(Received for publication, October 16, 1995; and in revised form, December 12, 1995)
From the
Deoxyuridine triphosphate nucleotidohydrolase (dUTPase; EC
3.6.1.23) was purified from HeLa cells by immunoaffinity
chromatography. Based on SDS-polyacrylamide gel electrophoresis, two
distinct forms of dUTPase were evident in the purified preparation.
These proteins were further characterized by a combination of
NH-terminal protein sequencing, mass spectrometry, and mass
spectrometry-based protein sequencing. These analyses indicate that the
two forms of dUTPase are largely identical, differing only in a short
region of their amino-terminal sequences. Despite the structural
difference, both forms of dUTPase exhibited identical binding
characteristics for dUTP.
Each form of dUTPase has a distinct cellular localization. Cellular fractionation and isopycnic density centrifugation indicate that the lower molecular weight form of dUTPase (DUT-N) is associated with the nucleus, while the higher molecular weight species (DUT-M) fractionates with the mitochondria. The DUT-N isoform is approximately 30-fold more abundant in HeLa cells than DUT-M as determined by densitometry.
The NH-terminal protein
sequence of both DUT-N and DUT-M did not match previous reports of the
predicted amino-terminal sequence for human dUTPase (McIntosh, E. M.,
Ager, D. D., Gadsden, M. H., and Haynes, R. H.(1992) Proc. Natl.
Acad. Sci. U. S. A. 89, 8020-8024; Strahler, J. R., Zhu X.,
Hora, N., Wang, Y. K., Andrews, P. C., Roseman, N. A., Neel, J. V.,
Turka, L., and Hanash, S. M.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4991-4995). A cDNA corresponding to the DUT-N isoform
was isolated utilizing an oligonucleotide probe based on the determined
NH
-terminal sequence. The cDNA contains a 164-amino acid
open reading frame, encoding a protein of M
17,748. The DUT-N cDNA sequence matches the previously cloned
cDNAs with the exception of a few discrepancies in the 5` end. Our data
indicate a 69-base pair addition to the 5` end of the previously
reported open reading frame.
Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) ()is a ubiquitous enzyme that functions in the hydrolysis of
dUTP to dUMP and pyrophosphate. This reaction is thought to occur
primarily to limit pools of intracellular dUTP in order to prevent
significant dUMP incorporation into DNA during replication and
repair(3) . A second role of dUTPase is to provide substrate
(dUMP) for the de novo synthesis of thymidylate. The effects
of a compromised dUTPase activity have been well documented in
prokaryotes(4) . Mutations in Escherichia coli dUTPase, which lower enzyme activity to 5% of wild type levels,
cause an increase in the intracellular dUTP pools. The result of
elevated dUTP pools is an increased incorporation of dUMP into DNA.
Uracil-DNA glycosylase initiates the base excision repair pathway in a
reiterative, self-defeating repair process, which results in removal
and reincorporation of dUMP. This ultimately leads to DNA fragmentation
and cell death(4) .
The consequences of a reduced dUTPase function in eukaryotes are not as well documented because of a lack of mutants. A dUTPase null mutant in the yeast Saccharomyces cerevisiae was shown to be inviable (5) , a result similar to what is observed in the bacterial system. In the mammalian system, indirect evidence has shown that anti-folate analogs and other inhibitors of de novo thymidylate biosynthesis cause an increase in the ratio of dUTP to dTTP resulting in DNA fragmentation and cell death(6, 7, 8) . Recently, Canman and co-workers (9, 10) demonstrated that, in certain human tumor cell lines, increased levels of dUTPase are responsible for an increase in resistance to the cancer chemotherapeutic agent fluorodeoxyuridine (FUdR), a thymidine synthase inhibitor. Together, these studies provide substantial evidence suggesting that dUTPase, the chief regulator of dUTP pools, mediates a critical step in FUdR toxicity.
