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From the
Received for publication, July 23, 2001, and in revised form, August 23, 2001
Purification from calf thymus of a DNA
N-glycosylase activity (HMUDG) that released
5-hydroxymethyluracil (5hmUra) from the DNA of Bacillus
subtilis phage SPO1 was undertaken. Analysis of the most
purified fraction by SDS-polyacrylamide gel electrophoresis revealed a multiplicity of protein species making it impossible to
identify HMUDG by inspection. Therefore, we renatured the enzyme after
SDS-polyacrylamide gel electrophoresis and assayed slices of the gel
for DNA N-glycosylase activity directed against 5hmUra. Maximum enzymatic activity was identified between molecular mass markers 30 and 34 kDa. Protein was extracted from gel slices and subjected to tryptic digestion and analysis by mass spectrometry. Analysis revealed the presence of 11 peptides that were homologous or
identical to the sequence of the recently characterized human single-stranded monofunctional uracil DNA N-glycosylase
(hSMUG1). The cDNA of hSMUG1 was isolated and expressed as a
recombinant glutathione S-transferase fusion protein that
was shown to release 5hmUra with 20× the specific activity of the most
purified bovine fraction. We conclude that hSMUG1 and HMUDG are the
same protein.
5-Hydroxymethyluracil
(5hmUra)1 is formed in DNA
exposed to ionizing radiation both in solution and in cells. Its
formation has been posited to be caused by the indirect action of
ionizing radiation under aerobic conditions via the hydroxyl
radical-mediated abstraction of a hydrogen radical from the
5-methyl-moiety of thymine followed by oxidation via molecular oxygen
(1). 5hmUra has also been identified in human urine and in the DNA of
white cells, and the amount has been found to vary under differing
conditions of oxidative stress (2-4). Human autoantibodies directed
against 5-hydroxymethyl-2'-deoxyuridine in DNA have been identified,
and the titer has been correlated with relative risk for certain types of cancer (5).
Despite identification as a radiation product nearly 20 years ago, the
biological consequences of the formation of 5hmUra from thymine in DNA
remain uncertain. 5hmUra is a normal constituent of SPO1 Bacillus
subtilis phage DNA (6), functioning as a coding partner for
adenine in place of thymine, and is present in large amounts in the DNA
of dinoflagellates (7). Nonetheless, 5hmUra is a substrate for a DNA
N-glycosylase activity, 5hmUra DNA N-glycosylase (HMUDG), which releases 5hmUra from the DNA backbone (8, 9). Compared
with most other DNA N-glycosylases, HMUDG activity is unusual in that no comparable activity has been detected in bacteria (10) or yeast (9).
We undertook a study of the phylogenetic distribution of HMUDG activity
(11), and our results indicate that the phylogenetic distribution of
HMUDG was linked to the use by the organism of 5-methylcytosine
(5meCyt) as a mediator of gene expression. We suggested that 5hmUra
could be formed in DNA via two pathways. One pathway was the oxidation
of the 5-methyl group of thymine yielding 5hmUra directly.
Alternatively, 5hmUra may arise via oxidation of the 5-methyl group of
5meCyt yielding 5hmCyt followed by deamination of 5hmCyt to
5hmUra (12). The propensity of 5hmCyt residues to deaminate to 5hmUra
has long ago been observed in the DNA of the Escherichia
coli T-even phages. These phages utilize glucosylated 5hmCyt as a
normal constituent of DNA and exhibit a very high rate of C to T
transitions upon heating (13). Thus, in cellular DNA the oxidation and
subsequent deamination of 5meCyt, if left unrepaired, may result in a C
to T transition in CpG islands.
We previously reported the partial purification of HMUDG from calf
thymus and demonstrated that HMUDG activity was distinct from the major
uracil DNA N-glycosylase (UDG) activity of the cell (12).
However, we have been unable to achieve a degree of purification
sufficient to identify the HMUDG protein species with certainty from
examination of an SDS-PAGE analysis of our most purified calf thymus
fraction. In this report we describe the methods by which we were able
to unequivocally demonstrate that HMUDG is the recently characterized
single-stranded monofunctional uracil DNA N-glycosylase
(SMUG1) (14).
