted by centrifugation, washed, treated with trypsin, and washed again (see "Experimental Procedures"). The mitochondria were disrupted by sonication and fractionated by 15% SDS-PAGE. The gel was dried and exposed to x-ray film for 48 h. Lane 1, DUT-M left untreated; lanes 2-5, DUT-M protein incubated with mitochondria for 10, 20, 30, and 40 min, respectively, and subsequently treated with trypsin; lane 6, left blank; lane 7, DUT-N protein left untreated; lane 8, DUT-N protein incubated with mitochondria for 60 min and subsequently treated with trypsin.
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To demonstrate that the DUT-M processing event correlates with import into the mitochondria, radiolabeled DUT-M protein was incubated with purified mitochondria for various times, and mitochondria were subsequently harvested by centrifugation. The mitochondria were washed and then treated with trypsin to degrade protein that was not imported into the organelle. The mitochondria were washed and subsequently fractionated by SDS-PAGE followed by autoradiography. DUT-N protein was used as a negative control. The results of these experiments are shown in Fig. 5B. Lane 1 represents DUT-M without treatment. Lanes 2-5 represent DUT-M protein incubated with mitochondria for 10, 20, 30, and 40 min, respectively, followed by treatment with trypsin. Lane 6 was left blank. Lane 7 shows DUT-N protein that was left untreated. Lane 8 represents DUT-N protein that was incubated with mitochondria for 60 min and subsequently treated with trypsin. These results indicate that the processed form of DUT-M (23 kDa) becomes resistant to tryptic proteolysis, indicating that import into the mitochondria results in protection from digestion. The residual DUT-M precursor protein remaining outside of the mitochondria and the treated DUT-N protein were subsequently degraded by treatment with trypsin.

Differential Expression of Nuclear and Mitochondrial Isoforms of dUTPase in Serum-stimulated and Serum-starved Human Fibroblasts

It has been shown previously that members of the de novo thymidylate biosynthetic pathway, including TS, dihydrofolate reductase, and thymidine kinase, are induced in response to serum stimulation of quiescent human fibroblasts (Ref. 26 and references therein). To investigate dUTPase isoform expression in a similar manner, we decided to use 34Lu human lung fibroblasts as a model cell culture system. 34Lu cells were serum-starved for 72 h and subsequently serum-stimulated by the addition of 10% fetal bovine serum to the media. DNA replication status was monitored by [3H]thymidine incorporation at the indicated time points. Cells were harvested at time points correlating with G0, G1, and S phase (as monitored by [3H]thymidine incorporation), and equivalent amounts of total cellular protein were fractionated by 15% SDS-PAGE. The protein was transferred to nitrocellulose, and dUTPase isoforms were detected by immunoblot analysis. Time course experiments presented in Fig. 6, A and B, illustrate dUTPase expression in response to serum stimulation. The Western blot presented in Fig. 6A illustrates dUTPase expression status in cells 0 h post-stimulation (lane 1), in cells 12 h post-stimulation (lane 2), and in cells 30 h post-stimulation (lane 3). It is clear from this data that the lower molecular weight DUT-N isoform is induced in response to serum stimulation, correlating with the onset of DNA replication (see Fig. 6B). In contrast, the higher molecular weight DUT-M isoform is expressed in a constitutive manner, independent of proliferation status.


Fig. 6. Expression of dUTPase isoforms during serum stimulation and serum starvation of 34Lu human lung fibroblasts. A, 34Lu cells were driven into G0 by serum starvation for 72 h. Cells were subsequently stimulated by the addition of serum, and protein samples were collected at time points correlating to G0, G1 phase, and S phase. Protein samples were fractionated by 15% SDS-PAGE, and dUTPase protein was detected by Western blot analysis utilizing dUTPase-specific polyclonal antibodies. Lane 1, 0 h (G0); lane 2, 12 h (G1 phase); lane 3, 30 h (S phase). Equivalent amounts of protein were loaded in each lane. B, [3H]thymidine incorporation was utilized to monitor DNA replication status during serum stimulation of 34Lu cells. At the indicated time points post-stimulation, cells were pulsed for 20 min with [3H]thymidine, and subsequent incorporation of radioactivity into DNA was determined. C, cycling 34Lu cells were driven into G0 by serum starvation. Protein samples were collected at 0, 24, and 48 h post-starvation. Samples were fractionated by 15% SDS-PAGE, and dUTPase protein was detected by Western blot analysis utilizing dUTPase-specific polyclonal antibodies. Lane 1, 0 h; lane 2, 24 h; lane 3, 48 h. D, thymidine incorporation was utilized to monitor DNA replication during serum starvation. At the indicated time points post-starvation, cells were pulsed for 20 min with [3H]thymidine, and subsequent incorporation of radioactivity into DNA was determined.
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A similar time course experiment was performed to establish dUTPase isoform expression as a consequence of serum starvation (Fig. 6, C and D). Cycling cells were serum-starved, and dUTPase expression was monitored by Western blot. In Fig. 6C, lane 1 illustrates dUTPase expression at 0 h, lane 2 shows expression at 24 h post-starvation, and lane 3 shows expression at 48 h post-starvation. These data indicate that DUT-N is preferentially degraded within 48 h post-starvation, a rate that corresponds with the cessation of nuclear DNA replication (Fig. 6D). DUT-M levels, however, remain constant as the cells transition into the G0 state.