In addition to prokaryotes and eukaryotes, a number of viruses are known to encode a dUTPase function. A diverse group of viruses including herpesviruses(11, 12, 13, 14) , poxviruses(15) , and retroviruses (16, 17) encode a viral dUTPase activity. A specific subset of the lentivirus group encodes dUTPase as part of the pol gene product in addition to the reverse transcriptase, integrase, and protease functions(16) . In contrast, the human immunodeficiency virus types 1 and 2 (HIV1 and HIV2) do not contain a virus-encoded dUTPase function (17) and may rely on the dUTPase of the host cell. The question of whether dUTPase is essential for viral replication has been addressed in both herpesvirus and retrovirus groups(16, 18, 19) . Null mutants of viral dUTPases demonstrate that this enzyme is required for successful viral replication in nondividing cells in which the cellular levels of dUTPase are exceptionally low. In contrast, virus-encoded dUTPase is not required for replication in actively growing cultured cells where dUTPase levels are high(16, 18) . It has been postulated that virus-encoded dUTPase expands the tropism of certain viruses by allowing viral replication in nondividing cell types with low cellular dUTPase activity(16) .
Our laboratory has undertaken a detailed biochemical characterization of the dUTPase enzyme function in human cells. In this report, evidence is presented identifying and characterizing two distinct forms of dUTPase that exist in humans. Cellular fractionation experiments suggest that the more abundant, lower mass form of dUTPase (DUT-N) localizes in the nucleus, while the higher mass form (DUT-M) is associated with the mitochondria. We also present the full-length cDNA encoding the DUT-N isoform.
Purified nuclei were obtained according to the hypotonic shock procedure described by Dignam et al.(23) . The resulting nuclei were further purified by isopycnic density gradient centrifugation on Nycodenz as described by Ford and Graham(24) . The resulting nuclear extract was utilized for subsequent immunoblot analysis or purification of the nuclear-associated dUTPase by the method described above.
Structural information may be discerned
from MALDI-MS by analysis of metastable ions that decompose in the
field-free drift region of the time-of-flight
analyzer(29, 30, 31, 32, 33) .
The (M + H) ions presumably become activated in
the ion source through multiple collisions with matrix and analyte ions
but do not decompose until they are in the first field-free drift
region after having been fully accelerated. These so-called metastable
ions decompose, producing fragment ions that have essentially the same
velocity as the parent ion but have energies proportional to the ratio
of the fragment-to-parent ion
mass(29, 30, 31, 32, 33) .
These product ions may be analyzed in the reflecting mode of operation
by stepping down the reflecting voltage to bring the lower mass
products into energy focus at the reflecting detector. A resolution of
>2000 (full-width half-maximum definition) has been achieved for
mass-selected product ions produced by
MALDI-MS(28, 47) . This resolution is sufficient to
determine monoisotopic mass up to at least m/z 1800
in the product ion analysis mode, and it greatly reduces uncertainty in
the mass assignment and structural interpretation of fragment peaks.
Metastable ion mass spectra (also referred to as postsource decay
spectra) were acquired in eight consecutive, overlapping mass scale
segments, each representing a 25% mass change from the previous
segment. The segments were combined and externally mass-calibrated (versus a metastable ion spectrum of a model peptide such as
renin tetradecapeptide or substance P, residues 2-11) by the data
system. A Bradbury-Nielsen ion gate was used for precursor ion
selection(28, 34, 35, 47) . The
resolution of precursor ion selection is in excess of
100(28, 47) .
Figure 1: Silver staining and Western blot analysis of HeLa S3 dUTPase. HeLa dUTPase was purified to apparent homogeneity by immunoaffinity chromatography. Approximately 300 ng of protein was fractionated by 15% SDS-PAGE and visualized by silver staining (lane 1). To further establish the identity of the two co-purifying proteins, 50 µg of total HeLa cell extract was fractionated by 15% SDS-PAGE, and dUTPase protein was detected by Western blot analysis utilizing dUTPase specific polyclonal antibodies (lane 2).
Figure 2:
Nucleotide and amino acid sequence of the
major form of dUTPase. The more abundant, lower molecular weight form
of dUTPase (DUT-N) was purified and subjected to
NH-terminal protein sequencing. The 25 amino-terminal
residues identified in this manner are underlined. An
oligonucleotide probe was generated based on the
NH
-terminal sequence and used to isolate a dUTPase-specific
cDNA from a human T cell cDNA library. A 164-amino acid open reading
frame is encoded beginning at position 30. The position of the
polyadenylation signal in the 3`-untranslated region is underlined.