Purification of HMUDG from Calf Thymus--
Fresh calf thymus
obtained from local abattoirs, generally weighing between 1.2 and 2 kg, was trimmed of connective tissue. 600-g portions were
homogenized in a Waring Blendor together with 3 volumes of Buffer A (25 mM HEPES pH 7.4, 7.5 mM EDTA, 1 mM
dithiothreitol, and 100 µl of protease inhibitor mixture (Sigma) per
1.0 liter of buffer). The total volume of homogenate from a typical
preparation was between 5 and 7 liters.
To this homogenate 5 M NaCl was added to a final
concentration of 320 mM NaCl. The resulting gelatinous
precipitate of nucleoprotein was removed by spooling onto a 10-ml glass
pipette. The remaining liquid was filtered through cheesecloth followed
by clearing via centrifugation in the GS-3 rotor of a Sorvall RC-5B
centrifuge at 9000 rpm for 20 min to yield Fraction I (15).
Fraction I was precipitated with solid
(NH4)2SO4 to a final concentration
of 70%, the pH continually being adjusted with NH4OH to
maintain pH 7.5. After centrifugation, the pellets were washed with
100% (NH4)2SO4 and stored at
4 °C in aliquots representing the pelleted material from 500 ml of
the original 70% (NH4)2SO4 suspension, yielding Fraction II.
Fraction II was dialyzed overnight against Buffer A and applied to a
100-ml bed cation exchange resin in an XK 26/20 column (SP Fastflow,
Amersham Pharmacia Biotech). Aliquots of Fraction II applied to the
column averaged 3-4 g of protein/sample, which was half the maximal
binding capacity of the resin. The column was washed with Buffer A
until absorbance of the eluent at 280 nm was at a minimum. Protein was
eluted with a 1-liter gradient of Buffer A increasing in NaCl
concentration from 0 to 1 M at a flow rate of 5 ml/min.
Fractions were collected as 10-ml aliquots and were assayed for HMUDG
activity as described below. Fractions containing HMUDG activity were
pooled as Fraction III. Fraction III was adjusted to 70%
(NH4)2SO4 by the addition of solid
(NH4)2SO4 and then pelleted by
centrifugation to make Fraction IV, which was stored in aliquots at
4 °C.
Aliquots of Fraction IV, dissolved in Buffer A in 400 mM
NaCl, were applied to a Hiload chromatography Superdex-75 preparative column that had been calibrated with the gel filtration low molecular weight calibration kit (Amersham Pharmacia Biotech). The column was
eluted at a flow rate of 2.5 ml/min using 400 mM NaCl
Buffer A, and fractions were collected as 5-ml aliquots. Fifty
microliters of each aliquot were assayed for HMUDG activity, and
aliquots containing HMUDG activity were pooled as Fraction V.
Fraction V was pooled and concentrated using a Mono-S column (Amersham
Pharmacia Biotech, HR10/10) equilibrated with Buffer A in 20 mM NaCl. The protein was eluted with a 20 mM to
1 M NaCl 100-ml gradient of Buffer A and collected as 1-ml
aliquots. Aliquots containing HMUDG activity were pooled as Fraction VI.
Fraction VI, which was ~200 mM with respect to NaCl as
determined by measurement of conductance, was diluted 10-fold to 20 mM NaCl with Buffer A and then applied to an 80-ml
heparin-Sepharose CL-6B column (Amersham Pharmacia Biotech). The
protein was eluted with a 20 mM to 1 M NaCl
800-ml gradient of Buffer A at a flow rate of 2 ml/min and collected as
8-ml aliquots. Aliquots containing HMUDG activity were pooled as
Fraction VII.
Fraction VII was pooled and dialyzed in 20 mM NaCl Buffer A
and applied to a 5-ml single-stranded DNA cellulose column (Sigma). The
column was pre-equilibrated with dialysis buffer. Protein was eluted
with a 20 mM to 1 M NaCl 50-ml gradient of
Buffer A at a flow rate of 0.5 ml/min, and 1-ml aliquots were
collected. Aliquots containing HMUDG activity were pooled as Fraction VIII.