To determine if a similar regulatory phenomenon was occurring at the mRNA level, Northern blot analysis was performed on mRNA derived from serum-stimulated and serum-starved 34Lu cells. Cells were either serum-starved for 72 h or serum-stimulated for 36 h, and poly(A)+ RNA was isolated. The RNA (1 µg) was fractionated by agarose gel electrophoresis and then transferred to nitrocellulose. The resulting Northern blots were probed with the 3' end (935-bp SphI/EcoRI fragment) of the DUT-N cDNA, a region shared between both isoforms (Fig. 7A). To ensure equal loading of mRNA samples, a GAPDH cDNA was used to probe the identical blot (Fig. 7B). Fig. 7A, lane 1, contains dUTPase mRNA isolated from serum-starved cells, and lane 2 contains dUTPase mRNA isolated from serum-stimulated cells. In serum-starved cells (lane 1), a single 1.4-kb dUTPase transcript is evident. Upon serum-stimulation, however, a 1.1-kb message is induced, while the levels of the 1.4-kb transcript, observed in quiescent cells, remain the same (lane 2). This pattern of dUTPase mRNA expression mimics dUTPase protein isoform expression, with the inducible 1.1-kb transcript corresponding to DUT-N and the constitutive 1.4-kb transcript corresponding to DUT-M. To clearly demonstrate the identity of the transcripts, an isoform-specific probe was utilized for Northern blot analysis. The extreme 5' end of the DUT-M cDNA sequence is unique, providing for an isoform-specific probe. This 5' DUT-M-specific sequence (250-bp EcoRI/AluI fragment) was used to probe 34Lu mRNA isolated from cycling cells. The common probe (935-bp SphI/EcoRI fragment) was used as a control on an identical blot (Fig. 7C). As previously shown, the common probe hybridizes to both the 1.1- and 1.4-kb dUTPase transcripts (Fig. 7C, lane 1). The DUT-M-specific probe, however, specifically hybridizes to the 1.4-kb transcript, indicating that the 1.4-kb transcript encodes DUT-M (Fig. 7C, lane 2). Together, these data indicate that the DUT-N and DUT-M isoforms are transcribed as distinct mRNAs of 1.1 and 1.4 kb, respectively, in 34Lu cells. In addition, the 1.1-kb transcript is induced as a consequence of serum stimulation, while the 1.4-kb DUT-M transcript is maintained at constant levels regardless of cell growth status.


Fig. 7. Northern blot analysis of dUTPase in serum-starved and serum-stimulated 34Lu human lung fibroblasts. Poly(A)+ RNA was isolated from serum-starved and serum-stimulated 34Lu cells. mRNA (1 µg) was fractionated by agarose gel electrophoresis and transferred to nitrocellulose for subsequent Northern hybridization analysis. A, the 3' region of the DUT-N cDNA (935-bp SphI/EcoRI fragment), the sequence shared by DUT-N and DUT-M, was used as a probe for hybridization analysis. Lane 1, mRNA isolated from cells serum-starved for 72 h. Lane 2, mRNA isolated from cells serum-stimulated for 30 h. B, to assure equal loading of mRNA sample, the blot seen in panel A was stripped and reprobed with GAPDH. C, Northern blot analysis of 34Lu mRNA with a DUT-M-specific probe. Poly (A)+ RNA was isolated from cycling 34Lu cells, fractionated by agarose gel electrophoresis, and transferred to nitrocellulose. Hybridization analysis was performed with either a probe common to both DUT-N and DUT-M (lane 1) or a probe specific for DUT-M isoform as described under "Experimental Procedures" (lane 2).
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Genomic Organization of the DUT-N and DUT-M Isoforms