Figure 3: Cellular fractionation and Western blot analysis of HeLa-derived dUTPase. HeLa dUTPase from total cell extracts (lanes 1 and 4), cytosolic extract (lane 2), purified mitochondria (lane 3), or purified nuclei (lane 5) was detected by Western blot analysis. Approximately 10 µg of extract was loaded in each lane. The blot was probed with a immunopurified polyclonal antibody generated against recombinant human dUTPase. Bands were visualized by the ECL system of Amersham Corp.
Western blot analysis of purified mitochondrial protein (Fig. 3, lane 3) and cytosolic extract (Fig. 3, lane 2) demonstrates a complete lack of the more abundant, lower molecular weight form of dUTPase, DUT-N, suggesting that this form is localized exclusively within the nucleus. In order to verify the specific nuclear localization of this form, nuclei were purified (see ``Experimental Procedures''), and dUTPase protein was detected by Western blot analysis. Fig. 3, lane 5, indicates that the more abundant, lower molecular weight form of dUTPase, DUT-N, is associated with the purified nuclei as compared with total cell extract (Fig. 3, lane 4).
The overall sequences of DUT-N and DUT-M were confirmed, and the
sequence of the NH-terminal region of DUT-M was completed
by MALDI-MS and tandem MS. HeLa dUTPase was fractionated by SDS-PAGE,
and the individual protein bands corresponding to each form were
excised from the gel. The gel slices containing DUT-N and DUT-M were
reduced, carboxamidomethylated, and digested with trypsin. The peptides
generated from each form were then analyzed by MALDI-MS. The resulting
spectra obtained on approximately 5 pmol of each digest are shown in Fig. 4, and the corresponding sequence locations are shown by underlines in Fig. 6. Approximately two-thirds of each
protein was mapped in these experiments. Coverage is not expected to be
complete for several reasons. First, not all peptides are extracted or
recovered from the gel with equal efficiency. Second, not all peptides
are ionized with equal efficiency in the MALDI-MS experiment, and the
choice of matrix can have a significant effect on the specific
components of a mixture that are detected and their apparent relative
ratios(36) . Third, many of the peptides that were not detected
are relatively small and would, if present, have molecular ions in the
region dominated by the intense background from the liquid matrix used.
Finally, suppression can occur in complex mixtures such that only the
most easily ionized and/or most abundant peptides are detected.
Figure 4: Mass spectrometry analysis of dUTPase tryptic peptides. MALDIMS analyses of in-gel tryptic digests of reduced and carboxamidomethylated nuclear (top) and mitochondrial dUTPase (bottom). Spectra were recorded in the linear mode on a Fisons VG TofSpec SE mass spectrometer (Manchester, UK), and are the sum of 20-50 laser shots.
Figure 6:
Comparison of the structural differences
between the nuclear and mitochondrial forms of dUTPase. Comparison of
the sequences of nuclear (top) and mitochondrial (bottom) human dUTPase. Solid underlines indicate
tryptic peptides whose (M + H) ions were observed
in the MALDI-MS data (see Fig. 5). The calculated, monoisotopic
molecular weights of the peptides are shown. Dashed underlines correspond to the Edman sequencing data obtained on the intact
proteins; the gap in the sequence data for the mitochondrial form is
indicated by the absence of dashed underline. The sequence
within the box was obtained by MALDI-MS/MS of the tryptic peptide of
molecular weight 1776 (see Fig. 5and text). Residue 10 in the
sequence of the nuclear form is a phosphoserine (pS; see
accompanying article (48) for
discussion).