Assay of HMUDG Activity--
[3H]SPO1 phage DNA,
prepared as described previously, was used as substrate (12). 3 mCi of
[5,6-3H]uridine (PerkinElmer Life Sciences) were added to
a phage-infected 100-ml log phase bacterial culture. This yielded
between 30 and 50 µg of phage DNA of a specific radioactivity of
between 105 and 2 × 105 cpm/µg as
measured by A260 (1 OD unit is 50 µg of
double-stranded DNA). Enzymatic digestion of SPO1 DNA to
2'-deoxyribonucleosides followed by HPLC analysis attached to an
on-line liquid scintillation counter revealed 65-70% of the
radioactivity to be in Cyt moieties and 30-35% to be in 5hmUra
moieties. Non-radioactive Cyt and 5hmUra (Sigma) were used as
UV-absorbing migration standards (12, 16).
Assays of HMUDG activity were performed in 200-µl volumes in Buffer A
(12). The reactions were terminated after 30 or 60 min, and the
macromolecules were precipitated with 100 µl of BSA (50 mg/ml) and 5 ml of ice-cold acetone. After standing for 15 min at 4 °C the tubes
were centrifuged in a Beckmann 6KR centrifuge at 4000 rpm for 15 min
and the supernatant acetone decanted. The acetone was evaporated under
the hood, and the dried residue was redissolved in 500 µl of
H2O. The radioactive content of the aqueous solution
containing the redissolved residue was measured in a liquid
scintillation counter. The specificity of the HMUDG reaction was
confirmed by HPLC analysis coupled with an on-line liquid scintillation
counter, of an aliquot of the aqueous supernatant using non-radioactive
5hmUra as a UV-absorbing migration standard (12).
Purification of HMUDG by SDS-PAGE Followed by Renaturation of the
Enzyme--
Electrophoresis was performed according to the procedures
of Hilbert et al. (15) with minor modifications. The protein
sample to be analyzed was boiled for 10 min and then loaded and run on a 13% gel (5% stacking gel) with low range molecular size markers (Bio-Rad) at 80 mA. Samples were applied to duplicate lanes to allow
one lane to be used for mass spectrometric analysis. After electrophoresis the gel was divided, and the portion to be used for
molecular weight alignment was stained with Coomassie Blue. The
unstained portion was kept in buffer. The marker gel was then destained
and soaked in double-distilled H2O to allow it to expand to
its original size. The stained and unstained gels were aligned on a
sheet of parafilm. The unstained gel was cut into 0.4-cm slices.
One-third of each slice was reserved for mass spectrometry, and the
remainder of each slice was placed into 1.5-ml Eppendorf tubes with 400 µl of elution buffer (25 mM HEPES pH 7.9, 0.5 mM EDTA, 50 mM KCl, 1 mM
dithiothreitol, and 0.1 mg/ml acetylated BSA).
The following renaturation procedure was adopted from that of
Neddermann et al. (21). The slices were homogenized (using a
microsample Teflon pestle (Scienceware) attached to a hand drill) until
no large pieces of gel remained. The suspension was then vigorously
shaken overnight at 37 °C. The gel suspension was centrifuged at
14,000 rpm for 15 min at 4 °C in the GS-3 rotor of a Sorvall RC-5B
centrifuge. The supernatant was transferred to a 2-ml Eppendorf tube,
and 1.6 ml of ice-cold acetone (4 volumes) were added. The samples were
maintained in an ethanol/dry ice bath for 30 min and then centrifuged
at 14,000 rpm for 10 min at 4 °C. The supernatant was removed, and
the pellet was washed with 600 µl of 80% acetone, 20% dilution
buffer (100 mM HEPES pH 8.3, 2 mM EDTA, 10 mM NaCl, 2 mM dithiothreitol, and 1 mg/ml BSA).