Because the cDNA and amino acid sequences of the dUTPase isoforms are identical in their overlapping regions, we set out to determine if they were encoded by the same gene. An EMBL-3 human leukocyte genomic library was screened with the DUT-N cDNA to isolate the dUTPase gene. Three genomic isolates were subcloned and characterized by restriction digestion and Southern hybridization analysis. The three genomic isolates contained identical restriction fragment patterns in their overlapping regions, suggesting that the three isolates contained identical nucleotide sequence (data not shown). An oligonucleotide was designed from the 5' region of the DUT-N cDNA and was used as a probe to identify a restriction fragment containing the extreme 5' region of the dUTPase gene (see "Experimental Procedures" for details). A 2.7-kb BamHI/EcoRI fragment from the largest genomic clone (15 kb) was isolated and subcloned into pGEM-3Z. Sequence analysis of a region of the dUTPase gene spanning 1.6 kb is presented in Fig. 8 and illustrated in schematic form with respect to the DUT-N and DUT-M cDNA sequences in Fig. 9. The sequence data demonstrate that the dUTPase gene does in fact encode both nuclear and mitochondrial isoforms. Interestingly, the isoforms appear to arise by the use of alternative 5' exons, which then join the common sequence. The 5' exon of DUT-M is spliced to the common sequence, while the 5' exon of DUT-N is contiguous with the common sequence. Exon 1 of DUT-M correlates with positions 492-833 of the genomic isolate (Fig. 9). There is a DUT-M-specific intron between positions 834 and 1302, with a splicing event occurring between exon 1 of DUT-M and the first exon shared between the isoforms. Consensus donor and acceptor splice sites are located at positions 833 and 1303, respectively. In contrast, the DUT-N isoform is encoded beginning at position 1259 and is continuous with the first common exon (see Fig. 9). After the first common exon there is an intron corresponding to position 1441 of the genomic isolate. A consensus donor splice site is located at position 1441. An exon containing a portion of the common carboxyl terminus shared by both DUT-N and DUT-N lies approximately 15 kb further downstream (data not shown).


Fig. 8. Sequence analysis of the 5' region of the human dUTPase gene. Numbering of the nucleotide positions was arbitrarily chosen. The beginning of the DUT-M and DUT-N sequences are indicated by arrows. Consensus sequences for NF-kappa B, Sp1, and E2F binding have been underlined.
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Fig. 9. Schematic illustration of the 5' region of the human dUTPase gene. Exons are boxed, and open reading frames of DUT-M, DUT-N, and common sequences are distinguished by different shading. Intron/exon boundaries are indicated and numbered with respect to the complete nucleotide sequence in Fig. 8. The NH2-terminal DUT-M splice junction is indicated by joining lines.
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DISCUSSION

We have set out to extend the characterization of dUTPase isoforms and begin to elucidate basic molecular mechanisms governing dUTPase isoform expression in human cells. In this report, we describe the isolation and characterization of a cDNA encoding DUT-M, elucidate isoform-specific expression patterns in replicating and serum-starved human fibroblasts, and describe the isolation and characterization of the 5' region of the human dUTPase gene that encodes both nuclear and mitochondrial isoforms.

Mitochondrial dUTPase

Mass spectrometry and NH2-terminal protein sequencing demonstrate that the nuclear and mitochondrial isoforms of dUTPase are identical except for a short region of their respective amino termini (11, 15). Their common COOH-terminal regions contain five highly conserved motifs, characteristic of dUTPase proteins from various species, that contribute to active site formation (13, 27). Here, we describe the cloning and characterization of a cDNA encoding the DUT-M isoform. The DUT-M cDNA encodes a 252-amino acid precursor protein with a calculated molecular weight of 26,704 that migrates with an apparent molecular weight of 31,000 as judged by SDS-PAGE. Similar to many other mitochondrially targeted proteins, the DUT-M precursor contains a positively charged mitochondrial targeting presequence (24). In vitro mitochondrial import studies demonstrate that the 31-kDa precursor protein is proteolytically cleaved into a mature 23-kDa form and transported into mitochondria. Reports comparing mitochondrial and cytosolic isoproteins suggest that the mitochondrial counterparts generally have a more basic net charge (25). In agreement with this observation, the mature DUT-M protein has a measured pI of 8.1, while the nuclear isoform has a pI of 6.0. A report by Strahler and co-workers suggests that distinct phosphorylated and unphosphorylated forms of dUTPase exist in stimulated peripheral blood lymphocytes and are separable by two-dimensional PAGE (14). The pI values reported by these authors were 6.0 for the phosphorylated form and 8.06 for the unphosphorylated form. These observations are consistent with the pI measurements of DUT-N and DUT-M in this report and are in agreement with the phosphorylation status of DUT-N (phosphorylated) and DUT-M (unphosphorylated) determined in previous reports (15). In addition, the phosphorylated form of dUTPase is induced upon mitogenic stimulation of peripheral blood lymphocytes, similar to the induction of DUT-N observed in serum-stimulated 34Lu cells (14). Together, these observations suggest that the nonphosphorylated form of dUTPase described by Strahler et al. (14) is in fact DUT-M (pI 8.1), while the phosphorylated form is DUT-N (pI 6.0).