Figure 5:
MALDI-MS (MALDI-MS/MS) of tryptic peptides
derived from the mitochondrial form of dUTPase (DUT-M). Metastable ion,
MALDI-MS (MALDI-MS/MS) of m/z 1776 (top) and m/z 2066 (bottom) were recorded using a
Fisons VG TofSpec SE mass spectrometer (Manchester, UK) single-stage
reflectron instrument. Desorption/ionization was accomplished with a
337-nm pulsed N laser and extraction at 25 kV. An ion gate
was used to select the precursor ion from the mixtures shown in Fig. 5whose metastable (or product ions) we wished to
record(2, 3) . MALDI-MS/MS spectra were acquired in
eight consecutive, overlapping mass scale segments, each representing a
25% energy change on the reflectron from the previous segment. The
segments were combined and externally mass-calibrated (versus a metastable ion spectrum of a model peptide such as renin
tetradecapeptide) by the VG OPUS data system. Nomenclature is according
to Roepstorff and
Biemann(45, 46) .
With
the exception of the signal at m/z 1776, all of the
major peptide-derived signals fit the sequences shown (the signals at m/z 1300 and 2183 correspond to monooxidized forms of
the peptides of M 1284 and 2167, respectively,
each of which contains a Met residue that presumably has partially
converted to Met-sulfoxide). The 1776 peptide is unique to the
mitochondrial form of the protein. Furthermore, based on the
observation that it was also present in tryptic digests that had not
been reduced and alkylated prior to MS analysis (data not shown), it
could not contain Cys. Absence of Cys suggests that this peptide cannot
be a simple modification of the NH
-terminal peptides of
DUT-N.
The new technique of metastable ion analysis in MALDI-MS (MALDI-MS/MS) was used to provide the sequence of this peptide, and to confirm the sequence of several of the other peptides observed in the MALDI-MS data. The parent ion of the 1776 peptide was selected from the mixture shown in Fig. 5for further MS analysis using a Bradbury-Nielsen ion-gating device (34, 35) in the Fisons VG MALDI mass spectrometer(28, 47) . Fragment ions are produced from the highly activated, metastable peptide ions as they undergo decomposition (sometimes referred to as ``postsource decay'') during flight in the field-free portion of the time-of-flight instrument. The fragment ions formed have energies in proportion to their masses, and they may be analyzed in the reflectron portion of the reflecting time-of-flight instrument by purposefully bringing to focus at the final detector ions of energy lower than that of the parent(29, 30, 31, 32, 33) .
The MALDI-MS/MS spectra of the peptides of (M + H) = 1776 and (M + H)
= 2066 are
shown in Fig. 6. The dominant fragment ions observed in these
spectra correspond to y
ions
(H-(NH-CHR-CO)
-OH + H) and internal acyl ions denoted
by single-letter codes. The internal acyl ions are formed by two amide
bond cleavages, the first occurring NH
-terminal to Pro, and
the second involving any residue COOH-terminal to the Pro (e.g. PET, Fig. 6,
HN
CHR
-CO-NH-CHR
-CO-NH-CHR
-C&cjs0809;O
,
where R
= Pro, R
= Glu, R
= Thr, and
indicates the cyclization of
R
to the NH). The subsequence . . . PAPGPETP . . . is
defined by the mass gaps between the y
, y
, y
, y
, and y
ions (Fig. 5). Cleavage COOH-terminal to Pro to form a y
ion is strongly disfavored and results in
sequence ion gaps indicative of the presence of Pro. This subsequence
is further supported by the internal acyl ions series (e.g. PGPE, PGPET, etc.). In the case of the peptide of (M +
H)
= 2066, the sequence of residues 2-15
are defined by the fragment ions observed (Fig. 5).
The
subsequence for the peptide of M = 1776
determined by MALDI-MS/MS overlaps with the Edman data for the NH
terminus of DUT-M, indicating that the residues 22 and 23 missing
in the Edman data correspond to glycine and proline, respectively (Fig. 6). Based on the Edman and MS data, a M
of 1775.9 is predicted for the tryptic peptide
AGGSPAPGPETPAISPSKR that would overlap with the determined sequence of
DUT-N. This predicted M
corresponds very closely
to that observed in the MALDI-MS data ( Fig. 4and Fig. 6). In addition, other major signals observed in the
MALDI-MS/MS data can be assigned to internal acyl ions for the partial
sequences PETPAIS and PGPETPAI and to the y
ion for PSKR (Fig. 5).
Thus, the MS data define the region of greatest uncertainty in the Edman data and establish the junction of the isoforms. Together these data indicate that the nuclear associated DUT-N and the mitochondrial associated DUT-M variants have distinct amino termini but are identical after the junction site ETPAI (Fig. 6).