The pellet was drained and dried at room temperature for 10 min
followed by resuspension in 37.5 µl of guanidine buffer
(dilution buffer plus 6 M guanidine HCl). After incubation
for 30 min at room temperature, 1.875 ml of dilution buffer was added,
and the samples were incubated at 4 °C overnight to permit renaturation.
The samples were concentrated in Centricon-10 microconcentrators that
had been washed with 0.5 ml of dilution buffer containing 0.5 mg/ml BSA
by centrifugation at 4 °C at 6000 rpm for 45 min. The samples were
then loaded onto the microconcentrators and centrifuged at 4 °C at
6,000 rpm for 1 h. The resulting retentate (generally 75-125
µl) was assayed for HMUDG activity.
Identification of HMUDG by Mass Spectrometry--
Six gel slices
containing proteins of apparent molecular mass between 23 and 35 kDa
were excised from an unstained SDS-PAGE gel under a tissue culture hood
to minimize contamination. Two-thirds of each gel slice was assayed for
HMUDG activity as described above. The remaining third was fixed in
50% methanol, 5% acetic acid in water for 20 min and then washed with
50% methanol and water to remove the remaining SDS and acetic acid.
The gel slices were cut into 1 mm3 pieces, reduced,
alkylated using iodoacetamide, and digested in-gel with trypsin (17).
The peptides were extracted, dried under vacuum, and redissolved in 20 µl of 5% acetic acid. Samples were desalted and concentrated using
Zip-TipTM C18 micro-columns (Millipore).
Peptides were eluted with 2 µl of 70% acetonitrile. The final
solution was adjusted to contain 50% acetonitrile, 1% acetic acid and
then loaded into a gold-coated medium nanoelectrospray needle (Protana).
MS and MS/MS spectra were acquired on a quadrupole time-of-flight
tandem mass spectrometer equipped with a Z-spray nano-ESI ion source
(Micromass, Manchester, UK). The instrument was calibrated in MS/MS
mode using 1 pmol/µl [Glu1]-Fibrinopeptide B (Sigma) in
50% methanol/1% acetic acid. The capillary voltage used was 1400 V,
and the cone voltage was set to 40 V. Tryptic peptide precursor ions
(singly, doubly, or triply charged) were selected by the quadrupole
mass filter (MS1) and induced to fragment by collision with Argon, and
the resulting product ions were analyzed by the time-of-flight mass
analyzer (MS2). The collision energy was varied between 15 and 45 V
depending on the mass and charge-state of the peptides. The recorded
MS/MS spectra were processed using MassLynx MaxEnt 3 (Micromass).
The charged state deconvoluted MS/MS spectra were directly used to
search the public data base using the Mascot search program (Matrix
Science, UK). For peptides with no exact matches in the public data
bases, the MS/MS spectra were sequenced de novo either manually or with the aid of the PepSeq program (Micromass). The amino
acid sequences obtained in this way were searched against the NCBI
nonredundant protein data base using the BLAST search program (18).
Expression and Purification of hSMUG1--
The hSMUG1 coding
sequence was polymerase chain reaction-amplified as a
BamHI-XhoI fragment from a hSMUG1 cDNA clone
(ATCC 294606) purchased from ATCC. The polymerase chain reaction
product was cloned into pGEX6P-1 (Amersham Pharmacia Biotech) so as to express the protein as an N-terminal GST fusion protein for
purification on a glutathione column. Expression of the recombinant
protein was induced in E. coli BL21-DE3 cells by the
addition of isopropyl- Assay for Uracil DNA N-Glycosylase Activity by Cleavage of Ura
Containing 2'-Deoxyribose Oligonucleotides--
2'-Deoxyribose
oligonucleotides were synthesized in the core facility of the NYU
School of Medicine, Department of Cell Biology. All 2'-deoxyribose
oligonucleotides were deblocked, deprotected, and purified by 20%
denaturing PAGE. A 26-mer uracil-containing 2'-deoxyribose
oligonucleotide of the sequence d(CGCGAAACGCCTAGUGATTGGTAGGG) was
labeled at the 5'-end using [
Assays were performed at 37 °C in buffer containing 50 mM HEPES, pH 7.5, 20 mM NaCl, 0.1 mg/ml BSA, 1 mM EDTA, and 1 mM dithiothreitol. Enzyme,