Expression of DUT-N and DUT-M Isoforms

dUTPase is the major regulator of dUTP pools in human cells, and recent evidence suggests that relative levels of dUTPase protein may significantly influence the efficacy of chemotherapeutics that target thymidylate synthase (7, 10). In light of these observations, we have set out to establish the basic mechanisms of dUTPase isoform expression in humans.

Western blot analysis of dUTPase in serum-starved and serum-stimulated human fibroblasts suggests that DUT-N protein expression is tightly linked to nuclear DNA replication status, being induced during the G0 to S phase transition. During exit from the cell cycle into G0, DUT-N protein is degraded. In contrast, DUT-M protein is expressed in a constitutive manner with no regard for cell cycle status, mimicking the pattern of mitochondrial DNA replication.

Northern blot analysis utilizing common and isoform-specific probes demonstrates that DUT-N and DUT-M are encoded by distinct transcripts of 1.1 and 1.4 kb, respectively. Northern analysis of cycling, serum-starved, and serum-stimulated cells demonstrate that DUT-M-specific mRNA is expressed in a constitutive fashion, while DUT-N-specific mRNA expression is growth-dependent.

Analysis of DUT-N expression suggests that it belongs to a class of S-phase-specific genes that are expressed in a growth-dependent fashion such as members of the nucleotide precursor biosynthesis pathway (TS, dihydrofolate reductase, and thymidine kinase) in addition to members of the DNA replication pathway (DNA polymerase alpha  and proliferating cell nuclear antigen) (26, 28). Expression of S-phase genes is controlled at the transcriptional as well as post-transcriptional levels, and regulatory mechanisms appear to be complex and multileveled (26, 29-31). However, certain common features are emerging from both structural and functional characterization of these genes. For instance, it appears that the E2F transcription factor is responsible for a certain percentage of transcriptional activation as a consequence of growth stimulation. The above mentioned genes all retain E2F consensus binding sites in their promoter regions, and each gene is induced, to varying degrees, in response to overexpression of E2F1 in a cell culture model system (26). The growth-dependent increase of both DUT-N protein and mRNA levels suggests that DUT-N may also represent a target gene for E2F-mediated S-phase induction. It is reasonable to expect that, like many other S-phase genes, growth-dependent expression of DUT-N will involve several levels of regulation.

In contrast to the growth-dependent expression of DUT-N, the DUT-M isoform is expressed in a constitutive manner at both the mRNA and protein levels. This pattern of expression is consistent with current views of mitochondrial DNA replication. Studies reviewed by Clayton (32) demonstrate that mitochondrial DNA synthesis occurs randomly through the cell cycle, with no regard to nuclear DNA replication status.

The regulation of dUTPase isoforms in 34Lu cells suggests that both isoforms are largely expressed to coincide with the replication pattern of their respective genomes. However, certain developmental conditions break this rule. Strahler and co-workers demonstrated that high levels of the phosphorylated form of dUTPase (presumably DUT-N) are expressed in nonproliferating immature thymocytes (14). These authors speculate that the dUTPase function may be required during T cell development. These authors also note that proliferating cell nuclear antigen, another growth-regulated gene, is also expressed in the immature thymocytes. This may indicate that many S-phase genes are expressed in these nonreplicating cells through a common mechanism. Our data clearly show that DUT-N and DUT-M are regulated by dramatically different mechanisms; this is of particular interest because the isoforms are encoded by the same gene.