The dUTPase function has been shown to be important in DNA
replication (3, 4, 5) and is highly conserved
throughout evolution(14) . Our laboratory is investigating the
basic biochemical and regulatory aspects of human dUTPase. We have
previously described HeLa-derived dUTPase as a 22.5-kDa phosphoprotein
with a K value for dUTP of 2.5 µM and
a requirement for Mg
(12, 20) .
To further characterize the human enzyme, we set out to isolate a
dUTPase-specific cDNA. Evidence presented in this report demonstrates
that the cDNA sequence described corresponds to the major form of the
dUTPase protein from HeLa cells (DUT-N). cDNA and amino-terminal
protein sequence analysis indicates that the open reading frame of the
DUT-N isoform of dUTPase contains 24 more amino-terminal residues than
previously reported(1, 2) . The
NH-terminal methionine is removed in the mature DUT-N
protein. Utilizing the methods described in this report, there is no
evidence suggesting the existence of an expressed form of dUTPase in
HeLa cells corresponding to the predicted translation start site
reported by McIntosh, et al.(1) or Strahler, et
al.(2) .
Western blot analysis of partially purified cytosolic extract (Fig. 3, lane 2) demonstrates the presence of the mitochondrial associated dUTPase. This analysis also reveals another previously undetected dUTPase species of slightly greater molecular weight than the mitochondrial associated form. We speculate that this protein may represent a precursor form of mitochondrial dUTPase. Many proteins residing in mitochondria are encoded by nuclear genes. These proteins are typically translated as precursor proteins containing an extended amino-terminal leader region containing amphiphilic amino acids(37) . Upon transfer into the mitochondria, the signal sequence is proteolytically removed by a signal peptidase. It is feasible that the immunoreactive, larger mass protein species present in cytosolic extract represents an unprocessed precursor form of mitochondrial dUTPase. This also suggests that the DUT-M protein identified in this study corresponds to the fully processed mitochondrial form. Future delineation of the unprocessed mitochondrial dUTPase protein as well as cloning of a full-length mitochondrial dUTPase cDNA may reveal further attributes of a mitochondrially targeted protein.
Analysis of the DUT-N and DUT-M
protein species by mass spectrometry indicates that the two forms of
dUTPase are largely identical except for a short region at their amino
termini. The fact that the nuclear and mitochondrial forms are so
similar in amino acid sequence raises the possibility that they are the
result of alternative splicing or differential transcription from
separate promoters within the same gene. There are several examples of
proteins that are partitioned or distributed to different intracellular
compartments through the use of alternative splicing (for review, see
Smith et al.(38) ). The actin filament-severing
protein gelsolin is expressed as a plasma and a cytoplasmic protein.
The two proteins are identical except for 25 amino-terminal residues
and are expressed by different promoters within the same gene. In
addition, they undergo differential alternative splicing of 5` exons to
generate distinct amino termini(39) . It is possible that
expression of the nuclear and mitochondrial forms of dUTPase is
regulated through the use of an analogous alternative splicing
mechanism. The data presented in this report are consistent with this
model. Northern blot analysis of HeLa poly(A) mRNA reveals two messages
of 1.1 and 2.3 kb, respectively. ()The more abundant 1.1-kb
message appears to correspond in size to the nuclear dUTPase. It is
possible that the 2.3-kb mRNA species may correspond to the larger
mitochondrial dUTPase. It will be of interest to determine the genomic
organization of the two dUTPase isoforms as well as to uncover the
mechanisms of expression.
In addition to potential cancer chemotherapy, inhibition of human dUTPase may also hold promise as an antiviral therapy as well. There has been evolutionary pressure to conserve the dUTPase function in many viral genomes, and loss of the viral encoded enzyme lowers replication efficiency in certain viruses(16, 18, 19) . It has been postulated that viruses that do not encode a dUTPase function (such as HIV) must rely entirely on the host enzyme(20) . Thus, the human dUTPase enzyme should also be considered as a potential candidate in the search for new anti-HIV targets.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U31930[GenBank].