protein, and substrate were diluted to working conditions in assay
buffer and equilibrated at 37 °C. Assays contained 2 µM 32P-5'-end-labeled single-stranded or
duplex 26-mer uracil-containing 2'-deoxyribose oligonucleotide and 20 nM hSMUG1 with or without 0.3 units of uracil DNA
N-glycosylase inhibitor (New England Biolabs). The
concentration of hSMUG1 was determined using the Bradford assay with
albumin as standard and was assigned a molecular weight of 30,142.0 to
the fusion protein. The control assays for effectiveness of the uracil
DNA N-glycosylase inhibitor contained 0.3 units of uracil
DNA N-glycosylase (Life Technologies, Inc.). The enzyme solutions were incubated at 37 °C for 1 min prior to the addition of
substrate. The final volume was 10 µl. The reactions were incubated at 37 °C for 60 min and were terminated by snap-freezing in ethanol and dry ice and treated with 5 µl of 0.5 M putrescine, pH
8.0 (19). The treated reaction mixtures were then heated at 95 °C for 5 min followed by the addition of 15 µl of loading dye (95% deionized formamide, 10 mM EDTA, 0.05% bromphenol blue,
and 0.05% xylene cyanol). Samples were then heated at 95 °C for 5 min, and products were separated by 20% PAGE in 7 M urea
and Tris borate/EDTA (TBE). Products were analyzed via
phosphorimaging using a Molecular Imager FX System with Quantity One
Software (Bio-Rad) (20).
Purification of HMUDG from Calf Thymus--
A mammalian protein
with HMUDG activity was purified from fresh calf thymus on the basis of
the ability to excise [3H]5hmUra from
[3H]SPO1 DNA, as shown in Table
I. Calf thymus was used as the source of
enzyme because large amounts of fresh tissue are available, and our
previous studies revealed that the specific HMUDG activity of thymus
was higher than any other tissue except brain (12). The
purification scheme was similar to that used previously to purify human
endonuclease III homologue 1 (15), with the addition of an
(NH4)2SO4 concentration step. After
the final purification step, the enzyme had been purified ~500-fold,
with a yield of 0.2%. This estimate of degree of purity was based on
the specific activity of the HMUDG in the crude preparation after the
addition of 5 M NaCl and spooling out of the gelatinous
nucleoprotein precipitate resulting from the addition of NaCl. This
step actually increased the specific activity of HMUDG by 3-5-fold
from that of the crude homogenate, so the final degree of purification
(as measured by an increase in specific enzyme activity) was somewhere
between 1500- and 2500-fold.
Polyacrylamide Gel Electrophoresis of Purified Protein and
Renaturation of HMUDG--
The most purified fraction (Fraction VIII)
was analyzed by SDS-PAGE (Fig.
1A). The HMUDG protein could
not be definitively associated with a single Coomassie Blue-stained
band on the gel. To identify the region of the gel that contained
HMUDG, an identical gel lane was cut into fractions (as indicated in
Fig. 1A) and the proteins renatured. The renatured proteins
were assayed for HMUDG activity. The greatest amount of HMUDG activity
was present in slice 2 (Fig. 1B). The activity of renatured
protein corresponded to a protein with an apparent molecular mass of 34 kDa, reasonably consistent with the molecular radius of 29-30 kDa we
observed for the native bovine protein on the Superdex-75 column
(Fraction V).
Determination of Bovine HMUDG Amino Acid Sequence and Alignment
with hSMUG1 Amino Acid Sequence--
The nano-ESI mass spectrum
(m/z range of 300-1600) of peptides from slice 2 that showed the highest HMUDG activity is shown in Fig.
2A. In this complex spectrum,
more than 150 singly, doubly, or triply charged tryptic peptides were
observed, indicating that a mixture of proteins was present in this gel
slice. In the MS survey, we found that the signals of a number of
peptides observed in Fig. 2A were significantly weaker or
absent in the nano-ESI spectra of other gel slices (data not shown),
indicating that these peptides correlated with HMUDG activity.