As illustrated in Figs. 8 and 9, dUTPase isoforms appear to arise through the alternative use of 5' exons. An interesting question is whether the isoform-specific transcripts are regulated through single or multiple promoters. The genomic organization and the differential regulation of DUT-N and DUT-M, at both the protein and RNA levels, suggests that there may be distinct promoters driving differential transcription. Support for this speculation is found by examination of the genomic sequence directly upstream of the DUT-N isoform (Fig. 8). Comparison of this upstream region with known transcription factor consensus sequences reveals several interesting traits. Consistent with the growth-dependent control of DUT-N protein and mRNA expression, several consensus elements that typify growth-dependent promoters are evident, including E2F, Sp1, and NF-kappa B (see Fig. 8). Many S-phase genes contain E2F and Sp1 elements in their promoters that play central roles in their cell cycle (growth)-dependent expression (28, 36, 37). The c-myc gene contains all of these putative elements (E2F, Sp1, and NF-kappa B) that regulate the transcriptional activation of c-myc under a variety of physiologic conditions (38, 39). Recently, the C. albicans dUTPase gene was reported to contain an MluI cell cycle box element in its promoter that is critical for cell cycle-dependent transcriptional regulation (18). This cis-element is analogous to the human E2F transcription factor in that it confers S-phase-dependent transcriptional activation in many yeast genes associated with DNA replication (40). All of these examples provide support for the involvement of these putative elements in DUT-N transcriptional control; however, confirmation awaits formal promoter analysis of the human dUTPase gene.

Genomic Organization

A recent report by Cohen and co-workers assigned the human dUTPase gene to a single locus on chromosome 15 by fluorescence in situ hybridization (41). Consistent with this observation, we have discovered that both isoforms are encoded by the same gene. By comparing the known protein and cDNA sequences of the dUTPase isoforms with the 5' region of the dUTPase gene, it is evident that both DUT-N and DUT-M are encoded by a single gene with each isoform arising through the use of alternative 5' exons (see Fig. 9). As illustrated in Fig. 9, the first exon of DUT-M begins 767 base pairs upstream of the first exon of DUT-N. Exon 1 of DUT-M splices directly to the first common exon (splice donor site, 833; splice acceptor site, 1303). In contrast, the first exon of DUT-N is continuous with the first common exon shared between the isoforms. The genomic organization of DUT-N and DUT-M is of particular interest because it represents the first example in humans of a single gene encoding differentially expressed nuclear and mitochondrial isoproteins that arise by the alternative use of 5' exons to generate discrete transcripts. Studies comparing homologous nuclear encoded mitochondrial and cytosolic isoproteins suggest that only rarely are compartmentally distinct isoforms encoded by the same gene (25). The citric acid cycle enzyme fumarase is an important example of a gene that encodes both cytosolic and mitochondrial isoforms in a variety of different organisms including human, rat, and yeast. Interestingly, these isoforms arise through several distinct regulatory mechanisms in different organisms. In humans, for example, fumarase isoforms are translated as a single gene product without a targeting presequence; however, the cytosolic form is acetylated (33). In contrast, rat fumarase isoforms arise by alternative start codon usage within the same transcript, the mitochondrial form encoding a cleavable targeting peptide at the NH2 termini (34). Yeast fumarase isoforms were recently shown to be transcribed and translated as a single protein, containing a signal sequence that is cleaved in the mitochondria (35). The authors speculate that the cytosolic form arises from the release of an import-incompetent form of fumarase from the organelle into the cytoplasm. These examples demonstrate that partitioning of isoproteins in different cellular compartments that are encoded by the same gene can occur through one or more transcriptional, translational, or post-translational mechanisms. Human dUTPase represents a novel example for generating mitochondrial and nuclear isoforms that are differentially regulated.

In summary, we have confirmed the existence of a mitochondrial specific isoform of dUTPase by cloning and characterizing a DUT-M-specific cDNA. By analyzing quiescent and serum-stimulated 34Lu human lung fibroblasts, we have show that the isoforms are differentially expressed at both the protein and mRNA levels to coincide with the DNA replication patterns of their respective target compartments. Further, we show that DUT-N and DUT-M are encoded by a single gene, arising through the use of alternate 5' exons. Future studies will focus on the transcriptional mechanisms contributing to isoform-specific expression.


FOOTNOTES

*   This research was supported in part by Grant CA42605 from the NCI, National Institutes of Health (to S. J. C.).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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U90223 (DUT-M cDNA) and U90224 (human dUTPase gene).


Dagger    To whom correspondence should be addressed: Dept. of Molecular Biology, The University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, 2 Medical Center Dr., Stratford, NJ 08084. Tel.: 609-566-6043; Fax: 609-566-6232; E-mail: ladner{at}umdnj.edu.
1   The abbreviations used are: TS, thymidylate synthase; DUT-N, nuclear isoform of human dUTPase; DUT-M, mitochondrial isoform of human dUTPase; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); kb, kilobase(s).

ACKNOWLEDGEMENT

We thank Michael J. Hansbury for review of this manuscript.


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