Therefore, these peptides were further fragmented by collision-induced
dissociation to generate sequences or sequence tags. The recorded MS/MS
spectra were used for a data base search (see "Experimental
Procedures"). To illustrate this identification process, the MS/MS
spectrum of the doubly charged peptides at m/z
696.36 is shown in Fig. 2B. The complete y ion series
(y1-y14) could be assigned, allowing the
determination of the full peptide sequence as
SLAEGFLQELR (low collision energy MS/MS
analysis as used here cannot distinguish between the isobaric Ile and
Leu amino acid residues).
Direct data base searching with the uninterpreted MS/MS spectrum did
not result in a positive protein identification. However, searching the
NCBI protein data base using BLAST revealed that the bovine sequence
nearly matched a peptide sequence from human SMUG1, a recently
identified member of a family of uracil DNA N-glycosylases.
Two changed amino acids were Ser Isolation of the cDNA of hSMUG1 and Expression of the
Protein--
The human SMUG1 cDNA was isolated and expressed as a
GST fusion protein, which was purified and the GST moiety removed,
leaving 5 amino acids on the N terminus of the recombinant hSMUG1. When analyzed by PAGE, the purified recombinant protein was visualized as a
single band (Fig. 4) with an apparent
molecular mass slightly larger than that of the region of the gel where
the maximal activity of purified bovine HMUDG was recovered (Fig.
3).
Release of 5hmUra by hSMUG1 Compared with That of Purified Bovine
HMUDG (Fraction VIII)--
Purified expressed hSMUG1 was assayed for
its ability to release [3H]5hmUra from
[3H]SP01. At the concentrations assayed, there was a
linear increase of release of 5hmUra with the increase in hSMUG1
concentration (Fig. 5). The identity of
the released material was confirmed by HPLC. The specific activity of
purified recombinant hSMUG1 was ~20× greater than the most purified
fraction (Fraction VIII) of bovine HMUDG.
Confirmation of Uracil DNA N-Glycosylase Activity of Recombinant
hSMUG1--
Recombinant hSMUG1 was analyzed for its ability to remove
Ura from single- and double-stranded 2'-deoxyribose
oligonucleotides containing a single Ura residue (Fig.
6). The recombinant protein showed
maximal activity in a cleavage assay against single-stranded DNA.
hSMUG1 also displayed activity against double-stranded DNA containing a single Ura residue. As reported by Haushalter et al. (14), hSMUG1 activity was greater against mismatches (U:G) than against matches (U:A).
The activity of the recombinant hSMUG1 was distinct from UDG (in this
case recombinant bacterial UDG). UDG had greater activity against
double-stranded DNA containing U opposite A (Fig. 6, lane 7). Furthermore, UDG activity was suppressed by UGI in contrast to
hSMUG1 activity, which was not (Fig. 6, lane 8).
SMUG1 was identified by binding to synthetic uracil DNA
N-glycosylase inhibitors and has been definitively shown to
be distinct from the primary uracil DNA N-glycosylase UDG
and a second protein, thymine DNA N-glycosylase (TDG)
which preferentially repairs U:G and T:G mismatches (14). In
this study we have shown that a bovine protein with HMUDG activity, as
defined previously by Hollstein et al. (8) and this group
(9, 12), has peptide sequences homologous to those of hSMUG1, whereas
recombinant hSMUG1 is shown here for the first time to have HMUDG activity.
Previous observations of HMUDG activity in cells and tissues can now be
reevaluated in light of the determination of the identity of HMUDG as
SMUG1. The HMUDG activity was first searched for in bacteria, where it
could not be found (10), and it was subsequently shown to be absent in
yeast but present in more complex multicellular organisms (11). This
unique phylogenetic distribution has been confirmed for SMUG1 (14) in
contrast to UDG, which is highly conserved throughout phylogeny. The
uracil DNA N-glycosylase activity of SMUG1 is not inhibited
by the phage UGI. Our most purified fractions of bovine HMUDG also
displayed uracil DNA N-glycosylase activity that was not
inhibited by UGI (data not shown), further confirming the identity of
HMUDG as SMUG1. Additional support for the identity of HMUDG as SMUG1
comes from the finding that SMUG1 ESTs are present in a wide variety of
tissues (14, 22), consistent with our previous observation that HMUDG
activity is present in all tissues, and is most abundant in brain and
thymus (11).
The biological role of HMUDG/SMUG1 is still uncertain. Haushalter and
co-workers (14, 22) speculated that SMUG1 might play a role in the
repair of Ura residues resulting from deamination of Cyt residues in
DNA that is transiently single-stranded during transcription,
recombination, or replication. Recent work by the same group has
suggested that SMUG1 functions as an antimutator, with preference for
repair of Ura opposite guanine (22).
We previously proposed that HMUDG might repair 5hmUra residues arising
from oxidation and deamination of 5meCyt residues in DNA that, if
unrepaired, would be mutagenic. Such a hypothesis is supported by the
common phylogenetic distributions of the use of 5meCyt as a regulatory
element in transcription, HMUDG enzyme activity, and SMUG1 EST
sequences. Recent evidence further supports this hypothesis. It was
shown that 5hmUra, as part of 5hmUra:G mismatches, was repaired to a
significantly greater extent than 5hmUra:A in identical sequences using
partially purified HMUDG from Hela cells (23).
Regardless of which of the two putative functions is of greater
significance to the maintenance of genomic integrity by HMUDG/SMUG1, it
should be noted that a mutant Syrian hamster cell line (V79) deficient
in HMUDG activity, which we developed, is viable and grows normally
(24). Although it seems evident that HMUDG/SMUG1 protects cells from
mutations arising from conversion of 5meCyt to 5hmUra and deamination
of Cyt to Ura, the possibility remains that HMUDG/SMUG1 is also
involved in a sequence of controlled oxidation of 5meCyt followed by
deamination and removal of 5hmUra as a mechanism of changing the
methylation status of specific regions of DNA.
Full understanding of the biological significance of HMUDG/SMUG1 will
require an explanation of both its unique in vitro substrate specificities and its phylogenetic distribution.
We thank Dr. Lawrence C. Sowers for valuable
discussions and remarkable insight.
*
This work was supported by National Institutes of Health
Grants CA 16669 and CA 49869 (to G. W. T.), CA 16087 (to Kaplan
Comprehensive Cancer Center) and 5T32 CA-09161 (to D. R. M.), and a
Whitehead Presidential Fellowship (to T. A. N.).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.
Published, JBC Papers in Press, August 28, 2001, DOI 10.1074/jbc.M106953200
The abbreviations used are:
5hmUra, 5-hydroxymethyluracil;
HMUDG, 5hmUra DNA N-glycosylase;
UDG, uracil DNA N-glycosylase;
SMUG1, single-stranded
monofunctional uracil DNA N-glycosylase;
hSMUG1, human
SMUG1;
5hmCyt, 5-hydroxymethylcytosine;
5meCyt, 5-methylcytosine;
UGI, uracil glycosylase inhibitor;
ESI, electrospray ionization;
EST, expressed sequence tag;
PAGE, polyacrylamide gel electrophoresis;
HPLC, high performance liquid chromatography;
MS, mass spectrometry;
BSA, bovine serum albumin;
GST, glutathione S-transferase.
Definitive Identification of Mammalian 5-Hydroxymethyluracil DNA
N-Glycosylase Activity as SMUG1*
,,,,
Department of Pathology and Kaplan
Comprehensive Cancer Center, the § Department of
Pharmacology and Skirball Institute of Biomolecular Medicine, and the
¶ Department of Cell Biology, New York University School of
Medicine, New York, New York 10016
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D thiogalactopyranoside to a
final concentration of 0.5 mM. A 1-liter bacterial culture
at an A600 of 0.6 was induced at 30 °C
for 5 h and harvested by centrifugation at 4 °C at 3200 × g in a Beckman 6KR centrifuge. The recombinant protein was
purified from the induced cell pellet using glutathione-Sepharose 4B
affinity medium (Amersham Pharmacia Biotech) per the manufacturer's
protocols for batch purification of fusion proteins. Following
purification, the glutathione S-transferase moiety was
removed according to the manufacturer's instructions using Prescission
Protease (Amersham Pharmacia Biotech), leaving five amino acid residues
remaining on the N terminus of the recombinant hSMUG1 (20).
-32P]ATP (PerkinElmer
Life Sciences) and T4 polynucleotide kinase (Life Technologies, Inc.).
For assays involving DNA duplexes, the 5'-end-labeled 26-mer
uracil-containing 2'-deoxyribose oligonucleotide was annealed to the
corresponding complementary strand (containing either an A or G at
position 12) in a 1:2 molar ratio.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Summary of purification of a 5-hydroxymethyluracil DNA N-glycosylase
activity from calf thymus
View larger version (12K):
[in a new window]
Fig. 1.
Purification and renaturation of HMUDG from
SDS-PAGE. A, gel electrophoresis of the most purified
fraction of HMUDG. Indicated regions were cut out for analysis by mass
spectroscopy and for assay of HMUDG activity. B, HMUDG
activity of renatured protein. Renatured protein from the indicated
regions of the gel was assayed as described under "Experimental
Procedures." Assays were run for 12 h.
View larger version (30K):
[in a new window]
Fig. 2.
Mass spectrometry analysis of a tryptic
digest of gel slice 2 (33-34 kDa) of an unstained SDS-PAGE gel.
A, nano-ESI MS spectrum in the m/z
region from 300 to 1600. A number of peptide ions were selected for
fragmentation (MS/MS), and 13 of those peptides (marked with a
point and summarized in Table I) corresponded to peptides
from the protein SMUG1. The inset demonstrates the
monoisotopic resolution of a doubly charged ion at
m/z 696.36. B, nano-ESI MS/MS spectrum
of the m/z 696.362+ ion. The complete
y-ion series (y1-y14) and assigned amino acid
sequence are indicated.
Gly(G) and
GluGln(Q). The probability that this identification was
correct was high because a total of 13 peptide ions in Fig.
1A, corresponding to 11 peptide sequences obtained by MS/MS
(Table II), were found to match or nearly
match sequences from hSMUG1. The alignment of these bovine sequences
with the hSMUG1 sequence is shown in Fig.
3.
Peptide sequences obtained by MS/MS and matched to SMUG1
View larger version (46K):
[in a new window]
Fig. 3.
Alignment of peptides from tryptic digests of
purified bovine HMUDG with the sequence of hSMUG1. The amino acid
sequences were obtained by mass spectroscopic analysis.
View larger version (19K):
[in a new window]
Fig. 4.
Expression of the cDNA of hSMUG1.
The cDNA was isolated, and hSMUG1 was expressed as described under
"Experimental Procedures." An aliquot was analyzed by
SDS-PAGE.
View larger version (17K):
[in a new window]
Fig. 5.
Comparison of the release of 5hmUra from SPO1
DNA by hSMUG1 and purified bovine HMUDG. Indicated amounts of
purified recombinant hSMUG1 (black bars) and Fraction VIII
(gray bars) of bovine HMUDG were analyzed for the ability to
release [3H]5hmUra from [3H]SPO1 DNA.
View larger version (20K):
[in a new window]
Fig. 6.
Activity of hSMUG1 against uracil containing
single- and double-stranded DNA containing matched and mismatched
uracil. Recombinant hSMUG1 was assayed for the ability to cleave
uracil containing 2'-deoxyribose oligonucleotides in the presence or
absence of UGI (lanes 2-7). The oligonucleotides were
either single- or double-stranded with uracil opposite A or G, as
shown. The left lane shows oligonucleotide in the absence of
enzyme. The right two lanes (8 and 9)
show the activity of recombinant UDG in the presence or absence of
UGI.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENT
FOOTNOTES
To whom correspondence should be addressed: Dept. of
Pathology, New York University Medical Center, 550 First Ave., New
York, NY 10016. Tel.: 212-263-5473; Fax: 212-263-8211; E-mail:
george.teebor@med.nyu.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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